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Phosphorus nutrition in farmed Atlantic salmon (Salmo salar): Life stage and temperature effects on bone pathologies	Bone health is important for a viable and ethically sound Atlantic salmon aquaculture industry. Two important risk factors for vertebral deformities are dietary phosphorus and water temperature. Here, we explore the interplay between these two factors during a full production of Atlantic salmon. Salmon were fed one of three diets (low 4.4–5.0 g kg−1, medium 7.1–7.6 g kg−1, or high 9.0–9.7 g kg−1 soluble phosphorus) from 3 to 500 g body weight, followed by a common diet of 7.3 g kg−1 soluble phosphorus until harvest size at 4 kg. Additional groups were included to investigate the effects of water temperatures of 10 vs 16 °C (low and high diets only) and the switching of dietary phosphorus levels (from low to medium or high, from medium to low or high, from high to low or medium), starting at seawater transfer (~100 g body weight) and lasting for 4 months (~500 g body weight). During the experimental feeding period, the low phosphorus diet caused reduced bone mineralization and stiffness and a greater prevalence of vertebral deformities, compared to the medium and high phosphorus diets. However, the prevalence of severely deformed fish at harvest was reduced by switching from the low to either the medium or high phosphorus diets for 4 months after seawater transfer, followed by rearing on the standard commercial feed. Concurrently, switching from either the medium or high to a low phosphorus diet for the same period following seawater transfer had no effect on vertebral deformities at harvest. The higher water temperature for 4 months following seawater transfer increased the severity of deformities at harvest, irrespective of dietary phosphorus. Finally, low dietary phosphorus was associated with increased fillet damage, due to ectopic connective tissue around the spine, at harvest. In conclusion, dietary phosphorus levels of 5 g kg−1 for the initial 4 months in seawater are more of a risk factor for vertebral pathologies if preceded by low, but not medium or high, dietary phosphorus in freshwater. However, dietary phosphorus levels may not play a role in temperature induced radiologically detectable vertebral pathologies. Under the reported growing conditions and diet compositions, a combination of 7.5–7.6 g kg−1 soluble phosphorus during freshwater and 5.0 g kg−1 for the first 4 months in seawater, was sufficient for normal bone health and growth in Atlantic salmon.	[ "AGRI" ]	[ "Fillet damage", "Mineralization", "Vertebral fusion", "X-ray" ]	[ "1104" ]	[ "A normal developing skeleton is a prerequisite for sustainable production and animal welfare, but vertebral column deformities are a persistent concern for farmed Atlantic salmon (Salmo salar) (Fjelldal et al., 2012a).", "Vertebral deformities reduce growth performance (Hansen et al., 2010), increase an individual's energy demand (Powell et al., 2009), and result in downgrading at harvest (Fraser et al., 2013).", "In some cases, vertebra deformities may also result in problems during filleting due to tissue damage (Sullivan et al., 2007).", "Dietary phosphorus supply has been highlighted as one of the key factors in the aetiology of vertebra deformities in farmed salmon (Bæverfjord et al., 2018), although a phosphorus deficient diet alone in some cases may not induce skeletal deformities (Gil-Martens et al., 2012; Witten et al., 2016).", "Phosphorus is an essential nutrient for fish with many metabolic functions as it is a constituent of bone, scales, adenosine triphosphate, cell membranes, and nucleic acids (Skonberg et al., 1997).", "Due to the low phosphorus salt content and inefficient absorption from water, phosphorus is supplied via the diet (Lall, 2003).", "If too low, dietary phosphorus leads to insufficient bone mineralization and a reduced mechanical stiffness of the vertebrae (Baeverfjord et al., 1998), which can result in the development of compressed vertebrae (Fjelldal et al., 2009).", "Although many papers have dealt with dietary phosphorus requirements during both freshwater (Fjelldal et al., 2012b; Helland et al., 2005; Ketola, 1975; Smedley et al., 2018; Vielma and Lall, 1998; Åsgård and Shearer, 1997) and seawater (Albrektsen et al., 2009; Baeverfjord et al., 1998; Fjelldal et al., 2009, 2012c) life stages separately, the combined effect of phosphorus nutrition in both environments has never been investigated.", "Water temperature is also an important factor in the aetiology of vertebral column deformities in farmed salmon (Fraser et al., 2015; Grini et al., 2011; Ytteborg et al., 2010).", "However, the link between temperature and mineralization is not obvious.", "For example, elevated water temperature (16 °C) during the early seawater period resulted in more vertebra deformities than a lower seawater temperature (10 °C), but there was no temperature effect on mineralization (Grini et al., 2011).", "Therefore, the negative effect on normal bone development imposed by elevated temperature may be mediated via other mechanisms such as altered bone remodelling (Wargelius et al., 2010).", "So far, the combined effect of phosphorus nutrition and water temperature on the development of vertebra pathologies has not been studied.", "Our aim was to determine whether dietary phosphorus requirements, primarily regarding bone health, can interact with life stage or water temperature.", "Therefore, we investigated growth, markers of bone health, and vertebral deformities in Atlantic salmon fed different dietary phosphorus levels from 3 to 500 g body weight, first in 13 °C freshwater (3–100 g body weight) and then either 10 or 16 °C in seawater (100–500 g body weight), and then followed up on one common diet at 8 °C until harvest size of 4 kg.", "The temperatures used throughout the study are based on those that are encountered during a typical production cycle.", "The dietary soluble (≈digestible) phosphorus levels used ranged from 4.4–9.7 g kg−1, a range that is above and below the official recommendation of 8 g kg−1 available phosphorus for farmed Atlantic salmon (NRC 2011).", "All experiments were conducted in accordance with the laws and regulations of the Norwegian Regulation on Animal Experimentation 1996.", "Atlantic salmon larvae (n = 2280) with a mean weight of 2.8 g were provided by Marine Harvest (Mowi strain) and transported to Skretting ARC Lerang Research Station, from Vågafossen Settefisk AS, on the 13th March 2012 (day 0), whereupon they were kept on continuous light.", "A commercial feed was produced according to the ingredient composition shown in Table 1.", "All chemical analyses described below were carried out in duplicate.", "A method for fractional extraction and analysis of NaOH soluble phosphorus was used as a predictor of digestible phosphorus in the diets (Albrektsen et al., 2018), based on the assumption that the chemical form and solubility of phosphorus are the main criteria for intestinal phosphorus absorption (Groote, 1986; Nordrum et al., 1997).", "The diets were produced to contain 3 different levels of phosphorus; low, medium, high that corresponded to 4.0–4.5 g, 7.0–7.5 g, and 9.0–9.5 g soluble phosphorus per kg of diet, respectively.", "Total phosphorus was determined by a spectrometric method (ISO 6491–1998, https://www.iso.org/standard/12864.html), while NaOH-extractable (soluble) phosphorus was determined following incubation of duplicate ingredient samples (0.8 g) in 80 mL of 1 M NaOH for 16 h according to a procedure described in Ruban et al. (2001) and later modified by Hua et al. (2005).", "The method on soluble phosphorus was modified and validated by Hovde (2013).", "By using this method, it is not possible to differentiate between phytate phosphorus in the plant ingredients (unavailable) and other soluble and available forms of phosphorus.", "Therefore, digestible phosphorus levels in the diets (Table 1) may be slightly overestimated by about 15% due to variable phytate phosphorus levels in the plant ingredients.", "The chemical contents of the diets showed that the intended contents of nutrients and expected levels of phosphorus were largely achieved following the subtraction of phytate phosphorus.", "The low phosphorus diet fed in the period after transfer (100–500 g fish) contained 5 g kg−1 soluble phosphorus, which is slightly higher than expected.", "For a schematic of the experimental design and environmental conditions see Fig. 1.", "Briefly, juveniles were divided equally between 3 dietary regimes; low (LP), medium (MP), and high (HP) phosphorus (8 tanks/diet, 95 fish/tank).", "Experimental feeding began on day 14 and all fish were vaccinated on day 98 with a standard 6 component vaccine (Alphaject micro 6, Pharmaq AS, Oslo, Norway).", "On day 155, the fish (approx. 100 g) were divided between 33 tanks (31–37 fish/tank) supplied with full strength seawater.", "Of these, 27 tanks were maintained at 16 °C, whereas 6 tanks were maintained at 10 °C.", "On day 156, the fish were divided into 11 groups; each original dietary group was divided into three with one group remaining on the freshwater diet while the other groups were fed one of the two alternate diets creating nine dietary groups (LP-LP, LP-MP, LP-HP, MP-LP, MP-MP, MP-HP, HP-LP, HP-MP, HP-LP) that were reared on 16 °C.", "A further two groups of the LP-LP and HP-HP combination were reared on 10 °C.", "All fish were tagged with a passive integrated transponder between days 266–267 to allow for individual recognition.", "On day 278, all groups were divided equally between 12 common garden tanks (63–66 fish/tank) where they were maintained on 8 °C and fed a common diet (Skretting AS Spirit Pluss, 9.1 and 7.3 g kg−1 total and soluble phosphorus [analysed values, as above], respectively) until the end of the experiment on day 686.", "Fish were overfed by 20–30% throughout the experiment.", "Feeding was continuous from the start until day 174 whereupon it was switched to 3 feeds per day until day 234, when it was switched to 2 daily feeds until the end of the experiment.", "Individual fish weight and length were recorded on day 69, 266, 394, and at harvest on day 686.", "Body size data was used to calculate body condition (i.e. K factor) using the following equation; K = weight [g]/length3 [cm] × 100.", "Tank biomass was also recorded at the beginning of the experiment.", "Growth was calculated as the % mass gain/day (i.e. specific growth rate [SGR]) as follows; (eq − 1)100 (Houde and Scheckter, 1981), where q = [In(W2) − In(W1)](t2 − t1)−1 (Bagenal and Tesch, 1978), and W2 and W1 are average body mass at times t1 and t2, respectively.", "The feed conversion ratio (FCR) was calculated on a dry matter basis per tank (FCR = feed consumed/weight gain) between days 69–100, 100–153, 153–278.", "Prior to the fish being 20 g, the FCR data was not calculated as the food pellets were too small to collect.", "The data for one MP diet tank was lost for the period June to August 2012.", "The fish were anaesthetized with 0.1 g L−1 of finquel (FINQUEL® vet.; Western Chemical Inc., Washington, DC, USA) before measurement.", "Externally visible deformities, sexual maturation (assessed by visual examination), and the occurrence of cataracts were recorded on day 266, 394, and at harvest on day 686.", "The cataract score was recorded at harvest and the scoring based on Wall and Bjerkås (1999).", "In brief, the scoring was done under darkened conditions by use of a slit lamp (Kowa SL-5 with 15· magnification).", "The severity was scored on a scale from 0 (no changes) to 4 (complete cataracts).", "The cataract scores per individual are given as the sum of both eyes, ranging from 0 to 8.", "Fish were sampled for radiology on days 153, 274, and 686.", "Vertebral ash content was measured on days 69, 155, 266, and 686.", "Vertebral stiffness was measured on day 69.", "Whole body phosphorus and the Ca:P ratio was measured on days 69, 155, and 266.", "Analysis of vertebral body deformities is based on the examination of radiographs.", "At sampling, whole fish were used for lateral radiographs and evaluated for vertebra deformities according to Witten et al. (2009).", "The vertebral columns were radiographed (Porta 100 HF; Eickemeyer Medizintechnik für Tierärzte KG, Tuttlingen, Germany) onto a 35 × 43 cm image plate in a rigid cassette (Dürr Medical, Bietigheim-Bissingen, Germany) with 40 kV and 10 mAs at a distance of 70 cm.", "The image plate was scanned (CR 35 VET; Dürr Medical) and the resulting image converted into a TIFF file (Vet-Exam Plus Software, version 4.14.0).", "Vertebrae numbers 40–43 were dissected, the neural and haemal arches were removed, and the amphicoelus centra were used for mechanical testing and measurement of total ash (mineral) content.", "The vertebrae were compressed in the cranial-caudal direction using a texture analyser (TAXT2 Texture Analyser, Stable Micro Systems, Haslemere, UK) with a steadily advancing piston (6 mm min−1).", "The resulting load-deformation data were continuously recorded and the stiffness calculated for each vertebra according to Fjelldal et al. (2004).", "After mechanical testing, vertebrae number 40–43 of each fish were pooled, defatted in acetone and chloroform baths, dried overnight at 100 °C, and then incinerated for 11.5 h in a muffle furnace (Mod.", "L40, Nabertherm GmbH, Bremen, Germany) (115 °C for 0.5 h, 540 °C for 5 h, and 750 °C for 6 h according to Kacem et al., 2000).", "The dry and total ash contents of each individual were weighed to the nearest 10−2 mg.", "The total ash content was calculated as follows: Total ash content (% dry weight) = (total ash weight × 100) × (dry weight)−1.", "Total phosphorus in pooled samples of whole-body were determined spectrophotometrically (430 nm) after ashing and treatment in 6 M HCl (ISO 6491-1998).", "Freeze dried samples of whole-body were analysed for total phosphorus (P) and calcium (Ca), and the whole-body Ca:P ratio calculated (whole-body Ca/whole-body P).", "Whole-body Ca was determined by atomic absorption spectrometry (ISO 6869-2000, https://www.iso.org/standard/33707.html).", "Macroscopic damage to the fillet, due to the occurrence of connective tissue, was scored as either present or absent at harvest.", "The fish were carefully filleted by hand and both fillets were evaluated for occurrence of excessive connective tissue.", "The structure of the excessive connective tissue allowed normal hand filleting without applying extra force.", "Excessive connective tissue was white and covered the area of the musculature closest to the vertebra column caudally to the anus (Fig. S1).", "Fish that had excessive connective tissue present in both fillets were classified as “damaged”.", "Based on the radiological examination, spine sections were selected for biopsies and histological examination.", "From each group, 4 fish with typical malformations and 2 fish with non-deformed spines (controls) were selected, 24 biopsies in total.", "Histological samples were decalcified and embedded in paraffin.", "Para-sagittal serial sections (periphery of the vertebral body to the mid line) of 5 μm thickness were stained with Masons trichrome as described in Presnnell and Schreibman (1998).", "Sections were analysed with a Zeiss Axio Imager Z1 microscope and photographed with a Zeiss Axiocam.", "The data were transferred to R version 2.15.0 (R Development Core Team, http://www.r-project.org).", "Significance was assigned at p < .05.", "Data were checked for normality following visual examination of plots (i.e. histograms and/or q-q plots).", "Linear mixed effect (LME) models were used for body mass, body length, and body condition.", "The random effect being the individual fish to account for repeated measures.", "LME models were used to assess associations between cataract score, sexual maturity or external deformities and body mass at harvest, whereas linear models (LM) were used to assess group effects on bone strength, vertebral mineral content, whole body phosphorus, the Ca:P ratio, and FCR.", "General linear models (GLM) with a binomial distribution were used to assess the prevalence of cataracts, sexual maturation, externally visible deformities, fish with ≥1 deformed vertebra, fish with ≥10 deformed vertebra, and fillet damage.", "For a comparison of sexual maturation in the temperature comparison, the exact binomial test was used.", "Cataract score was non-parametric, and therefore analysed using the Kruskal Wallis test.", "Post-hoc tests included lsmeans for parametric data or the Dunns test for non-parametric data.", "The R libraries used were “nlme”, “MuMIn”, “multcomp”, “car”, “lsmeans”, and “dunn.test”.", "For models in which more than one explanatory variable was investigated, all possible 2-way interactions were initially allowed based on previous findings demonstrating that sex (Fraser et al., 2014), water temperature (Grini et al., 2011), and dietary phosphorus (Albrektsen et al., 2009; Deschamps et al., 2014; Fjelldal et al., 2016; Ketola, 1975; Prabhu et al., 2013; Åsgård and Shearer, 1997) can all influence growth in salmonids, and these effects can be transient.", "In addition, dietary phosphorus (Fjelldal et al., 2012d) and water temperature (Fjelldal et al., 2011) have been found to affect the level of early male sexual maturation leading to sex effects on growth.", "However, the final model was selected based on a comparison of all possible model combinations using the “dredge” command within the “MuMIn” library, with the final model being the one with the lowest akaike information criterion (AICc), i.e. the best data fit weighted against the number of variables.", "The “Anova” command within the “car” library was used to extract the results for the main effects.", "Type II sum of squares were used for models without interactions, whereas main effects were calculated using type III sum of squares when interactions were present within the final model.", "The body mass and body length data were natural log transformed prior to analysis.", "All the raw data (Fraser et al.xlsx) and the R script (Fraser et al.R) used to analyse the data can be found in Supplementary material.", "Due to the large number of groups within the experiment, the data was analysed in four ways; i) a comparison of being fed continuous LP, MP, or HP throughout the whole experiment, ii) a comparison of switching diet at seawater, iii) a comparison of being fed either the LP or HP throughout, but being exposed to different water temperatures at seawater transfer, and iv) general associations between harvest size and cataract score, sexual maturity, or external or radiologically identified deformities.", "This approached allowed for a more focused analysis and discussion of the research questions.", "The data on body size, condition, sexually maturity, and cataracts can be found in Table 2, while parameters regarding vertebral deformities can be found in Table 3.", "Significant explanatory variables for each parameter can be found within Tables 2 and 3.", "The full statistical output for the final models, as well as relevant post-hoc results can be found in Supplementary Tables S1–6.", "Data on specific growth rates can be found in Supplementary Table S7, but was not included for statistical analysis as only tank means were available prior to pit tagging and the body mass data indicated no major biological effects of diet.", "There was no effect of dietary phosphorus or sex on body mass or length at any time point in freshwater or seawater, but MP fish had lower body condition than the LP fish in freshwater and the HP fish had lower condition factors than the MP and LP groups throughout the seawater period.", "Dietary phosphorus had no effect on FCR, cataracts, or the prevalence of sexual maturation.", "Compared to the MP and HP groups, the vertebra of the LP fed fish had a lower mechanical stiffness and whole-body phosphorus and Ca:P ratio, and the mineral content was lower at all time points except at harvest.", "Compared to the MP and HP groups, those fish fed the LP diet had a higher prevalence of externally visible deformities and fish with ≥1 and ≥10 deformed vertebra at all time points.", "It was also noted that those fish fed the LP diet in freshwater had an increased number of deformed vertebrae in the tail fin region (v50–60) at day 266, whereas deformed vertebrae in this region were not as evident until harvest (day 686) in those fish fed the MP and HP diets (Supplementary Figs. S2–5).", "There was no difference in vertebra strength, mineral content, whole body phosphorus and Ca:P ratio, or the prevalence of deformed fish between MP and HP fed fish throughout.", "There was a tendency for the LP diet to increase the prevalence of fillet damage due to connective tissue, but this was not significant.", "There were some transient effects of switching diet at seawater transfer on body mass and condition, but not length.", "The only dietary effect at harvest was that the LP-MP fed fish had a lower body condition than the MP-MP fed fish, with the MP-HP fish being intermediary.", "For both the MP and the HP diets, there was a significant drop in FCR when switched to LP vs those that were on MP or HP.", "Generally, the early seawater diet had no effect on cataract prevalence or severity, with the exception that those fish fed the HP-MP diet combination had a significantly lower prevalence of cataracts than those fed HP-LP.", "There was a trend for reduced levels of grilsing at harvest in those fish fed the HP diet in seawater, but this was not significant (linear model, p = .06).", "Sex had only minor transient effects on body condition within the HP and LP dietary phosphorus groups (data not shown).", "Switching the diet for the first 4 months in seawater had no effect on the prevalence of fish with ≥1 deformed vertebra.", "However, switching those fish fed the LP diet in freshwater to either the MP or HP diet in seawater led to a significant reduction in the prevalence of fish with ≥10 deformed vertebra.", "Switching from the HP diet to the LP diet for the initial 4 months in seawater led to a significant reduction in vertebral ash content while in those fish fed the LP diet in freshwater, switching to MP and HP diets in seawater led to an increase in the whole-body Ca:P ratio compared to those fish fed the LP diet continuously.", "There was no effect of switching diet at seawater transfer on fillet damage due to connective tissue, although there was a tendency (GLM, p = .05) for a negative association with dietary phosphorus in those fish fed the HP diet during freshwater.", "Irrespective of diet, those fish maintained on 10 °C for the initial 4 months of seawater rearing weighed significantly less on day 266, but became significantly heavier at termination of the experiment, than those fish kept at 16 °C.", "Furthermore, the fish maintained at 10 °C had lower body condition on day 394 and at harvest, but not on day 266.", "There was also an interaction between diet and temperature on body condition, but not mass, with the HP fed fish having a significantly lower body condition than the LP fish at 16 °C, but not at 10 °C.", "Those fish maintained on 16 °C had a significantly higher prevalence of cataracts and sexual maturation than those kept on 10 °C, as well as a higher cataract score.", "There was no effect of dietary phosphorus on cataracts or the prevalence of sexual maturation.", "Water temperature at seawater transfer had no effect on the whole-body phosphorus or the Ca:P ratio, ash content, or the prevalence of fish with one or more deformed vertebrate.", "However, those fish raised at 16 °C did have a higher prevalence of externally visible deformities and fish with ≥10 deformed vertebra, irrespective of diet.", "Here, it was also observed that those fish fed the LP diet had significantly more fillet damage than those fed the HP diet, irrespective of temperature.", "In total, 13 different deformity types were recorded.", "Here, it was notable that homogeneous compressions were more evident at termination, whereas compression with reduced intervertebral space was more evident on day 266 (500 g fish).", "Otherwise, those fish given the LP diet were the only groups to show compressed vertebrae without the X structure (type 4, Witten et al., 2009) whereas compressed and fused vertebrae were observed in all groups (see Supplementary Fig. S6).", "The variety of bone cell types and structural components identified on bone sections are shown in Fig. S7, based on control material from this study.", "Osteoblasts are abundant, osteocytes are present in low numbers, and bone resorption by multinucleated osteoclasts is common.", "The bone is rich in Sharpey fibres.", "To choose samples for histological examinations, a sub sample of x-rays were analysed to locate and dissect deformed and non-deformed vertebral bodies, respectively.", "In freshwater, 8 (×7 LP, ×1 MP) out of 22 fish were diagnosed for a low mineralised spine phenotype based on X-rays.", "The histological analysis shows that all fish with this phenotype developed ectopic cartilage adjacent to the vertebral body growth zone of the vertebral body endplate.", "Ectopic cartilage is located dorsal and ventral (Fig. S8).", "Fig. S9 shows the regular, non-pathological situation of bone formation without the presence of ectopic cartilage.", "A less commonly observed pathology was the development of ectopic cartilage in the region of the bone trabeculae that connects the vertebral body endplates.", "In post-smolts, 6 (×5 LP, ×1 MP) out of 18 fish showed a low mineralised spine phenotype based on x-rays.", "These fish displayed ectopic cartilage adjacent to the vertebral body growth zone, but more of this cartilage has been developed.", "The cartilage also acquires a more hyaline phenotype.", "Fig. S10 compares growth zones in a regular (non-deformed) and a malformed vertebral body.", "In fish without malformations, only bone is formed in the vertebral body growth zone.", "In fish with radiological signs of malformations, abundant ectopic cartilage was present.", "At harvest, we selected 4 fish with skeletal malformations, and 2 without, from each of the 11 groups.", "Fish with deformities showed primarily different compression and fusion phenotypes (Fig. S11).", "There was a strong anterior posterior gradient, as only few fish had deformities in the anterior part of the spine whereas deformities in the posterior part of the spine were more abundant (Fig. S3–5).", "The majority of diagnosed deformities related to low mineralised vertebral body endplates (x-ray diagnosis based).", "Typical was the development of ectopic cartilage, the replacement of regular (notochord-based) intervertebral tissue by ectopic cartilage, and the extension of ectopic cartilage as chondroid into adjacent musculature.", "Only few fish with low mineralised vertebral body endplates (x-ray diagnosis based) showed alterations of the intravertebral body bone spongiosa.", "Fig. S12 shows examples in comparison to regular intravertebral body bone spongiosa: Hyper-dense bones with a large amount of chondroid bone and soft, undulating, intervertebral bone trabeculae.", "In fish with pronounced vertebral body compression, ectopic cartilage completely replaced the notochord tissue in the intervertebral spaces (Fig. S13).", "The vertebral body growth zone displayed the transition of bone forming cells (osteoblasts) into cartilage forming cells (chondroblasts).", "This transition suggests that the cells from ectopic (pathological) cartilage derive from osteoblasts that undergo osteoblastic to chondroblastic transdifferentiation.", "Accordingly, a gradient from bone to cartilage tissues was recorded.", "This tissue is not present in regular vertebral bodies.", "Ectopic cartilage extends from compressed and fused vertebral bodies into a tissue that is intermediate between cartilage and connective tissue: chondroid (Fig. S14).", "In areas of vertebral fusion, fibrous chondroid tissue has an amorphous extracellular substance with staining properties similar to extracellular cartilage matrix.", "This tissue is present in the musculature where it replaces regular muscle fibres to a large degree (Fig. S14).", "In models with all groups pooled, there was a significant negative association between cataract score, external deformities, and sexual maturation on body mass at harvest.", "Generally, body mass increased with cataract score up until a score of 4, but then decreased between scores of 5–8.", "Here, those fish with a score of 8 were the only group significantly smaller (LME: χ2 = 67, df = 8, p ≤ .001; −1050 ± 321 g) than those fish without cataracts at harvest.", "Fish that had matured as either post-smolts or grilse were significantly smaller (LME: χ2 = 35, df = 1, p ≤ .001; −860 ± 144 g) than those fish that remained immature.", "Finally, fish with an externally visible spinal deformity were significantly smaller (LME: χ2 = 62, df = 1, p ≤.001; −653 ± 72 g) than those fish with no externally visible deformity.", "Our objective was to study the effects of dietary phosphorus level on vertebral column bone health in response to feeding regime and water temperature.", "The LP diet significantly affected indicators of bone health and increased the prevalence of severe vertebral body deformities.", "However, this could be partially rescued by switching the diet to a higher (≥7.1 g kg−1 soluble phosphorus) phosphorus content for 4 months following seawater transfer.", "However, high dietary phosphorus could not prevent the increase in vertebral body deformities observed in fish reared at high water temperatures for 4 months following seawater transfer, suggesting dietary phosphorus may not be a risk factor for water temperature induced vertebral body deformities.", "Fish fed from 3 to 500 g body weight the 4.4–5.0 g kg−1 (LP) soluble phosphorus diet, which is below the recommended level of 8 g kg−1 available phosphorus for Atlantic salmon (NRC 2011), had an elevated prevalence of vertebral deformities as typically found in salmonids (Baeverfjord et al., 1998; Deschamps et al., 2014; Fjelldal et al., 2012b, 2016; Taylor et al., 2015), although not always (Gil-Martens et al., 2012; Witten et al., 2016).", "The LP diet was associated with numerous markers of impaired bone mineralization such as reduced vertebral ash content (Albrektsen et al., 2009; Deschamps et al., 2014; Fjelldal et al., 2012b; Helland et al., 2005; Witten et al., 2016), reduced whole-body phosphorus (Baeverfjord et al., 1998) and Ca:P ratio (Albrektsen et al., 2009), and the induction of markers of bone mineralization (Fjelldal et al., 2012c, 2016; Ytteborg et al., 2016).", "However, switching LP fish to either the MP or HP diet at seawater transfer for 4 months did mitigate the prevalence of fish with severe vertebral deformities.", "This is similar to previous work where adequate dietary phosphorus at seawater transfer was found to mitigate deformities in a stock already suffering from a high prevalence of tail deformities as post-smolts compared to a low phosphorus diet (Fjelldal et al., 2009).", "However, we found that switching the diet for 4 months after seawater transfer had no effect on the prevalence of vertebral deformities at harvest in the MP-LP or HP-LP fish, despite a reduction in whole-body phosphorus to those levels found in the LP-LP fish.", "In another study, bone and scale mineral content and alkaline phosphatase (ALP) also indicated early phosphorus deficiency signs in smolts fed 5 g kg−1 soluble phosphorus (Albrektsen et al., 2018).", "The dietary phosphorus requirement of salmon is expected to be lower at later life stages due to a decrease in the utilisation of dietary energy as the growth rates decline (Shearer, 1995).", "Evidence of this may be found in the mineral content of the bone, as we found that although the LP diet had consistently lower values throughout the study, there was no significant difference between the LP and MP groups at the end of the different feeding regimes (day 266).", "This could also be explained by the slightly higher soluble phosphorus content in the LP diet during the seawater compared to freshwater phase, 5.0 vs 4.4–4.6 g kg−1, respectively.", "However, Deschamps et al. (2014) found that a phosphorus deficient diet (0.5% total phosphorus) reduced ash content and bone mineralization in small rainbow trout (Oncorhynchus mykiss), to a greater extent than in larger trout.", "We found a combination of 7.5–7.6 g kg−1 (MP) soluble phosphorus during freshwater and 5.0 g kg−1 (LP) during the early seawater phase, was sufficient for bone health in Atlantic salmon under the current conditions.", "Similarly, a dietary requirement of 8 g kg−1 soluble phosphorus was needed to reach the highest whole-body mineral reservoirs in pre-smolts (Ytteborg et al., 2016) in accordance with the requirement for available phosphorus set by NRC (2011), whereas the soluble phosphorus requirement for normal bone development was met at 6.5 g kg−1 (Ytteborg et al., 2016).", "In fast growing 0+ smolts, the phosphorus requirement for normal bone development was met between 5.1 and 7.4 g kg−1 soluble phosphorus (Albrektsen et al., 2018).", "In seawater, Fjelldal et al. (2012c) found that post-smolts (230 g) fed 4 g kg−1 available dietary phosphorus for 49 days had poorer bone health, as assessed by bone ALP expression, bone tartrate resistant acid phosphatase (TRACP), matrix metallo-proteinase 13 (mmp13) gene expression, and vertebral mineral composition and stiffness.", "In contrast, we found salmon on the MP-LP diet had no differences in vertebral ash, whole body phosphorus or Ca:P ratios, or vertebral deformities, compared to those salmon fed the MP diet throughout.", "Increasing the water temperature at seawater transfer led to an increase in the prevalence of severely deformed fish.", "This is similar to a previous report whereby an increased prevalence of vertebral deformities, particularly in the tail region, was observed after keeping Atlantic salmon on higher (16 °C) vs lower (10 °C) temperatures at seawater transfer (Grini et al., 2011).", "Previous work in salmon has shown that serum phosphorus drops during seawater transfer, suggesting fish may be deficient for phosphorus during this period (Grini et al., 2011).", "However, we found no significant interaction between diet and water temperature on the level of vertebral deformities, suggesting that either dietary phosphorus is not a factor in the effect of water temperature at seawater transfer, or the dietary phosphorus levels used were not high enough to mitigate the temperature effect.", "Previous work has found that high water temperature (16 vs 10 °C) alters the dorsal ventral diameter of vertebra (Grini et al., 2011).", "Therefore, temperature induced bone remodelling/growth together with the higher growth rates achieved by the 16 °C group during the early seawater phase, could explain the increase in severity of deformities observed in the 16 vs 10 °C groups rather than insufficient dietary phosphorus.", "Fish with low-mineralised vertebral bodies were diagnosed for the presence of ectopic cartilage.", "Freshwater fish and post-smolts showed ectopic cartilage adjacent to the vertebral growth zones before compression became visible on x-rays.", "In contrast, at harvest ectopic cartilage and fibrocartilage was present in the intervertebral space in vertebral bodies that are visibly compressed on x-ray, and fibrocartilage extended into the musculature.", "Pauwels model (Pauwels, 1960) could explain the development of cartilage and fibrocartilage.", "Here, mechanical forces determine the nature of skeletal cells (Hall, 2015); strain favours the formation of ligaments and intramembranous bone and compression favours the development of cartilage.", "Combinations of strain and compression generate intermediate tissues, such as fibrous connective tissue and fibrocartilage (Weinans and Prendergast, 1996).", "One can well imagine that the bending of under-mineralised vertebral body endplates transmits compression to adjacent structures and this compression triggers the development of ectopic cartilage and fibrocartilage cartilage.", "Ectopic cartilage in the intervertebral space of compressed Atlantic salmon vertebral bodies has been reported previously (Kvellestad et al., 2000; Witten et al., 2005) and is known to be caused by compression of vertebra body spongiosa trabelculae (Helland et al., 2006).", "We found fibrocartilage to extend from deformed vertebral bodies into the musculature; a pathology that affects animal welfare and production.", "Previous studies about compressed vertebral bodies only reported cartilage in intervertebral spaces.", "Whether this more severe pathology relates to the size and age of the fish, or if particular risk factors exist that promote muscle infiltration, needs to be evaluated.", "Inflammation is a factor that has been associated with vertebral fusion and alterations of the musculature because large numbers of granulocytes can occur in the vicinity of the vertebral column (Kvellestad et al., 2000; Munday et al., 2017).", "Other studies have found no indications for inflammation (Witten et al., 2005).", "Granulocytes that surrounded the spinal cord and thus appear in the vicinity of vertebral bodies are indeed common in farmed Atlantic salmon, but also in fish that have no deformities (Boglione et al., 2013).", "Using a potent local inflammation inducer, Gil-Martens et al. (2012) did not observe a connection between vertebral body compression and inflammation.", "Similarly, no connection was observed between vertebral body compression, phosphorous deficiency, and inflammation.", "This suggests that mechanical forces according to Pauwels (1960) model rather than inflammation are causative for the occurrence of ectopic fibrocartilage.", "Although not consistent across all comparisons, low dietary phosphorus tended to be associated with an increased occurrence of connective tissue within the fillet.", "This condition reduces the value of the fillet and in severe cases decreases processing efficiency, as these fish cannot be machine filleted (Sullivan et al., 2007).", "The occurrence of such connective tissue has been linked to compressed vertebrae/compressed or missing inter-vertebral space (Haugarvoll et al., 2010), therefore one would expect the low dietary phosphorus group to show the highest prevalence of fillet damage as this group had the highest prevalence of deformed vertebra.", "However, it was noted that there were fish with no deformed vertebra that still had connective tissue within the fillet, and fish with up to 48 deformed vertebra that had no occurrence of connective tissue.", "Therefore, further work is required into the aetiology of this condition.", "There was a notable effect of temperature on growth and cataract development, but no effect of diet.", "Higher temperatures following seawater transfer can lead to an increase in growth, but also have a negative effect on long-term performance (Grini et al., 2011).", "Indeed, the optimum temperature for growth in post-smolts has been found to be between 12.8 (Handeland et al., 2008) and 13 °C (Handeland et al., 2003), whereas salmon reared at 10 and 16 °C were predicted to grow at similar rates (Handeland et al., 2008).", "We found the 16 °C group were larger after 4 months in seawater, but smaller at harvest size, a similar finding to Grini et al. (2011).", "The reasons behind this result are unknown, but sexual maturation, skeletal deformities, and cataracts are likely to reduce long-term growth rates and were higher/more severe in the 16 vs 10 °C groups.", "Indeed, all 3 factors were negatively associated with harvest weight.", "High water temperature around seawater transfer (i.e. 16 °C) is a known risk for cataract development (Bjerkås et al., 2001).", "The causes of this may be multifactorial, but include a deficiency of dietary histidine, an important amino acid for cataract development, and/or osmoregulatory stress around smoltification (Bjerkås and Sveier, 2004).", "In terms of diet, low (Albrektsen et al., 2009; Deschamps et al., 2014; Ketola, 1975; Prabhu et al., 2013; Åsgård and Shearer, 1997) or even high (Fjelldal et al., 2016) dietary phosphorus levels have been associated with reduced growth, however, salmonids can maintain growth at pathologically low dietary phosphorus levels (Baeverfjord et al., 1998; Fjelldal et al., 2012b; Helland et al., 2005) as found in the current study.", "There was no effect of dietary phosphorus on the level of sexual maturation, in contrast to a previous report of a negative association (Fjelldal et al., 2012d).", "However, a lower water temperature at seawater transfer was associated with reduced levels of early sexual maturation.", "This is of particular interest to those wishing to produce larger smolts through temperature manipulation as high-water temperature around seawater transfer has recently been identified as a risk factor for the incidence of post-smolt maturation, particularly in males (Fjelldal et al., 2011; Imsland et al., 2014).", "The exact mechanism is unknown, but is expected to be related to energy reserves at or prior to specific life history events (Adams and Thorpe, 1989).", "Here, it is noted that body condition (a.k.a. condition factor), a proxy for energy reserves (Herbinger and Friars, 1991), was much higher in LP fish that were reared at 16 vs 10 °C, but no such pattern was evident in the HP fish.", "Therefore, further work is required into the role of energy reserves and temperature manipulation on the prevalence of early sexual maturation.", "Similar to recent work (Fraser et al., 2019), we found that those fish that sexually matured as post-smolts survived in seawater, but were significantly smaller at harvest than fish that remained immature throughout.", "Atlantic salmon suffer from a higher risk of developing of vertebral deformities when fed 4.4–4.6 g kg−1 soluble phosphorus during the freshwater life stage compared to 5.0 g kg−1 during the first 4 months in seawater.", "However, the effects of the LP diet could be partially mitigated by feeding fish ≥7.1 g kg−1 soluble phosphorous in seawater.", "Fish fed the LP diet also tended to have more fillet damage than those fed the HP diet, irrespective of water temperature.", "Furthermore, high water temperatures for 4 months following seawater transfer resulted in an increase in vertebral deformities, but this temperature effect appears independent of dietary phosphorus levels.", "Therefore, further work is required into understanding the mechanisms behind temperature induced vertebral deformities.", "This project was funded by the Fiskeri- og Havbruksnæringens Forskningsfond ([FHF] Fisheries and Aquaculture Industry Research Fund), project number 900798." ]	[ "Dietary phosphorus levels in salmon following seawater transfer, and in respect to seawater temperature, were evaluated", "Low soluble phosphorus (LP) levels of 4.4–5.0 g kg−1, from first feeding to post smolts, resulted in poor bone health", "Switching from LP to ≥7.1 g kg−1 soluble phosphorous from sea transfer mitigated some of the effects on bone health", "Dietary soluble phosphorus levels of 5 and 9 g kg−1 did not interact with sea temperatures of 16 and 10 °C, on bone health." ]
Lab-on-a-Chip hyphenation with mass spectrometry: Strategies for bioanalytical applications	The Lab-on-a-Chip concept aims at miniaturizing laboratory processes to enable automation and/or parallelization via microfluidic chips that are capable of handling minute sample volumes. Mass spectrometry is nowadays the detection method of choice, because of its selectivity, sensitivity and wide application range. We review the most interesting examples over the last two-and-a-half years where the two techniques were used for bioanalytical applications. Furthermore, we discuss the merits and limitations of such hyphenated systems. We inventorize the reported applications and approaches. We see an ongoing trend towards chip-based liquid chromatography-mass spectrometry usage and small volume analysis applications, particularly in the field of proteomics where bottom-up approaches profit from chip-based technologies and hyphenation with complex cell cultures.	[ "BIOC", "CENG", "ENGI" ]	[]	[ "1305", "1502", "2204" ]	[ "Current day challenges in bioanalysis include automation, throughput, small volume handling and disposability.", "These challenges are addressed with increasing success through miniaturized laboratory processes, so-called Lab-on-a-Chip (LOC).", "LOC systems use microfluidics (see Supporting information) to handle minute volumes and can be manufactured as cost-effective disposables.", "Moreover, it is possible to integrate laboratory protocols and/or analysis methods into a single cartridge.", "Typical detection techniques that are combined with LOC techniques include optical detection (e.g. UV/Vis spectrophotometry), nuclear magnetic resonance, and electrochemical detection [1].", "To date, none of these sensing techniques can match the selectivity and sensitivity of mass spectrometry (MS).", "LOC systems and MS match particularly well.", "LOC operates in the small volume domain and provides (limited) separation.", "MS detection improves when downscaled; it has a high resolving power and is sensitive.", "One of the first hyphenated LOC-MS systems was reported by Xue et al. [2] At the time a key driver for this combination was to increase throughput in diagnostics and screening applications by using multiple fluidic channels and ionization sprays.", "The authors also perceived the potential for sample preparation, capillary electrophoresis (CE) and liquid chromatography (LC) on chip coupled to MS.", "We inventorized developments in the last 2.5 years in the LOC-MS field from two perspectives: analytical approach (Figure 1a) and application area (Figure 1b).", "The most commonly used approach is LC and the most commonly used application area is proteomics.", "The review is structured on approaches to sample preparation, direct infusion MS, separation and the total analysis system principle.", "Comprehensive reviews on LOC-MS have recently been published by Gao et al. [3••] and Feng et al. [4••].", "In this critical review we argue that the combination of LOC and MS will prove to be the ideal combination for bioanalytical applications and we discuss the, in our view, crucial steps forward and the most dominant trends.", "Common sample preparation techniques are liquid–liquid extraction and solid-phase extraction; only one example of the latter was reported on LOCs in the last 2.5 years.", "Solid-phase extraction was integrated with in vitro cell culturing and will be discussed later in the review.", "In bottom-up proteomics proteolysis is an important part of the sample preparation workflow; the majority of LOCs focussed on this.", "Several devices integrating the proteomics workflow into one LOC were presented.", "One example is a fully integrated electrowetting-powered LOC capable of automated performance of the whole proteomics workflow (from sample preparation to acquisition).", "MALDI was enabled by removing the top cover of the LOC after addition of the MALDI matrix.", "Then the open LOC was placed into a custom-made MALDI plate and analysis was performed [10].", "A device with similar functionality was created using Quake valves to generate and control droplets in an LOC coupled to MS via an integrated nano-ESI emitter [11].", "Furthermore, a droplet microarray plate for the proteomics workflow was developed.", "This microarray was interfaced to ESI-MS via an L-shaped capillary with a tapered tip that served as sampling probe and ESI source [12].", "Tryptic digestion for proteomics after LC-based fractionation is normally performed off-line and suffers from low throughput.", "On-line methodologies involving immobilized trypsin have aspecific adsorption, which leads to carry-over.", "These problems were solved via an LOC in which LC effluent droplets were trypsinized and consequently quenched.", "The LOC was interfaced to MS via an integrated stainless steel emitter [13].", "Another device interfaced droplet microfluidics with a microarray plate containing hydrophilic and hydrophobic spots for the observation of enzyme kinetics (angiotensin II to angiotensin I conversion) in a massive parallel format — 8265 droplets were deposited on the plate — as shown in Figure 2d — and dried using N2.", "Afterwards MALDI matrix was deposited and, because each dried spot represents a time-point, the reaction kinetics could be observed via MALDI-MS [8•].", "An LOC with an integrated proteolysis microreactor to monitor hydrogen-deuterium exchange in proteins was coupled to MS via a custom ESI emitter for both continuous [14] and pulse [15] labelling experiments, enabling the characterization of conformational changes that cannot be probed by conventional techniques.", "In this work, a push–pull probe was coupled to ESI-MS and a droplet micro-array.", "This probe was used to analyse dry surfaces via both scanning electrochemical microscopy and droplet deposition on a MALDI plate.", "It was also used to image immobilized enzymes under a fluid layer by delivering para-aminophenyl phosphate via the microfluidic probe and analysing the para-aminophenol products by ESI-MS (see also Figure 2b) [6•].", "An application where electrowetting-based LOCS are applied in combination with ESI-MS is dried blood spot (DBS) screening.", "Succinylacetone in DBS samples was quantified using a fully automated, nine-step analysis on an LOC.", "Interfacing to MS was achieved via a removable pulled glass capillary emitter nano-ESI source, inserted between the chip substrates.", "No statistically significant differences (95% confidence interval) were found between results obtained with conventional methods and the LOC [16].", "Direct infusion-MS (DI-MS) refers to introducing sample into the MS without prior separation.", "DI-MS chips are marketed by Advion.", "These systems are capable of delivering robust data, can be used for high-throughput analyses, and utilize disposable tips, thereby removing carry-over.", "Recently it has been used for ganglioside analysis from the human caudate nucleus [17], DBS alpha-galactosidase assaying to diagnose Fabry's disease [18] and determination of unusual glycosaminoglycans sulphation patterns in murine brain tissue [19].", "A competitor for this system is the capillary gap sampler (Figure 2e).", "Analyte droplets are introduced from a 384-well plate into a liquid junction between a glass capillary supply line and ESI needle [9•].", "The main advantage of this system is its ability to sample nanoliters, which is a three-order magnitude improvement over the Advion system.", "The vast majority of papers reporting the use of chip-based LC (chipLC) utilized commercial systems, for example from Agilent Technologies, Waters Corporation and Eksigent.", "Miniaturized LC–MS exists in a variant known as nanoLC–MS, which provides extremely sensitive analysis, but can have issues with robustness due to dead volumes and leaks.", "ChipLC offers a solution for these challenges.", "ChipLC interfacing to MS is often achieved via tubing to ESI sources or on-chip integrated sprayers coupled to a (special) interface.", "Monolithically integrated sprayers are also emerging [20].", "ChipLC–MS has been used for, amongst others: pharmacokinetic studies in rodents [21–23], detecting aflatoxins in peanut products [24], detecting gangliosides and sulfatides in murine brains [25], glycomic [26] and proteomic biomarker discovery [27], quantification of matrix metalloprotease-9 in bronchoalveolar lavage fluid [28] and quantification of stable isotope dimethyl-labelled phosphopeptides [29].", "ChipLC–MS steroid analysis [30,31] demonstrated improved LOD than conventional LC–MS. ChipLC was also coupled to MALDI-MS, using EOF-based pumps.", "After separation the proteins were transported orthogonally via electroosmosis in microchannels to MALDI reservoirs [32].", "Another important development is that, to our knowledge, chipLC–MS was used for the first time on patient samples in a phase II clinical trial.", "ChipLC–MS was used to monitor incorporation of deuterated leucine into an apolipoprotein(a)-derived peptide [33].", "This indicates that ChipLC–MS is currently at a level of robustness that pharmaceutical companies are willing to employ it during drug development.", "Other significant developments indicate maturation of chipLC–MS are the appearance of validated chipLC–MS methods for analysis of illegal drugs [34], monitoring of fluoxetine and norfluoxetine in rat serum [21] and 7-ethy-10-hydroxycampotothecin in murine plasma [22].", "A commercial application by Newomics Inc. is the multinozzle emitter array chip (Figure 2c), which can be used for parallel DI protein analysis and enhanced throughput chipLC–MS analysis of tryptic digests, thanks to the sensitivity enabled by the multiple nozzles per emitter [7•].", "The main challenge in chip-based electrodriven separation systems lies in MS interfacing.", "Recent chip-based capillary electrophoresis (chipCE) works have focused on increasing the robustness of interfacing to MS, for example through monolithic integration of ESI tips [35,36].", "Also, an integrated make-up flow chip design and its effect on separation, LOD and robustness of amino acid analysis was demonstrated [37].", "Furthermore, chips utilizing zero, one and three make-up flows were compared.", "The authors conclude that, while LODs for cardiac drugs are improved without make-up flow, the LOCs with make-up flow are more robust and easier optimized [38].", "Optimal chipCE–MS conditions for proteins and peptides are challenging: a low ionic strength background electrolyte and acidic pH are required for efficient ESI.", "Under these conditions silica is prone to electro-osmotic flow (EOF) instability due to protein–wall interactions.", "Batz et al. coated silica channel walls with aminopropyl silanes, ensuring stable EOF between pH 2.8 and 7.5, and an inter-device EOF reproducibility of 2.6% RSD.", "Protein analysis showed 0.7% RSD migration time reproducibility and plate numbers up to 400 000; peptide separation efficiency was over 600 000, the highest reported for any CE–ESI-MS.", "ESI was achieved from the corner of the chip aided by electroosmosis-driven make-up flow [39•].", "In another electro-driven separation, capillary isoelectric focusing (cIEF), ampholytic analytes are separated according to their isoelectric point in a pH gradient.", "Wang et al. developed a methodology to hyphenate chip-IEF to MS using nanowells in glass bottom and top plates, which, when oriented in a semi-overlapping fashion, form a separation channel.", "After separation the glass plates were moved, resulting in MADI-MS-ready nanowells containing separated analytes.", "Eleven amine metabolites were putatively identified in CSF using this method [5•].", "Li et al. integrated cell culturing and chiral chipCE–MS analysis in one LOC.", "Cell culturing was performed on a 0.22 μm filter on top of the sample inlet channel; downstream the separation channel, chiral selectors (moving opposite to the net flow) were introduced and periodically the extracellular matrix was sampled.", "ESI took place at corner of the chip, aided by a make-up flow.", "The enantioselective catabolism of racemic DOPA by neuronal cells was monitored [40], showing that chipCE is a feasible technique for analysis of in vitro cell models.", "Hyphenating in vitro cell models to MS is attractive as the information level provided by MS exceeds traditional optical detection techniques.", "Furthermore, on-line analysis allows following kinetics.", "Several LOC devices integrating biological experiments and sample preparation have been developed by the Jin-Ming Lin group.", "In these devices, micro-solid phase extraction is integrated.", "The interfacing to MS is achieved via tubing connected to an ESI needle.", "Applications include: measuring acetaminophen metabolism via cultured microsomes [41], quantitative analysis of tumor cell metabolism of genistein [42], testing of absorption of various concentrations of methotrexate and its cytotoxic effects [43] and the uptake of curcumin by CaCo2-cell lines [44].", "One system was used for studying signalling molecules in cell-cell communications [45].", "Emerging trends involving 3D cell culture and organ-on-a-chip will likely increase the attention for these types of systems.", "An overview incentives and pre-requisites for adoption of LOC-MS systems is presented in Table 1.", "The incentives to use LOC-MS are to enable small volume analysis, high throughput/parallelization and automation, time-continuous monitoring and on-line sample preparation.", "Several of these pre-requisites have already been fulfilled.", "Commercialized systems as well as cartridge-integrated set-ups are present especially in the chipLC–MS field.", "The added value and benefit of sample preparation on LOC are clear, especially in the proteomics field.", "The perfect match between the scaling efficiencies of enzymatic reactions with the decreasing volumes provided by droplet-sized microreactors, proteomics, and MS’ ability to deal with low-volume samples make it an ideal candidate for wide-spread usage within the proteomics community.", "However, robust datasets, are demonstrated sparsely, one example is continuous monitoring of enzyme kinetics on a micro-array plate.", "We foresee chipLC–MS becoming commonplace in upcoming years, especially since several commercial systems that offer increased throughput, sensitive analysis and allow easy operation are already available.", "The main advantage of chipLC–MS lies in its reduced sample usage and therefore it will be predominantly used for small sample volume applications.", "ChipCE–MS systems need further improvements in robustness before they can be applied on a larger scale.", "Work is currently focussed on make-up flows, which we expect to lead to more robust systems.", "Lastly, we foresee increasing attention for coupling in vitro cell models (such as organ-on-a-chip and 3D cell culture) to MS. Pharmaceutical companies are increasingly interested to make use of such devices to gain additional information efficacy and toxicity of their compounds in the discovery and pre-clinical stage.", "Papers of particular interest, published within the period of review, have been highlighted as:", "• of special interest", "•• of outstanding interest" ]	[ "The state-of-the-art of coupling Lab-on-a-Chip to mass spectrometry is provided.", "Applications using Lab-on-a-Chip coupled to mass spectrometry are discussed.", "We look at the role of miniaturized analysis systems in the analytical workflow.", "Perspective on integration of miniaturized analytical systems in the lab is provided." ]
A pilot study of scanning acoustic microscopy as a tool for measuring arterial stiffness in aortic biopsies	This study explores the use of scanning acoustic microscopy (SAM) as a potential tool for characterisation of arterial stiffness using aortic biopsies. SAM data is presented for human tissue collected during aortic bypass graft surgery for multi-vessel coronary artery disease. Acoustic wave speed as determined by SAM was compared to clinical data for the patients namely, pulse wave velocity (PWV), blood pressure, cholesterol and glucose levels. There was no obvious trend relating acoustic wave speed to PWV values, and an inverse relationship was found between systolic and diastolic blood pressure and acoustic wave speed. However, in patients with a higher cholesterol or glucose level, the acoustic wave speed increased. A more detailed investigation is needed to relate SAM data to clinical measurements.	[ "MEDI" ]	[ "Acoustic wave speed", "Arterial stiffening", "Blood pressure", "Cholesterol", "Glucose", "Pulse wave velocity", "Scanning acoustic microscopy" ]	[ "2702", "2705", "2737" ]	[ "Scanning acoustic microscopy (SAM) is a useful tool for determining the mechanical properties of vascular tissue.1–6", "The image contrast in a SAM derives from differences in the propagation speed of acoustic waves (typically with a frequency of 100 MHz–1 GHz) through a material; this wave speed is related to the elastic properties of the material, in particular Young's modulus.7", "The advantage of SAM over other in vitro mechanical property characterisation techniques for vascular tissue is that it is an imaging method.8", "The contrast in an acoustic image in the frequency range 100 MHz–1 GHz is comparable to that obtained from optical microscopy and is sufficient to resolve distinct features such as collagen fibres.", "Obtaining both histological and mechanical information9 should be useful for studies on arterial stiffening, given the structural changes in collagen and elastin as the main load-bearing components of (large) arteries.10", "Therapies have been proposed which aim to target the assembly, degradation or stiffness of elastic fibres in large arteries11 and hence may be ideal to determine the efficacy of such treatments.", "An additional advantage is that SAM can be applied to small biopsy samples, for example <1 cm2 of tissue.", "In our previous work, we used SAM to examine age-related changes in an ovine model of ageing.", "There we found that the acoustic wave speed, as determined by SAM, increased with age, and that the increased acoustic wave speed was localised to the inter-lamellar region of the medial lamellar unit in the aorta.10", "We also used SAM as a tool to examine the role of a hypertrophy-inducing cytokine, Cardiotrophin 1 (CT-1), on vascular stiffening in a rat model12 and in an experimental model of diabetes in rats.5", "In CT-1 treated rats, we reported an increase in acoustic wave speed in the aorta, matching in vivo mechanical property measurements.", "In the diabetic model, we also found profound changes in the aorta with diabetes could be localised to the inter-lamellar region of the vessel wall and linked to increased extracellular protease activity.5", "SAM has previously been used for characterisation of human aortic tissue, for example to examine atherosclerosis, the stress distribution in atherosclerotic plaques, and aortic aneurysms.1–3", "Here, we showcase some pilot data collected from human aortic biopsies and explore the use of the technique as a tool in human arterial stiffening, we believe for the first time.", "Aortic biopsy samples were obtained from 8 patients undergoing aortic bypass graft surgery for multi-vessel coronary artery disease at the cardiothoracic surgery department, Klinikum Wels-Grieskirchen, Austria.", "The study was approved by our regional ethics committee (Ethics committee of Upper Austria, EK number E-2-10), and all patients gave written informed consent.", "Details for the patients are presented in Table 1.", "The biopsy samples were prepared for cryosectioning by freezing in optimal cutting temperature (OCT) resin (Sakura Fintek Europe BV, Alphen aan den Rijn, The Netherlands) in pre-cooled isopentane and stored at −80 °C.", "The OCT resin is water-soluble and does not affect the mechanical properties of the tissue.", "The frozen tissue blocks were subsequently sectioned to nominal 5 μm thickness sections.", "The sections were imaged with SAM (SAM2000 microscope, PVA TePla Analytical Systems GmbH, Herborn, Germany) as has been reported in detail previously with the Multi-Layer Phase Analysis (MLPA) method.13", "This allows the determination of the acoustic wave velocity from each pixel in the image.", "Imaging was conducted at 760 MHz which provided a nominal spatial resolution of ∼1.3 μm.", "A series of 200 × 200 μm images were captured with the SAM and the mean acoustic wave speed was determined from each image.13", "Each image compromised 512 × 512 pixels and three locations in the media were measured for each sample.", "Hence, the mean acoustic wave speed was averaged from 786,432 pixel measurements for each sample.", "For each sample, serial sections were stained with Haemotoxylin and Eosin (H&E) and imaged with optical microscopy (Nikon Eclipse 50i, Surrey, UK) to locate the medial region of the vessel for subsequent imaging with SAM (Fig. 1).", "Pearson's correlation coefficient was used for all statistical analysis with the following parameters presented: Pearson's r. adjusted r2 and p-values for each group.", "All statistical analysis was conducted with OriginPro 9 (OriginLab Corporation, Northampton, MA, USA).", "A typical SAM image is shown in Fig. 2a.", "The SAM data not only allows extraction of quantitative data (Fig. 2b) from the images but also allows qualitative assessment of the biopsy sample from the histological information, which is clearly visible (Fig. 2a).", "The range of acoustic wave speed values determined in this study (approximately 1500–1700 ms−1) matched those reported by Saijo et al.1", "The SAM data were plotted firstly against invasive, catheter-based measures of PWV and BP, and then against blood concentrations of total cholesterol and glucose in the patients.", "In this small sample study, PWV values were not obviously related to acoustic wave speed (Fig. 3a), while invasive BPs were, inversely (r = 0.22 systolic, 0.23 diastolic), with p values 0.13–0.14, (Fig. 3b and c).", "It should be noted that PWV data were only available for 6 of the patients hence the total sample size was 25% smaller than the other variables that are presented in this study.", "Total cholesterol and plasma glucose values tended to be higher with higher acoustic wave speeds (Fig. 4a and b).", "In the Moens-Korteweg equation there are a number of inherent assumptions, which mean it cannot be used to make a robust measurement of mean elastic modulus of the arterial wall in an individual.14", "However, in the clinical setting PWV is easy to measure15 and is considered to be both reproducible16 and a strong predictor of cardiovascular events and all-cause mortality.17", "In contrast, SAM provides highly localised data and does not represent the in vivo state of the entire aortic wall.", "Counter-intuitively, we found an inverse relationship between SBP and acoustic wave speed.", "This is surprising given that blood pressure plays a significant role in determining changes in the aortic wall structure18 However, this is most likely related to the small sample size with only one single measurement of blood pressure.", "The negative relationship with DBP would be expected, as in this elderly group of individuals, an increase in aortic stiffness is associated with a decrease in DBP.", "However, the problem with sample size and single blood pressure value applies to DBP as well.", "Furthermore, we noted that the correlation of SBP and DBP with PWV in this data are also weak (r2 = −0.07 and r2 = −0.11).", "The systemic effect of elevated cholesterol and glucose in blood appears to be associated with measureable changes in the localised mechanical properties of the medial layer with SAM.", "The mechanisms linking arterial stiffness with high cholesterol and glucose are related to enzymatic degradation of elastin,5,19 which may explain the relationship here with these parameters and micromechanical properties in the medial layer.", "A more substantive study would be required to investigate the relationship between these parameters further in human tissue biopsies.", "If SAM is to become more widely used, a number of technical challenges will apply.", "For example, because high spatial resolution techniques such as SAM are highly sensitive to sample preservation techniques, surgeons will require access to facilities to orientate the samples carefully, before embedding them in the appropriate medium and rapidly freezing at −80 °C.", "Such sample preparation is of paramount performance given that the structural integrity of the aortic tissue is highly affected by preservation technique.20", "However, the ability to be able to target stiffness measurements to very specific components of the aortic wall is appealing for many applications.", "In conclusion, SAM is a useful tool for characterizing small volumes of aortic biopsy tissue.", "A more substantive study is needed to test whether SAM data are related to clinical measurements of arterial stiffness.", "There are no conflicts of interests to declare." ]	[ "Scanning acoustic microscopy (SAM) is a potential tool for arterial stiffness.", "SAM provides a measure of the acoustic wave speed.", "In this pilot study, no clear trend was observed with pulse wave velocity.", "Blood pressure was inversely related with acoustic wave speed.", "Trends observed with other clinical markers such as glucose and total cholesterol." ]
Different strokes for different folks? Revealing the physical characteristics of smartphone users from their swipe gestures	Anthropometrics show that the lengths of many human body segments follow a common proportional relationship. To know the length of one body segment - such as a thumb - potentially provides a predictive route to other physical characteristics, such as overall standing height. In this study, we examined whether it is feasible that the length of a person's thumb could be revealed from the way in which they complete swipe gestures on a touchscreen-based smartphone. From a corpus of approx. 19,000 swipe gestures captured from 178 volunteers, we found that people with longer thumbs complete swipe gestures with shorter completion times, higher speeds and with higher accelerations than people with shorter thumbs. These differences were also observed to exist between our male and female volunteers, along with additional differences in the amount of touch pressure applied to the screen. Results are discussed in terms of linking behavioural and physical biometrics.	[ "COMP", "ENGI", "SOCI" ]	[ "Behavioural biometrics", "Physical biometrics", "Touchscreen gestures" ]	[ "1708", "1709", "1712", "2200", "3304", "3307" ]	[ "The ability to accurately link a physical person to their activities conducted in the digital realm is an increasing challenge for our intelligence and law enforcement agencies.", "The present work was inspired through collaboration with the large multidisciplinary project Super Identity1 whose overarching aim is to model links between a wide range of physical/digital identity measures in order to build new identity attribution techniques.", "The theme of this paper was inspired by a need to address a specific real-world problem: how to identify criminals who conduct their activities using temporary burner mobile phones that are unregistered to them by name.", "Our main objective is to establish whether touch interaction dynamics can be leveraged to provide usable evidence as to the physical characteristics of their creator.", "In this paper, we present initial findings from an exploration into whether it is feasible to infer a single specific physical characteristic of a person – specifically the length of their thumb – from the way in which they perform a common smartphone interaction gesture, the ‘swipe’.", "If such a link could be made, we suggest that by following a route through known proportional relationships between that digit length and other human measurements, we would conceivably be able to infer various other physical characteristics of the person who created the gestures.", "To provide an example, to possess a long thumb suggests a longer hand length, a longer hand length suggests a longer forearm length and a longer forearm length suggests a taller standing height.", "As classically illustrated by Da Vinci in ‘Vitruvian Man’ (Fig. 1), studies of anthropometrics (e.g. Drillis et al., 1964; Doczi, 1981; Gordon et al., 1989; Fromuth and Parkinson, 2008) have shown that numerous segment lengths of the human body follow a common proportionality, approximating the ‘golden ratio’ of 1:1.618.", "This ability to predict the length of one body segment length (e.g. a persons height) from another (e.g. the length of their forearm) has found utility in multiple domains, most notably forensic science and ergonomics.", "Such is the importance of access to accurate anthropometrics data for the purposes of human tool and clothing design, large corpura of detailed body measurement data have been collected over the last century.", "Larger datasets include works conducted by the U.S. Military (e.g. Gordon, 1989; White, 1980; Garrett, 1971) and NASA (e.g. Webb Associates, 1978).", "The anthropometric data provided by ‘Kodak׳s Ergonomic Design for People at Work’ (E.K. Company, 2004) is a frequently cited classic resource within the field of ergonomics and product design.", "Analysis of the proportional relationships between various body segment lengths continues to produce the support to forensic and medical science, often to assess their suitability as a usable predictive tool.", "The ability to predict the length of a missing body segment (e.g. a femur bone) is for example useful in forensic anthropology, where the need to perform reconstructions of bodies from incomplete skeletal remains is a frequently encountered task.", "Results of a number of studies that have specifically considered the hand offer promising evidence that a predictive route from thumb length to larger body segment lengths is available.", "The close relationship between hand length measurements and standing height has been reported by Ilayperuma et al. (2009), Abdel-Malek et al. (1990), Meadows and Jantz (1992) among others, while similar analyses examining the relationship among hand length, foot length and standing height can be found in Sanli et al. (2005).", "When held one-handed, interacting with the touchscreen of a smartphone is naturally restricted to the use of the thumb only.", "In this paper, we hypothesise that when a smartphone is used in this way, evidence as to the likely length of its user׳s thumb is revealed by dynamics of their gestural touch interactions (such as their length and drawing speed).", "If this were indeed the case, it would provide a useful link between what would be considered a behavioural biometric, to a soft physical biometric for that individual.", "In the present study, we obtained an accurate measure of the length of each of our participants’ thumb, before then capturing a number of their touchscreen swipe gestures using an instrumented smartphone.", "Swipe gestures were drawn and captured in multiple directions.", "To maximise any potential correspondence between thumb length and the resultant characteristics of swipes created using that thumb, we restricting our participants’ interaction with the device to one-handed use only, with the device held in portrait orientation.", "The swipe gesture (sometimes referred to as flick or fling) involves placing a finger or thumb on the screen and – while maintaining contact with the screen – quickly swiping it in the desired direction.", "Akin to flicking through the pages of a book, the swipe gesture is frequently used to support content navigation, such as navigating a photo album or paging through a list.", "The swipe gesture was selected for this investigation over other touch gestures (e.g. drag, or multitouch gestures such as pinch-to-zoom) primarily because of its high frequency of use, but also for its high range of extractable features across a number of dimensions, including length, drawing speed and touch pressure.", "Biometrics are measures of human characteristics and traits.", "Biometrics can be physical or behavioural based.", "Physical biometrics relate to something a person is, and can be subclassed into either soft or hard depending on the degree to which they are unique to a particular group of individuals or a specific individual respectively.", "An example of a hard physical biometric would be the unique pattern associated with a person׳s fingerprint or their DNA profile.", "An example of a soft biometric would be the colour of a person׳s hair or iris.", "A behavioural biometric relates to something a person does; the identification of an individual through some unique aspect of their behaviour.", "Like physical biometrics, behavioural biometrics can also take hard and soft forms.", "Human–computer interaction research has explored potential sources of behavioural biometric material from a variety of angles, including the way in which individuals type on a keyboard (keystroke dynamics e.g. Maxion et al., 2010; Killourhy et al., 2009; Banerjee and Woodard, 2012; Monrose and Rubin, 2000; Zahid et al., 2009), how they use their mouse (Zheng et al., 2011; Shen et al., 2013) and even the way that they walk (Derawi et al., 2010; Ho et al., 2012).", "In recent years, the interactive touchscreen has become the dominant input mechanism for a range of mobile computing devices, most notably smartphones and tablets.", "The ubiquity of these devices, along with rapid advances in the sensitivity and accuracy of touchscreen technology has attracted renewed interest from researchers for their potential as a source of new behavioural biometrics.", "Of particular interest is whether the dynamics of a given person׳s touch interaction style are unique enough to provide new user authentication mechanism that are both secure and usable (Kolly et al., 2012).", "This effort is a response to the increasing concerns of both security researchers including Azenkot et al. (2012) and Ben-Asher et al. (2011) and end-users (Chin et al., 2012) that the security/usability tradeoff of existing user authentication schemes for these new mobile devices is not optimal, particularly when taking into account the broad ways in which these devices are used.", "For example, a common setup of these devices is to rely upon a single active authentication scheme (such as a pin-lock or password) that is entered every time the device is activated for use.", "A 4-digit pin lock – while easier to enter quickly – offers little actual security value compared to most passwords (Chang et al., 2012), yet passwords – and particularly ‘strong’ passwords of sufficient length and complexity – are hamstrung by the difficulty of their swift and accurate entry on small ‘virtual’ keyboards (Angulo and Wästlund, 2012; Findlater et al., 2011).", "The need for the development of effective user authentication mechanisms that offer actual security value while also addressing the needs of users and how these mobile devices are used is an issue of increasing significance, particularly given the value of personal data that is stored or accessible via these devices.", "However, and as a number of researchers have noted (e.g. Herley, 2014; Adams and Sasse, 1999) this effort must take a two-pronged but much more user-centric approach: users must be made aware of the importance of protecting their devices, but they must also be provided with the means of doing so that is appropriate for the ways in which these devices are used.", "By way of example, as these devices tend to be used frequently on the move and/or in short bursts, lengthy authentication procedures that require concentration or dexterity (e.g. one-handed virtual keyboard entry) are inappropriate.", "A consequence of authentication procedure design that fails to take into account the end user׳s needs is an entirely understandable but worrisome tendency for users to turn user authentication measures off (Clarke and Furnell, 2005).", "Given the increased interactive affordances of touchscreens relative to physical keypads, many novel graphically based active authentication systems have been proposed (Sae-Bae et al., 2012; De Luca et al., 2012; Azenkot et al., 2012) as usable and secure alternatives to pin-locks and passwords.", "Oftentimes however, instead of reducing the potential for unauthorised access, many of these methods serve instead only to increase the range of attack vectors available to criminals.", "Notable recent examples include the ‘smudge’ attack (Aviv et al., 2010; Airowaily and Alrubaian, 2011) and the observation of key entry via ‘shoulder surfing’ (De Luca et al., 2012).", "Even the use of dedicated fingerprint scanners has been demonstrated as being easily defeated (e.g. Zhang et al., 2012, often by exploiting the same skin-oil deposits upon which the smudge attack relies.", "An alternative solution is to include features of touch interaction dynamics as a behavioural biometric, for the principle reason that it is assumed that such behaviours would be more difficult for an attacker to consistently mimic.", "Some researchers, including De Luca et al. (2012), Angulo and Wästlund (2012) and Frank et al. (2013) have explored this in depth, reporting high levels of success using touch-behaviour based material to augment existing active authentication mechanisms.", "Others have presented entirely unique systems that are based on single or multi-touch gesture dynamic profiles (e.g. Sae-Bae et al., 2012; Feng et al., 2012) or through fusion with other sensory data such as accelerometers (e.g. Jain and Kanhangad, 2015) with similarly positive findings.", "Finally, researchers, including Shi et al. (2011), Bo et al. (2013), Damopoulos et al. (2013) and Li et al. (2013) have gone further still by developing touch-behavioural authentication systems that are entirely passive in nature, constantly monitoring touch interaction behaviour as a background process, invoking active authentication requests only when sufficient evidence has mounted that the active device user has changed.", "Our work here however takes the use of touchscreen behavioural dynamics in a slightly different direction.", "While other work has considered the degree to which touchscreen gestural dynamics might offer value as a behavioural biometric for user authentication, relatively little attention has been given to what touchscreen gestures are able to reveal about the physical characteristics of their creator.", "Here then, instead of using touchscreen dynamics as a means of directly identifying an individual, we seek to establish whether links exist between these behavioural characteristics and other physical characteristics of the person behind the behaviours.", "Such a technique would be considered a ‘soft’ biometric in that we are seeking to identify a feature that is related to a group of individuals as opposed to a feature that is unique to an individual.", "While the potential for such techniques have been demonstrated using physical keystroke dynamics to predict a user׳s gender (e.g. Fairhurst and Da Costa-Abreu, 2011), to our knowledge, there currently exists no equivalent research on touchscreen gestural dynamics for this purpose.", "In this initial exploration, our overarching research question was to identify the degree to which a relationship exists between the length of a person׳s thumb and the resultant characteristics of touchscreen swipe gestures made with that thumb.", "To investigate this question, we first assumed that there would exist variance in swipe gesture characteristics.", "Further, we assumed that this variance would have two main sources: (1) the physical physiology of the user and (2) their individual level of touchscreen interaction skill/experience.", "The swipe gesture can be completed in one of four directions (left-right, right-left, down-up, up-down)2.", "For one-handed use, the performance of a swipe gesture requires various manoeuvres of the thumb, dependent upon the direction of the swipe and the hand in which the device is held.", "Specifically, two types of thumb movement are required for one-handed swipe gestures.", "These are flexion (bending)/extension (straightening) at the interphalangeal joint (the first joint from the thumb-tip), and palmar abduction (towards palm)/adduction (away from palm) at the carpometacarpal joint – the joint closest to the wrist.", "Again, assuming one-handed use, to swipe horizontally primarily demands palmar abduction/adduction, but will also involve some degree of supporting movement at the interphalangeal joint for precision i.e. to maintain a reasonably straight horizontal line.", "Conversely, vertical swiping demands more involvement of the interphalangeal joint.", "It is somewhat difficult (though not impossible) to perform vertical swipes using palmar abduction/adduction in isolation.", "We expected that these differences in manoeuvre would be detectable in the characteristics of swipe gestures on aggregate, hence:", "Hypothesis 1: Different manoeuvres required for the thumb to swipe in different directions will result in differences in the characteristics of swipe gestures made in each direction.", "Differences in the length of thumb also afford more or less ability to manoeuvre across the screen.", "Most obviously, a longer thumb can naturally and more easily reach further across the screen both horizontally and vertically.", "This we hypothesised would also be detectable, hence:", "Hypothesis 2: People with longer thumbs will create swipes with different characteristics to people with shorter thumbs.", "Supplementing hypothesis two, we also consider the fact that males have – on average – hands that are larger and thumbs that are longer than females.", "Given this known difference in thumb length across the biological genders, we hypothesised that males and females would generate swipes that are detectably different, hence:", "Hypothesis 3: The characteristics of swipe gestures made by males will be different to those made by females.", "Finally, and following our previous hypothesised differences in thumb manoeuvre, the hand in which the device is held is also potentially a factor in the eventual characteristics of a swipe gesture made in a given direction.", "Using the right hand, swipes in the D–U and R–L direction demand an initial flexion of the thumb at the interphalangeal joint prior to a ‘pushing away’ motion towards extension, whereas U–D and L–R swipes demand an initial extension of the thumb before ‘pulling’ the thumb across the screen.", "Using the left hand, these ‘pushing’ and ‘pulling’ manoeuvres for horizontal strokes will of course be reversed, hence:", "Hypothesis 4: There will be differences in the characteristics of horizontal swipe gestures between left-handed device users and right-handed device users.", "In the last ten years, touchscreen-based smartphones have rapidly become a ubiquitous feature of modern urban life.", "Interacting with a touchscreen efficiently is however a skill, and one that takes some time to develop fully.", "In light of this, and in combination with our main investigation into physiological differences of device users, we also considered the effect of user experience with current touchscreen technologies.", "For finger-based operation of screens with ever increasing pixel densities, almost all smartphones now use capacitive touchscreen technology to provide a much higher sensitivity and accuracy than previous stylus-based resistive screen technologies.", "We expected that people with less experience with capacitive touchscreens (or indeed touchscreens in general) would exhibit a higher range of variance in their swipe patterns.", "Conversely, we expected experienced users to display a more habituated/stable gesturing pattern with smaller, more efficient movements and lighter pressures:", "Hypothesis 5: People with higher self-reported experience with touchscreens will produce more consistent swipe patterns, with lower levels of variance across their features on aggregate.", "A small software application was created to facilitate the systematic and controlled capture of swipe gesture samples in four directions.", "This application was deployed on a single reference touchscreen-based smartphone running the Google Android operating system.", "178 participants (m=87, f=91) were recruited from the Universities of Bath and Southampton.", "The age range of our participant pool was between 18 and 59 years old, with 71% aged between 18 and 24 years old.", "At Bath (n=61), an opportunity-based sampling method was used and no reward was given for participation.", "At Southampton (n=117), participants volunteered their data as part of a larger data collection exercise for which they were paid.", "The collection methodology was identical in both cases.", "All participants were over 18 years of age, but no other participation eligibility criteria were applied.", "Of our participants, 161 used their right hand to complete the study and 17 used their left.", "Ethical approval for the study was sought and approved independently by the experimental ethics committees of the Universities of Bath and Southampton.", "The requirements of our study demanded that we develop a systematic method for eliciting and capturing touchscreen swipe gestures made in four directions from a reference smartphone.", "While it is possible to log all touch interaction events on an Android-based smartphone as a background process (i.e. regardless of the application currently in use), to do this requires maximum privilege, or ‘root’ level access to be made available on the device.", "As the process of obtaining root level access (‘rooting’) invalidates the device warranty, this method was not available to us.", "Without root level access, the recording of touchscreen interaction events is – for reasons of security – limited by the Android OS to a single application that must be custom built for this purpose.", "For the purposes of our study, we required an application that would utilise multi-directional swiping as its primary interaction dynamic (so as to maximise our data yield).", "Common tasks, such as text messaging and internet browsing, do not utilise the swipe gesture to any great extent and were thus not considered appropriate options for further investigation.", "The potential of modifying an existing application such as the Android homescreen, photo browser or calendar systems was explored as they all use swiping for the purposes of navigation, but these options were later abandoned – primarily due to their very limited use of vertical swiping.", "The final system that we developed for our data capture was a standalone Android application that used a tile sliding paradigm similar to that used to control the popular puzzle game 2048.3", "Our chosen method presents a trade-off between ecological validity and experimental control that we recognise.", "However, we felt that this was the most appropriate method of collecting a controlled number of genuine swipe gestures from a large number of people without contaminating the data with other gestures (e.g. tap/double-tap), or by inadvertently directing participants to swipe from a particular point on the screen.", "Further, as all measures collected used standard methods of the Android SDK with no transformation or filtering applied (including details of the screen size, orientation and resolution of the reference device used), we are confident that the data collected here can be repeated, reproduced and built upon by other researchers who wish to use devices with more advanced screens than the Samsung i9100 used in this study.", "To elicit swipe gestures in as unconstrained a manner as possible while still forcing the participant to swipe in several directions (specifically L–R, R–L, U–D, D–U), our gesture capture system was augmented with a simple reading task that was designed to produce a controlled number of swipe requests over all four directions.", "The task set to participants was to use the application to read a series of short jokes.", "Each joke (e.g. what is the fastest thing in water?)", "and its punch line (a motor pike!)", "were presented separately as simple slides.", "To progress through the slides, participants were required to perform a swipe gesture in the direction indicated on the screen.", "For the purposes of user feedback, upon completion of each slide the content of the slide would exit the screen in the direction that the swipe was made (Fig. 2).", "For the operating system of our reference device (Google Android), a swipe gesture interaction is triggered (and subsequently recorded by our application) upon satisfaction of two parameters that must be specified at the application level.", "These are (1) a minimum value for length (measured in pixels) and (2) a minimum value for velocity (measured in milliseconds).", "As screen sizes and pixel density vary widely across smartphones, there currently exists no standard value for these parameters.", "For the purposes of the study, the minimum length was set at 120 pixels and the minimum velocity was set at 200 ms. These values were suggested by the Android software development community as being commonly used values, and are based upon those used on the default Android OS home-screen.", "A series of pilot studies of gesture captures conducted with six users indicated that, in practice, the distance travelled during a typical swipe gesture is typically several times this minimum figure, and had no identifiable impact on the way that participants completed their gestures.", "Upon capture of each gesture sample, all raw data that could be captured about it was recorded and sent immediately to a central database via Wi-Fi.", "All measure were captured using the standard touchEvent methods provided by the Android software development toolkit.", "Custom built web-based software automatically analysed the incoming gesture data, producing a set of calculated metrics along with the ability to immediately reconstruct each gesture to scale as a vector graphic (Fig. 3).", "The ability to immediately and visually inspect each swipe gesture proved to be extremely useful in accurately identifying software recording errors and other potential data contaminants.", "Each swipe gesture generates a series of time-stamped points that together trace the path of the gesture as it travels across the screen.", "From each recorded gesture, six features were extracted and/or calculated.", "These feature were felt to adequately cover the main dimensional features of the swipe gesture and are described thus:", "Gesture length (pixels): calculated by summing the Euclidean distances between each of the internal path-points.", "Gesture completion time (ms): i.e. from initial screen contact to finger release.", "Avg.", "gesture thickness (px): thickness here refers to the approximated amount of skin surface area that was detected as being in contact with the screen as the swipe gesture path was created.", "A final value was obtained for each gesture by obtaining the averaged thickness value across the gesture path as a whole.", "Avg.", "applied touch pressure: the Android SDK reports a normalised value between 0 and 1 for pressure applied to the screen at each path point.", "As with gesture thickness, we took the averaged value for the entire gesture path for this measure.", "Max.", "speed achieved (cm/sec): a simple conversion from pixels to cm was obtained via measures of the screen dimensions and known dots-per-inch.", "Max.", "acceleration achieved (cm/sec2): calculated as final velocity initial velocity/time.", "For the purposes of reproducibility, all data relating to touch interaction events, including timing information, were collected via methods supplied by the Android OS motionEvent class.4", "X and Y co-ordinates were collected using the getRawX and getRawY methods respectively.", "During data collection, all user controllable applications on the device were shut down prior to launching our custom gesture recording application.", "Each participant was first provided with written instructions detailing the requirements of the study, after which their signed consent was obtained.", "Participants were then asked to provide details of their biological gender, phone handedness5 and the length of their thumb (mm).", "The length of the thumb was measured from the carpometacarpal joint (closest the wrist) to the thumb tip, with the thumb at full extension.", "Finally, we asked all participants to self-report their prior experience with touchscreen-based smartphones, using a 6 point Likert scale (1: no prior experience to 6: highly experienced).", "The participant was then presented with the instrumented smartphone and the researcher demonstrated the swipe gesture.", "In order to maximise consistency in our sample set and to provide the best possible conditions for linking our known physical measurement of thumb length to subsequent touch screen interactions, for this study the orientation of the screen was fixed to portrait mode and interactions with the screen were restricted to single-handed use only (participant׳s preference), using only the thumb of that hand to interact with the screen.", "Future work will seek to establish the degree to which two handed operation impacts on our findings here.", "Prior to completing the study, each participant was allowed a few moments to familiarise themselves with the device and the generation of swipe gestures by navigating menus on the home-screen and browsing the Internet.", "These applications were then shut down, and the participant was then instructed to launch the gesture recording application, following the instructions provided on the screen.", "Participants were instructed to complete the task using only the thumb of one hand (their choice) to generate each swipe gesture.", "The order of slide presentation (and by extension the direction of swipe required to progress through the slides) was randomised for each participant.", "In total, each participant submitted 120 swipe gestures (30 samples in each direction).", "The total duration of the study was between 10 and 15 min, after which all participants were fully debriefed.", "A total of 21,360 individual swipe gestures were collected from 178 participants, with each participant providing 30 swipe gestures for each of four directions (U–D, D–U, L–R and R–L).", "Prior to analysis, 2207 samples were identified as containing extreme outliers on at least one measure and were removed, leaving a final dataset of 19,153 samples.", "Exploration of the data revealed a non-normal distribution for all six of our feature extractions.", "Normalisation of the data for each feature was attempted using logarithmic and square transforms to reduce skew where appropriate.", "However, subsequent examination of the Kolmogorov–Smirnov and Shapiro–Wilk tests of normality indicated that both remained highly significant, and Levene׳s test for homogeneity of variance also failed for all features, regardless of transform.", "Consequently we chose to employ non-parametric data supportive statistical tests in all subsequent analyses of swipe gesture characteristics.", "Spearman rank order correlation revealed no significant relationship between participant age and touch screen experience (rs(178)=−0.06, p=0.40 n.s.), though this result needs to be considered with caution given that the majority of our participants were less than 25 years of age.", "To examine the relationship between self-reported touchscreen technology experience and swipe gesture consistency, we first calculated the total variance for each of the six features across all of the gesture samples provided by each participant.", "These values were then examined against their self-reported levels of touchscreen-based smartphone experience using the Spearman RHO correlation coefficient procedure.", "A Kruskal–Wallis test was conducted to determine if there were significant differences in our six measures across the four directions of swipe captured.", "Post hoc pairwise comparisons were performed using Dunn׳s procedure, with a Bonferroni correction for multiple comparisons applied.", "Our Hypothesis 1, different manoeuvres required for the thumb to swipe in different directions will result in differences in the characteristics of swipe gestures made in each direction, was partially supported.", "The Kruskall–Wallis test revealed a significant omnibus effect of direction on all six of our gesture feature extractions.", "Post hoc analysis further revealed statistically significant differences at the 0.05 level for all features between almost all direction pairs.", "These differences suggest that the direction in which a swipe is drawn is an important factor in its resultant characteristics, and that any further analysis of swipe gestures should take these directional differences into account.", "However, there were instances where particular characteristics did not differ across specific direction pairs that are important to note: gesture lengths for swipes drawn in the vertical directions (U–D and D–U) were not significantly different from one another, and completion times for swipes drawn in the horizontal directions (R–L and L–R) were also statistically indistinguishable from one another in our sample.", "Median values for all six measures were compared between participants who held the device in their left hand and participants who used their right hand using the Mann–Whitney U Test procedure.", "All pairwise comparisons were again performed using Dunn׳s procedure with a Bonferroni correction for multiple comparisons applied.", "Our Hypothesis 4, there will be differences in the characteristics of horizontal swipe gestures between left-handed device users and right-handed device users was supported.", "Mann–Whitney revealed that swipes created by left-handed device users exhibited lower completion times and higher speeds than right-handed device users for swipes made in the R–L direction (where completing the gesture demanded that the thumb be extended first before being pulled across the screen), whereas right-handed device users outperformed left-handers on the same measures with swipes made in the opposite L–R direction.", "These differences were observed as being statistically significant at the 0.05 level.", "Median completion times and maximum speeds for left and right handers across the two directions of horizontal swipe are presented in Figs. 4 and 5 respectively.", "Post hoc analysis also revealed several other statistically significant differences in gesture characteristics that were related to handedness but limited to specific directions of swipe.", "Right handers were observed to create longer L–R ‘pulling’ swipes than left-handed device users (Mdn[left]=250.00, Mdn[right]=252.00, U=860823.5, Z=−2.095 p=0.04), and right handers created thicker R–L ‘pushing’ swipes than did left handers (Mdn[left]=43.4, Mdn[right]=44.4, U=819043, Z=−2.823, p=0.005).", "Conversely, left handers applied more pressure on L–R ‘pushing’ swipes (Mdn[left]=0.225, Mdn[right]=0.214, U=999993.5, Z=2.960, p=0.003).", "No differences were observed for maximum accelerations achieved.", "A revised and more accurate method for collecting thumb lengths was used in the second data collection at Southampton (n=116).", "Our revised thumb measurement protocol was developed in collaboration with forensic anthropologist Prof. Sue Black (University of Dundee, U.K.).", "Under her advisement, in the second (Southampton) data collection, our measurement of thumb length was taken from the carpometacarpal joint (closest the wrist) to the joint tip.", "For each participant in the Southampton data collection, high resolution scaled photographs of both hands were collected under controlled conditions for reference, and all measurements were completed by the same person.", "In the first collection (Bath) we took only a manual measurement from the adjacent metacarpophalangeal thumb joint to the tip, without collecting a photographic reference.", "This change in procedure rendered the two measurement sets incompatible with one another.", "As result of this, thumb length measures from the Bath dataset (n=61) have been excluded from this analysis.", "To examine the relationship between thumb length and swipe gesture characteristics, we compared each of our six features with thumb length using the Spearman RHO correlation coefficient procedure.", "Our Hypothesis 2, people with longer thumbs will create swipes with different characteristics to people with shorter thumbs, was supported by three of our measures.", "Spearman׳s RHO revealed a statistically significant relationship between thumb length and the following features of swipe gesture:", "No significant relationship was observed between thumb length and gesture thickness, avg.", "pressure or gesture length.", "A comparison of the median values for our six measures between our male and female participants was performed using the Mann–Whitney U Test.", "Pairwise comparisons were again performed using Dunn׳s procedure with a Bonferroni correction for multiple comparisons applied.", "Our Hypothesis 3, the characteristics of swipe gestures made by males will be different to those made by females, was partially supported.", "Building upon the correlations that we observed for thumb lengths, the Males in our sample completed swipe gestures faster than the females in all directions, and this was again observed in terms of shorter completion times (Fig. 9), higher speeds (Fig. 10) and higher accelerations (Fig. 11).", "An additional difference between the genders was also observed in that males were found to apply more pressure to the screen than did females, across all directions of swipe (Fig. 12).", "No statistically significant differences were observed for gesture lengths or gesture thicknesses between males and females in any direction of gesture.", "In this paper, we present an initial investigation of the relationship between six touchscreen swipe gesture characteristics and a specific physical characteristic of their creator: the length of their thumb.", "Should such a relationship exist, we suggest that this could – via following the known proportional relationships between human segment lengths – offer a route to inferring other measures of a given user׳s physical characteristics, such as their standing height or foot size.", "This work was inspired by a known problem in contemporary criminal investigations: how to identify criminals who conduct their activities using temporary ‘burner’ mobile phones that are unregistered to them by name.", "Within this scenario, we suggest that the ability to link physical characteristics to behavioural metrics captured from the way in which a person interacts with a touchscreen device could prove useful to law enforcement analysts as they begin eliminating groups of potential suspects towards the identification of a specific individual.", "From an analysis of approximately 19,000 swipe gestures supplied by 178 participants, we show that there is a relationship between thumb length and three features of swipe gestures.", "Our analysis thus far has shown that users with longer thumbs complete swipe gestures with shorter completion times, higher speeds and higher accelerations than users with shorter thumbs.", "Further supporting these findings, these differences in swipe composition were also observed to exist between our male and female participants, where there is a known difference in thumb length within the general population (males on average having longer thumbs than females).", "Finally, while no relationship was found between thumb length and touch pressures applied during swipe gesture composition, we did observe a statistically significant difference between males and females (males apply more touch pressure than females, regardless of swiping direction), suggesting that other physiological factors relating to larger hands – such as variance in grip strength – might also be detectable.", "That our two remaining features (swipe gesture length and thickness) were not found to be useful indicators of the physiological characteristics of their creator׳s thumb was unexpected.", "Gesture length in particular was surprisingly unfruitful given the reasonable assumption that a longer thumb affords an increased range of motion that we expected would result in longer swipes.", "However, from what we have seen of the swipe gesture in practice, the length of a swipe gesture is actually too short for longer thumbs to be of any detectable benefit.", "Also contrary to our initial expectations, participants in our sample did not create swipe gestures as efficiently or as consistently as we assumed they would.", "However, we did find some evidence to suggest that increased experience with touchscreens reduced intra-participant variance in some the features we examined, namely that higher self-reported experience was weakly correlated with swipe gestures of a higher efficiency (shorter lengths, faster completion times and higher accelerations).", "However, we must currently treat this finding with some caution given that our participant pool was heavily skewed toward younger smartphone users.", "Through the course of the collection and analysis of the large dataset collected during the study, there were a number of lessons learned from our experience that will inform our future work and would be of benefit to other researchers and practitioners working in this area.", "Firstly, both intra- and inter-participant swipe gesture data is somewhat noisy, with all of our measures following a non-normal distribution.", "The non-normal nature of the data placed limitations upon the statistical tools that could be appropriately used to perform our analysis, particularly with regard to predictive modelling techniques such as regression.", "While machine learning algorithms potentially offer an alternative predictive route (and one we are actively investigating), there exists significant risk of both type I and II error in datasets of this type if inappropriate statistical tools are applied.", "Researchers are encouraged to be mindful of this.", "Further, researchers are encouraged to be mindful to pay particularly close attention to the impact of extreme outliers in touchscreen interaction data at the level of gesture capture.", "Though rank-order based statistical analysis that focus upon median values rather than means are better suited to mitigating the effect of outliers, examination of our data prior to analysis indicated that we needed to discard some 10% of our results due to the presence of extreme outliers on at least one of our measures.", "Identifying reasons and developing countermeasures for these extreme values to maximise data yields is the focus of continuing investigation.", "Some are certainly due to the intermittent software level recording error that we mentioned in our results.", "These are relatively easy to identify and discard as they occur as they all have clearly identifiable breaks in their internal path sequence identifiers.", "However, others appear to be valid swipes in all respects other than that they have characteristics that fall far outside the ‘normal’ range of their creator.", "A full appreciation of the reasoning behind these intermittent ‘extreme’ swipe patterns is far from complete, but we suspect that at least some are the result of an over-compensation immediately after a recording error (i.e. as the participant received no feedback that the swipe gesture was recognised, they immediately re-doubled their efforts in the next swipe attempt leading to a far longer/higher pressure swipe gesture).", "More work is required both to finesse the gesture collection technique we have developed and to evaluate the impact that these errors have.", "Secondly, our analysis thus far indicates clearly that the direction in which the swipe is drawn does affect its characteristics, thus swipe gesture patterns should be analysed separately on a per-direction basis.", "Similarly, left and right handed device users exhibit differences in swipes made horizontally.", "Both of these factors should be controlled for when analysing swipe data characteristics at the aggregate level.", "Thirdly, and though care was taken to clean the screen of our reference device regularly between collection sessions, a rapid build up of skin oils on the screen was still observed.", "Indeed, we suspect that a source of some of the noise encountered in our data was due to variances in skin/screen friction as a result of the build up of skin oils.", "Further, and though we ourselves did not use any aftermarket screen protectors, we recognise that many device users do fit such devices.", "Further investigation is required to understand the degree to which these factors affect the characteristics of swipe gesture composition and – if necessary – how these effects be detected and controlled for.", "Finally, we must accept that our initial methodology for collecting measurements of thumb length lacked sufficient rigour, resulting in the loss of a significant amount of data.", "It is apparent that techniques for collecting anthropometric data vary widely, and a discussion of the relative accuracy of various techniques (e.g. manual calliper vs. imaging, the identification/selection of external joint/skinfold landmarks) is ongoing.", "Researchers are strongly encouraged to seek advice from the medical and forensic research communities before gathering data of this type.", "In discussing the conclusions of our research thus far, we must of course be mindful of the potential future applications of any system that relies upon indirect measures in order to draw inferential conclusions about people.", "This is of particular importance here given that there is obvious potential for gesture data collection to be conducted covertly without knowledge or consent.", "While a full discussion of the privacy implications involved in developing such techniques is outside the scope of this paper, it is important to be very clear that this work – currently in its infancy – has significant privacy issues attached to it and is some considerable distance from deployment in a real-world scenario.", "Therefore, while we feel that our results presented here offer an encouraging first step towards our ultimate goal, we stress the limits of our findings thus far, particularly given the limited choice of hardware examined and the restricted interaction mode imposed upon our participants.", "To restate our main objective, the aim of the present work was to explore whether it is feasible to link touchscreen gesture characteristics to the thumb length of their creator, as a precursor to the detailed and significant amount of further work that would be required to operationalise such links for use in a forensic context.", "So far, we have found evidence to support the presence of relationship between several features of the swipe gesture and thumb length, but note that this relationship is currently only useful as an indicator when considered relative to population averages (as opposed to revealing the actual length of a particular user׳s thumb).", "Further, the data collection method was conducted under controlled laboratory conditions.", "Much more research is required to establish a means of inferring actual thumb length to any degree of accuracy, particularly when taking into account the myriad devices available and the many ways in which people choose to interact with these devices.", "To this end, we highlight below several limitations of our research that will be the focus of attention in our future work.", "Firstly, we accept that our findings are currently limited to one specific mobile device that was held and operated in a very specific way (our participants were forced to hold our instrumented smartphone in one hand, using only their thumb to interact with the device).", "This we argue was necessary at this early stage to maximise any potential correspondence between thumb length and touch gesture characteristics.", "However, we do of course recognise that these devices can and often are operated with both hands and with other digits than the thumb.", "Further work will seek to address this by allowing participants to choose the way in which they hold the device and the digit that they use to interact with the screen.", "Secondly, there are limitations to our sampling method.", "While the effect of both physiological differences and performance factors related to past experience with touchscreens was considered in our analysis, as a result of using an opportunity-based sampling method, the majority of our participants were young adults.", "Most of our participants were highly experienced touchscreen operators.", "Despite this however, we did still observe a weak but statistically significant signal in our less confident participants, indicating that lower touchscreen technology experience may well effect the stability and consistency of swipe gestures over time – particularly as novices become more adept at using the technology.", "The development of our future recruitment strategy will address this issue by substantially increasing the range of age and experience in our population sampling method.", "Finally, in this study we collected only 120 gesture samples from each participant within a single session.", "Given that we now know that direction is important, future work would certainly seek to increase the number of samples that we collect in each direction, perhaps by instrumenting a game (such as the previous puzzle game 2048 mentioned previously) that uses multi-directional swipe as its main control.", "Future work will seek to capture multiple gesture samples over a number of sessions in order to develop a better understanding of individual gesture consistency over time.", "Our results thus far offer positive signals that a route between a behavioural biometric and a physical biometric is potentially available via features of a common touchscreen interactional gesture.", "Our work so far has uncovered some evidence to support the notion that a physical characteristic of a person – namely their thumb length relative to population trends – could be detectable from characteristics of their behavioural touchscreen interactions.", "Future work will build upon the relationships we have observed here, and will seek to examine other feature extractions along with assessing the predictive capability of the features we have examined thus far.", "Future studies will also seek to increase our population sample and to examine free input (using finger instead of thumbs) along with devices of different form factors." ]	[ "We examine if the length of a person׳s thumb can be revealed from how they interact with a smartphone.", "From thumb length, we can infer other physical characteristics such as likely standing height.", "We examined 19,000 swipe gestures captured from 178 volunteers.", "People with longer thumbs complete swipe gestures faster than those with shorter thumbs.", "Males and females differ in the amount of pressure applied to the screen." ]
A conceptual review on polymer electrolytes and ion transport models	This review article provides a deep insight into the ion conduction mechanism in polymer electrolytes (PEs). The concepts of different categories of polymer electrolytes are discussed. The significance of the existence of functional (polar) groups on the backbone of host polymers, which are used in polymer electrolytes, is well explained. The working principle of electrical impedance spectroscopy (EIS) is overviewed. The relationship between impedance plots and equivalent circuits, which are crucial for electrical characterization, is extensively interpreted. Based on the patterns of dc conductivity (σdc) versus 1000/T, the ion transport models of Arrhenius and Vogel–Tammann–Fulcher (VTF) are discussed. Effects of coupling and decoupling between ionic motion and polymer segmental relaxation are analyzed. The important role of dielectric constant on cationic transport in PEs is also explained. The relationships existing between electrical and dielectric parameters are elucidated, which help interpret and understand the ion conduction mechanism. From the reported empirical curves of dc conductivity vs. dielectric constant, the reformulated Arrhenius [σdc(T)=σ0exp(−[Formula presented])] equation is proposed. Finally, other important phenomena, occurring in polymer electrolytes, are shown to be understandable from the dielectric constant studies.	[ "MATE" ]	[ "Arrhenius model", "Electrical impedance spectroscopy", "Impedance plots", "Polar polymers", "Polymer electrolytes", "Reformulated Arrhenius model", "Vogel–Tammann–Fulcher (VTF) model" ]	[ "2501", "2502", "2503", "2504" ]	[ "Various sources of alternative energy are continuously evolving to reduce the long-term dependence on oil, nuclear and other fossil fuels.", "The other environmentally friendly fuel cells, such as batteries, super capacitors and dye sensitized solar cells, are strong candidates for this reason [1].", "The conception of polymer electrolytes (PEs) is a highly specialized and multidisciplinary field that covers the disciplines of electrochemistry, polymer science, organic and inorganic chemistry [2].", "Dry solid polymer electrolytes (SPEs) have attracted great attention as safer alternatives to liquid electrolytes [3].", "In the field of SPEs, a pioneering work was carried out by Wright et al. and cited by Singh and Bhat [4].", "In their work, the dc conductivity of order of 10−5 S/cm at 330 K in highly crystalline polyethylene oxide-sodium thiocyanate (PEO–NaSCN) complexes was reported [4,5].", "The SPEs are formed by inorganic salts dissolution in a polar polymer matrix.", "The choice of PEs in modern applications, such as high energy density batteries, electrochromic devices, sensors and fuel cells, was justified by studying their structural, morphological and electrical properties [6,7].", "On the other hand, the choice of polymer hosts for PEs largely depends on two factors: first, the existence of polar (functional) groups with a large power of sufficient electron donor to form coordination with cations and, second, a low hindrance to bond rotation [2].", "Fig. 1 shows the chemical structures of some important polymers that are widely used as host polymers in PEs.", "The good mechanical strength, ease of thin film fabrication with desirable shapes and the ability of forming good electrode/electrolyte contact are the main advantages of dry SPEs [6,8,9].", "From the economical and commercial viewpoints, a low-cost membrane with good ionic conductivity, enhanced dimensional and mechanical stabilities are recent challenges to be invented.", "The main drawbacks of SPEs are their high crystallinity and low ionic conductivity [10].", "Polymer electrolytes comprise both crystalline and amorphous regions.", "It has been well reported that the ion transport occurs mainly in the amorphous region rather than the crystalline region, but the polymers host materials that used in PEs are often semi-crystalline [4,11].", "Thus, to overcome the disadvantages and improve SPEs' conductivity, plasticizer, as one mostly applied method, has to be added to improve the ambient ionic conductivity.", "Through using plasticizers, the amorphous region and ion aggregates in PEs can be increased and dissociated, respectively, causing the dc electrical conductivity of SPE to be improved [12].", "It has been established that the ionic conductivity in plasticized polymer electrolytes can be increased at the expense of decreased mechanical strength and vice versa [4,13].", "In addition to high conductivity and a broad electrochemical stability window, PEs must exhibit good thermal and mechanical properties.", "These performances can be achieved by dispersing nanosized fillers into polymer electrolyte.", "Following the creative work of Weston and Steele [4,14], who have improved the ionic conductivity and mechanical stability of polymer electrolytes by adding Al2O3 particles, nanocomposite SPEs have been broadly studied.", "A complete understanding of the effects of inorganic fillers on the ion transport, thermal, mechanical and electrochemical properties of PEs is still not reached [15].", "From the above survey, it is clear that the dc electrical conductivity can be improved by incorporating the plasticizer or inorganic fillers into the SPEs.", "But yet, the ion conduction mechanism in solid plasticized and composite polymer electrolytes is not fully understood [4].", "The main goal of this review article is to shed light on different types of PEs and ion transport models.", "Additionally, the necessary of reformulation of Arrhenius equation based on recent experimental achievements in this field is elucidated.", "Polymer electrolytes have been proved to be promising materials in the research and development of electrochemical devices.", "Most of the research activities are devoted in the field of solid state electrochemistry, in which high ion-conducting materials are considered to be developed for the energy conversion and storage applications [16].", "In this sense, PEs are a class of materials, which have been witnessed in the last 20 years by massive research efforts, to achieve systems with a good conductivity and an electrochemical stability [16,17].", "On the basis of materials, the polymer electrolytes have been categorized into dry solid polymer electrolyte, plasticized polymer electrolytes, gel polymer electrolytes, and composite polymer electrolytes [18].", "The concept of dissolving inorganic salts in functional (polar) polymer, thus creating an ion conducting solid electrolyte is known as a solid polymer electrolyte (SPE) [19].", "The interactions of metal ions with polar groups of polymers are mainly resulting from electrostatic forces and accordingly the formation of coordinating bonds [20,21].", "There are some important factors that may have effect on the polymer-metal ion interactions, such as nature of the functional groups attached to the polymer backbone, compositions and distance between functional groups, molecular weight, degree of branching, nature and charge of metal cation, and counter ions [21].", "The cations can transfer from one coordinated site to another when subjected to an electric field.", "This is due to the weak coordinate of the cations to sites along the polymer chain.", "For a better understanding of these technologically important materials, further study has to be conducted in this field, with a particular emphasis on their complex chemistry and ionic transport properties [19].", "Plasticized polymer electrolytes, which are a branch of PEs, are prepared by incorporating the polymer host with low molecular weight compounds, such as ethylene carbonate, propylene carbonate and poly ethylene glycol (PEG) [22].", "Plasticizers can reduce the number of active centers and thus weaken the intermolecular and intramolecular forces between the polymer chains [23].", "Consequently, they result in lessening the rigidity of the three dimensional structure formed on drying, and changing the mechanical and thermo-mechanical properties of the prepared films [23,24].", "Therefore, the addition of low molecular weight plasticizers decreases the glass transition temperature of the PE system.", "Hence, the reduction of crystallinity and increment in salt dissociation capability are guaranteed, by which the enhancement of charge carrier transport is achieved.", "However, the resulting polymer electrolytes are predicted to obtain a low mechanical strength [25].", "Recent studies have confirmed that the amorphous fraction of composite polymer electrolytes can be increased due to the plasticizers.", "It has been reported in ref. [26] that the crystallinity can be decreased when the plasticizer (PEG200) is added to the polyethylene oxide (PEO) based nanocomposite polymer electrolytes, as shown in Fig. 2b and c.", "It is obvious that the micrograph of PEO25-NaClO4 + 5 wt.", "% DMMT complex system [see Fig. 2a] shows the presence of spherulites.", "The boundary between the spherulites can be attributed to the presence of amorphous fraction.", "The surface roughness in Fig. 2a was ascribed to the existence of large amount of crystalline fraction in the PEO based polymer electrolyte.", "It was observed that the surface roughness was decreased upon the addition of plasticizer [see Fig. 2b and c], exhibiting a smooth surface texture.", "These changes could be attributed to the effect of plasticization that resulted in the reduction of crystallinity of the host polymer PEO and the enhancement of the overall amorphous fraction in the materials.", "From the above discussion, it is understood that the increase of SPE conductivity by addition of plasticizer at room temperature results in the loss of mechanical strength.", "Furthermore, plasticized polymer electrolytes exhibit a number of drawbacks, such as inadequate mechanical properties at high level of plasticization, reactivity of the polar solvents with lithium electrode and solvent volatility [22,27].", "Recently, a substantial effort in the field of polymer electrolytes has been given to gel-type polymer electrolyte (GPE).", "This is due to the fact that the advantages of liquid-type electrolytes, such as high ionic conductivity, and solid-state electrolytes, such as high safety, can be combined.", "In gel-type PE, polymer as a host matrix has been used to trap the liquid constituents.", "Therefore, the GPE based products are considered to be much safer than liquid-based electrolyte products, particularly, when it is used in lithium ion (Li-ion) batteries [28].", "In the preparation of gel electrolytes, a large amount of organic solvent or plasticizer must be added into the polymer host [29].", "The incorporated plasticizer molecules can form a wide network whereby the ion conduction takes place along with the host polymer, which principally provides structural support.", "Gel electrolytes can exhibit high ambient conductivities, but still undergo some other disadvantages as mentioned in the plasticized polymer electrolytes, such as the release of volatiles and increased reactivity towards the metal electrode [30].", "Scheme 1 shows the influence of plasticizer on the percolative behavior of ion transport.", "It is clear that the portion of amorphous regions is well below a percolation threshold at room temperature (See the left hand side of Scheme. 1), resulting in the poor ionic conductivity.", "Plasticized polymer electrolytes with suitable liquid solvents that have high dielectric constant, ε, and low viscosity, η, are desired to form gel polymer electrolytes.", "Therefore, the amorphous regions grow larger in number and size, owing to the adsorption of liquid.", "This ultimately leads the percolation threshold to be accomplished at ambient temperature.", "The connected network of amorphous regions provides fast ion conducting pathways, which acts upon enhancing the ion mobility and hence the higher ionic conductivity [31].", "One of the major reasons behind the poor ionic conductivity of polymer electrolytes has been attributed to the presence of ion-pairs (or ion-association) and ion triplets.", "This is due to weak dielectric constant of the host polymers [32].", "Many approaches have been developed to avoid the occurrence of ion–ion association in polymer electrolytes.", "To solve these difficulties and improve the qualities of SPEs, inorganic inert fillers with high dielectric constant has been lately suggested to be dispersed in PEs [33].", "Dielectric permittivity can be properly adjusted, simply by controlling the type and the amount of incorporated inorganic filler material.", "Ceramic materials, which are classified as inorganic fillers, are typically fragile and possess low dielectric strength [34].", "Though, polymers have relatively low dielectric permittivity, they can undergo high fields, they are also flexible and easy to be processed.", "Therefore, by combining the advantages of these two materials, i.e., ceramic filler and polymer material, new hybrid composite materials with high dielectric constants can be fabricated [35].", "When the size of these inorganic fillers is in the nano-dimension, the newly formed composites are called nanocomposite polymer electrolyte (NCPE) [10,33].", "Composite polymeric materials containing fine ceramic particles are considered as heterogeneously disordered systems [34,36].", "Their electrical property depends on the dielectric constant and their constituents' conductivity.", "Additionally, the volume fraction, size and shape of the added filler particles have impacts on the electrical performance of composite materials [34].", "Li et al. [37] have successfully investigated the effect of in situ synthesized TiO2 on the morphology of poly (vinylidene difluoride-co-hexa fluoro propylene) (PVDF-co-HFP)) complexed with LiPF6.", "The surface and cross-section morphology of the PVDF-co-HFP polymer incorporated with different amounts of in situ synthesized TiO2 nanoparticles have been studied.", "They have observed many closed pores on the surface of the samples.", "They have found that the connected spherical pores are crucial in enhancing the ion mobility and ionic conductivity.", "Electrochemical impedance spectroscopy (EIS) is a technique currently used to study the electrical properties of the bulk materials and their interfaces (i.e., electrode–electrolyte interfaces) over a wide range of frequency and temperature.", "It is also known as ac impedance spectroscopy (or dielectric spectroscopy).", "The bulk and interface contributions can be separated by using this technique.", "It can also be used to study the ion conduction mechanism and dielectric relaxation in PEs.", "Electrical charge displacements in a bulk material produce two distinct physical phenomena: (i) if the charge motion in a localized volume of the matter is strictly confined then a polarization phenomenon takes place or (ii) if the electrical charges in the materials are collectively diffused over long distances then diffusion is possible and a dc conductivity, σdc, is established [38].", "In order to identify and overcome the effect of space charge polarization at the electrode/solid electrolyte interface, it is crucial to carry out the ac conductivity measurements by means of complex impedance spectroscopy (CIS), i.e., using an ac electric field [39].", "It is established that, dielectric spectroscopy depends on the tendency of ions and dipoles to orient along the electric field direction [40].", "When an ac electric field is applied to a parallel plate capacitor sandwich with polymeric materials, four categories of polarization are taken place, which are known as electronic, atomic, dipolar and migrating charge polarizations [41].", "Ions often originate as impurities in the raw materials.", "Dipoles result from atoms with unequal electro negativities, which are attached to each other on the backbone of polymeric materials [40].", "The dielectric relaxation processes are usually correlated to one or more polarization processes of the studied material.", "Dipolar polarization and polarization due to charge migration are the two main components of the dielectric responses in polymers.", "Dipolar and charge migration polarizations can be detected at frequencies less than 109 Hz [41].", "If the electric field is reversed in sign (or direction), the dipoles will realigned with the applied field and the ions start to diffuse (or migrate) to the other electrode.", "As the frequency of field reversal increases, the ions and dipoles become increasingly difficult to keep up with the field changes.", "In addition to that the functional groups with larger sizes will be much harder to be reorienting with the field [40].", "The investigation of conduction and different dielectric polarizations can eventually lead us to achieve more information on the dynamic behavior with regard to relaxation processes.", "The complex impedance plot corresponding to Eq. (7) is illustrated in Fig. 3c.", "The impedance plot of a parallel combination of a single resistor and a capacitor, ZR−C, has a semicircular shape on the complex impedance plane, as shown in Fig. 3d. Qualitatively, this behavior can be easily understood.", "The impedance of the capacitor at very low frequencies becomes very large.", "Thus the majority of the current is flown through the resistor, causing its properties to be dominated.", "On the other hand, the impedance of the capacitor at very high frequencies becomes very small, such that the resistor is effectively shorted out and gives rise to zero net impedance [48].", "Experimental evidence reveals that in complex impedance plane plots the low frequency tail is not truly vertical as illustrated schematically in Fig. 3e. Fig. 4 shows an experimental data example of this behavior.", "Most commonly, it is impossible for the ac response to be described by using simple ideal circuits.", "The semicircles showing in the complex (Z″-Z′) plane are often widened and deformed to asymmetrical arcs [49].", "One can see in Fig. 4 that the center of the semicircles lies below the real Z′ axis.", "The impedance response of PVA:AgNt (75:25) reveals the semicircular and spike regions at high and low frequencies, respectively.", "The semicircle at high frequency indicates the bulk response of the sample, whereas the spike at low frequency can be attributed to the accumulated double layer charges at the solid polymer electrolyte/electrodes interfaces.", "At low frequencies, the impedance plots should expose a straight line parallel with the imaginary axis; though the electrode polarization effect (double layer capacitance) at the blocking electrodes induces a curvature [50–52].", "A crucial feature that distinguishes ion conducting polymer electrolytes from other ionic conductors is that polymer electrolytes are formed by dissolving low lattice energy salts in a polar polymer matrix.", "For this reason, the cations are found to be responsible for the dc ionic conductivity.", "In agreement with the theories of cationic transport in high molar mass polymer electrolytes, long range cation transport only takes place by dissociative steps, in which cations can move between neighboring coordinating sites, whether situated on the host molecule or on a nearby host molecule [61].", "It might therefore be predicted that cations, forming non-labile bonds with polar groups of the host polymer, would not promote the dc conductivity of a polymer electrolyte [61].", "Fig. 5 shows the ionic motion of a lithium ion in a PEO-host.", "It has been established that polymer electrolytes comprise both amorphous and crystalline fractions at room temperature.", "It is known that ion transport principally occurs in the amorphous regions.", "Despite the fact that the conduction mechanisms are not fully understood yet, it is widely recognized that cations, which are interconnected with functional groups of the host polymer chains, can move through re-coordination along the polymer backbone [63].", "Based on recent review works, the polymer chains are reported to be folded to form cylindrical tunnels, in which the cations are located and coordinated by the functional groups [64,65].", "These cylindrical tunnels create channels, providing a pathway for the movement of cations.", "The study of dc conductivity vs 1000/T can be conducted to identify the crystalline and amorphous nature of solid polymer electrolytes as can be seen in later sections.", "This is related to the fact that the result of this investigation can be interpreted in terms of one of the following models:", "Most of polar polymers used in the preparation of polymer electrolytes are semicrystalline polymers, i.e., containing both crystalline and amorphous phases.", "For instance, host polymer PEO has a glass transition temperature, Tg, of −67 and melting temperature, Tm, of about 68 °C.", "The existence of a high crystallinity fraction of PEO below Tm, can prevent the movement of small chain segments (i.e., segmental motion) in the PEO polymer.", "However, the crystalline regions are completely absent above the melting temperature (Tm) and hence a relatively high extent of segmental motion would be expected, and result in a high dc conductivity [71].", "The two regions for the plot of dc conductivity versus inverse temperature (1000/T) have been outlined for the PEO based polymer electrolytes as can be seen in Fig. 7.", "At temperature below Tm (i.e., in region I), the dc conductivity was observed to increase gradually with the temperature up to 70 °C, as depicted in Fig. 7.", "On the other hand, above the Tm temperature (i.e., in region II), the conductivity was noticed to abruptly increase with temperature compared to that of region I.", "This is due to that, at high temperature, the energy would be large enough to overcome the potential barriers that created between the sites and thus leads to increase the free volume in the system, which facilitates the segmental motion of ionic charge carriers [72].", "Therefore, the segmental motion either allows the ions to be hoped from one site to another site or offers a pathway for ions to be moved [71,72].", "It is therefore understood that, in polymer electrolytes, the ionic motion can takes place through the transitional motion/hopping and dynamic segmental motion of the host polymer [72,73].", "As the amorphous phase progressively swells at high temperature (i.e., region II), the polymer chain gains faster internal modes in which bond rotation creates segmental motion.", "This, successively, favors the inter-chain and intra-chain ion hopping of ion movements and the conductivity of the polymer electrolyte accordingly becomes higher [73,74].", "Furthermore, Kim et al. [78] and Uma et al. [79] have explained the curvature feature of dc conductivity versus inverse temperature.", "They have confirmed that the VTF behavior can still be retained, in which the ion transport is correlated with the polymer segmental motion.", "However, in Ref. [80], the coupling between cation transport and segmental motion of the polymer has been demonstrated by the curvature of dc ionic conductivity versus 1000/T.", "As earlier mentioned, the free volume model can be used to understand the correlation between ion transport and segmental mobility.", "The extensive and intensive survey of literature revealed that most of researchers have applied the free volume model to interpret the abrupt increase in the dc conductivity at high temperatures (Arrhenius or VTF behavior).", "This agrees with the explanation that the polymer will expand and produce free volume as the temperature increases.", "Therefore, through the produced free volume, the polymer segments, ions or solvated molecules can easily move.", "Consequently, increased values are expected in ion and segmental mobility, which will assist the ion transport [81].", "The question is, despite the existence of polymer segmental motion, why does still the Arrhenius conductivity linearly behave at higher temperatures?", "This can be explained by the fact that, at higher temperature, the increase in conductivity can be attributed to the vibrational dynamics of the polymer backbone and side chains.", "The increase of vibrational amplitude can bring the coordination sites closer together and enable the ions to hop from their occupied site to an adjacent empty site, using less energy [8].", "In other words, the polymer segmental motion is decoupled completely from the ionic motion, i.e., the polymer segmental motion just brings the coordination sites closer together and hence ions can be easily hopped from one site to another.", "The pattern of dc conductivity versus 1000/T as a result becomes linear as occurred for ionic crystals.", "From the above discussion, it is understood that the linear and curvature behaviors of dc conductivity can be ascribed to the coupling and decoupling mechanisms between the ionic and polymer segmental motions, respectively.", "In Ref. [108], the effect of ethylene carbonate (EC) on the dielectric properties of PVA:LiBr:H2SO4 polymer electrolyte was studied.", "It was shown that the increment of storage charge was attributed to the increase of dielectric constant in the material, owing to the ion dissociation in the EC plasticized polymer system.", "Ethylene carbonate (EC) plasticizer has been shown that to have higher dielectric constant, PVA and thus more salts can be dissociated.", "This is consistent with those obtained from Eq. (11) as mentioned earlier.", "Various researches have been conducted to study the dielectric properties of polymer electrolytes and nanocomposite polymer electrolytes [50,109–112].", "It is commonly recognized that the electrical impedance spectroscopy is an essential experimental technique for studying the dynamics of ion transport.", "An ac device has been used to measure the capacitive (imaginary) and resistive (real) component as a function of frequency for the sample inserted between two electronically conducting electrodes [113].", "Several important phenomena in polymer electrolytes can be identified by studying their dielectric constant.", "In our previous work [8], the dc conductivity for the chitosan:AgTf solid polymer electrolyte at higher temperatures has been found to be decreasing due to the reduction of silver ions to silver particles.", "This silver ions (Ag+) transformation to silver particles (Ago) means reducing the number of involving silver ions in the conduction of charge as expected from Eq. (10).", "The existence of such silver particles induces grain boundaries and thus increases the resistance within the bulk of the material [8].", "In our previously reported study on dielectric constant for the chitosan:AgTf system at high temperatures (see Fig. 13), this transformation phenomenon and its effects on dc conductivity have been confirmed [115].", "Evidence for this transformation and its effects on the dc conductivity and dielectric constant in the chitosan:AgTf system has been given in detail in the Refs. [8] and [115].", "Moreover, in another recent work, we have demonstrated the importance of the dielectric constant study in understanding the electrical percolation phenomenon in ion conducting solid polymer composites (see Fig. 14) [116].", "Therefore, from the above reviews and findings, it can be noted that the study of dielectric constant is a viable informative technique to predict the conductivity behavior of polymer electrolytes and other physical phenomena as mentioned above.", "The concepts of different categories of polymer electrolytes have been discussed.", "Different materials' design in polymer electrolytes, such as plasticized polymer electrolytes (PPEs), gel polymer electrolytes (GPEs) and composite polymer electrolytes (CPEs), has been mainly aimed at developing dry solid polymer electrolytes (DSPEs) with a high amorphous content, in which a higher ion transmission can be provided.", "The significance of presence of functional (polar) groups on the backbone of host polymers, which are used in polymer electrolytes, has been revealed.", "A comprehensive explanation has been given on the working principle of electrical impedance spectroscopy, which is an important technique for electrical characterization of polymer electrolytes.", "The relationship between impedance plots and equivalent circuits, which are crucial for electrical characterization, has been analyzed in detail.", "The Arrhenius and Vogel–Tammann–Fulcher (VTF) models for ion transport have been shown to be inadequate in interpreting the ion conduction mechanism.", "It has been pointed out that the crystalline phases have a great influence on lowering the dc ionic conductivity in polymer electrolytes.", "Therefore, the researchers are encouraged to redirect their intention of research towards the development of new polar polymers with high amorphous contents.", "Effects of coupling and decoupling between the ionic motion and polymer segmental relaxation on the pattern of dc conductivity have been elucidated.", "In reference to our recent publications, the role of dielectric constant on cationic transport in polymer electrolytes has been shown to be crucial.", "The importance of the electrical and dielectric parameters relationships has been emphasized to understand the ion conduction mechanism.", "The presence of an empirical smooth curve between the dc conductivity and dielectric constant has revealed that the ion conduction mechanism in condensed matter physics is not a straightforward subject and the current ion transport models have to be reformulated.", "Some other important physics phenomena occurring in polymer electrolytes can be understood from the dielectric constant studies." ]	[]
Elaphostrongylus and Dictyocaulus infections in Norwegian wild reindeer and red deer populations in relation to summer pasture altitude and climate	Nematodes of the genera Elaphostrongylus and Dictyocaulus are associated with disease in semi-domesticated tundra reindeer and farmed red deer whereas less knowledge exists in the wild. Their first stage larvae (L1) develop to the infective third stage (L3) in the environment; Elaphostrongylus spp. within intermediate gastropod hosts and Dictyocaulus spp. as free-living larvae. Larval development of Elaphostrongylus is highly temperature dependent with a developmental minimum of 9–10 °C. Larval development of Dictyocaulus spp. may occur at low temperatures (5 °C) but the larvae are sensitive to desiccation. We examined the prevalence and intensity of Elaphostrongylus spp. and Dictyocaulus spp. infections in six wild reindeer and two wild red deer populations in relation to altitude, temperature and rainfall in their respective main summer pasture area over the 5 summers prior to sampling. The parasitological examination was based upon morphological identification of L1 in the faeces of hunted animals. Altitude was calculated from animal position data and temperature and precipitation by means of a nationwide gridded data set. Temperature decreased with increasing altitude, from 13.3 °C for the lowest located red deer population (300 m) to 6.1 °C for the highest located reindeer population (1400 m). No significant relationship between altitude and rainfall was identified. Elaphostrongylus spp. infection decreased in prevalence with increasing altitude, being identified in 89% of investigated samples from the lowest located population and in 3% of samples from the highest. The prevalence of Dictyocaulus spp. infection varied between 28 and 80% and no relationship with altitude was found. The intensity of Elaphostrongylus spp. infection was low in reindeer and moderate in red deer whereas the intensity of Dictyocaulus spp. infection was moderate in both species. Our results indicated that the climatic conditions in all areas studied were suitable for Dictyocaulus spp., whereas summer temperature was a restrictive factor for Elaphostrongylus sp. in reindeer.	[ "AGRI", "IMMU", "MEDI" ]	[ "Cervids", "Climate", "Deer", "Dictyocaulus", "Ecology", "Elaphostrongylus" ]	[ "1103", "2405", "2725" ]	[ "Nematodes of the genus Elaphostrongylus (brain worm) are parasites and a cause of neurological disease in cervids.", "Reindeer (Rangifer tarandus) are hosts for E. rangiferi and red deer (Cervus elaphus) for E. cervi (Gibbons et al., 1991).", "Dictyocaulus nematodes (lungworm) infect both cervids and domestic ruminants and cause bronchitis.", "Dictyocaulus spp. found in the lungs of cervids were previously often identified as D. viviparus (bovine lung worm) but according to recent studies, the valid names of Dictyocaulus in reindeer and red deer are D. eckerti and D. cervi respectively (see Pyziel et al., 2017).", "Both Elaphostrongylus spp. and Dictyocaulus spp. have a climate-related ecology.", "This is linked to the ambient temperature and moisture dependent development of first stage larvae (L1) shed in the faeces of an infected host, to infective third stage larvae (L3) in the environment.", "Larval development from L1 to L3 of E. rangiferi and E. cervi takes place in various intermediate gastropod hosts (Mitskevich, 1964; Panin, 1994; Prosl and Kutzer, 1980; Skorping and Halvorsen, 1980) and is highly temperature-dependent.", "Halvorsen and Skorping (1982) reported a developmental time in gastropods of L3 (E. rangiferi) of about 12 days at 24 °C and 2 months at 12 °C, with a developmental minimum of 9–10 °C.", "Hosts are infected following ingestion of gastropods containing L3 larvae that have developed during summer (June–August) (Halvorsen et al., 1980).", "The L3 develop to adult nematodes in the brain and spinal cord (CNS) and subsequently migrate to the skeletal muscles (Hemmingsen et al., 1993; Handeland, 1994; Handeland et al., 2000).", "Clinical elaphostrongylosis (neurological signs) is associated with the developmental phase in the CNS (Handeland, 1994; Handeland et al., 1994).", "The pre-patent period is 3–5 months (Mitskevich, 1964; Panin, 1964) and patency of infection is several years (Halvorsen et al., 1985).", "Due to the long pre-patent period, infected calves will normally not excrete Elaphostrongylus L1 larvae until after the autumn hunt.", "Thus infections found in hunted yearlings were generally acquired the previous year (as calves).", "Infections in adult animals may have occurred several years previously.", "Due to the high temperature requirement for larval development of Elaphostrongylus spp. in gastropods, low summer temperature is considered a restrictive factor for host infection at high latitudes e.g. for semi-domesticated tundra reindeer (Rangifer tarandus tarandus) in northern Norway (Halvorsen et al., 1980; Halvorsen and Skorping, 1982).", "In this area, epizootics of clinical elaphostrongylosis in reindeer have occurred after particularly hot summers (Handeland and Slettbakk, 1994).", "A close association between summer temperature and the level of Elaphostrongylus infection could also be expected for the wild tundra reindeer populations in southern Norway, and possibly in wild red deer.", "Wild reindeer populations live in alpine and sub-alpine mountain areas with short and cool summers (Punsvik and Frøstrup, 2016) whereas wild red deer usually have winter grounds at lower altitudes and a majority of the populations migrate to higher inland summer pastures during spring (Mysterud et al., 2011; Bischof et al., 2012).", "The level of Elaphostrongylus infection in areas with previously marginal summer temperatures may increase as a result of climate warming.", "Virtually no information exists about Elaphostrongylus infection in Norwegian wild reindeer populations while two studies have documented the presence of this parasite in wild red deer (Ottestad, 1983; Bakka et al., 2006).", "Unlike Elaphostrongylus spp., development of Dictyocaulus L1 to L3 is direct and can take place at low temperatures (5 °C) (Rose, 1956).", "However, the larvae are sensitive to desiccation and sufficient moisture (precipitation) is important for larval survival and host infection.", "Hosts are infected following ingestion of Dictyocaulus L3 present on the vegetation.", "The pre-patent period and patency of infection (D. viviparus) are around one and two months respectively (Taylor et al., 2007).", "While clinical outbreaks of dictyocaulosis have been registered in semi-domesticated reindeer herds in northern Norway (Kummeneje, 1977) no knowledge exists of this infection in wild reindeer populations.", "Dictyocaulus sp. has been recognized as the single most important nematode parasite in farmed red deer in Norway (unpublished data) and other countries, including the UK and New-Zealand (Charleston, 1980; Corrigall et al., 1980; Fletcher, 1982; Mason, 1995).", "Although commonly found in the lungs of autopsied wild red deer in this country, no systematic prevalence studies on Dictyocaulus infection have been carried out.", "In the present study, we examined the prevalence and intensity of faecal Elaphostrongylus and Dictyocaulus L1 excretion in six wild reindeer and two red deer populations sampled during autumn hunting in southern Norway.", "The results were analyzed in relation to average altitude of each population's main summer pasture, and the calculated temperature and rainfall in this area during the previous five summers prior to sampling.", "Climatic calculations were extended to assess trends during the last 60 years, to look for changes in the face of climate change.", "The wild tundra reindeer of southern Norway represent the last naïve remains of this species in Western Europe.", "Today they are distributed between 23 separate populations, totaling 25,000 to 30,000 wintering animals (WA).", "This study includes six of these populations (Fig. 1): 1 Forollhogna (2000 WA), 2 Snøhetta (2700 WA), 6 Rondane (3500 WA), 14 Nordfjella (2100 WA), 19 Setesdal Ryfylke (3500 WA) and 20 Setesdal Austhei (2000 WA) (Punsvik and Frøstrup, 2016).", "The Rondane population is divided into northern and southern subpopulations and this study concerned the southern subpopulation.", "During the last decade, the positions of each flock, with the exception of Forollhogna, have been monitored by means of GPS-tracked animals.", "For Forollhogna, standardized field position data is available (from 2015) based on a systematic “reindeer seen” (Sett-rein) registration system.", "GPS position data for June–August was used to calculate altitude in meters above sea level (m.a.s.l.) of the main summer grazing area of each reindeer population.", "The number of registered GPS-positions varied between 30,470 and 142,560 per population.", "For Forollhogna, the calculation was based upon 1009 recorded field positions.", "ArcMap from ESRI and Spatial Analyst/Kernel Density were used to create “heat maps\"/raster maps that visualized the main summer grazing area.", "We chose to define these maps with 30 classes and made the layer that indicated the least use transparent (Fig. 2).", "To calculate average altitudes, we ran analyses of the Point Data Sets (GPS and “reindeer seen”) against the Digital Terrain Model from the National Mapping Authority/GeoNorge.", "“Spatial Analyst/Extract values two points” was used to calculate m.a.s.l. for each position.", "The exported data set automatically gave us average m.a.s.l. of the main grazing area with standard deviation (SD), and min and max positions.", "Wild red deer are widely distributed in southern Norway with the highest densities in the western coastal municipalities.", "This study encompassed two of these municipalities: 24 Ørsta and 25 Kvinnherad (Fig. 1).", "GPS position data for June–August was used to calculate altitude in m.a.s.l. of the main summer grazing area.", "For the Kvinnherad population, position data (33,076 positions) was available from GPS-tracked adult animals in the period 2009–2012.", "For the Ørsta population, no radio-collar studies have been carried out and therefore position data (51,193 positions) from GPS-tracked adults in three neighboring municipalities Stranda, Norddal and Sykkylven (2014–2015) was used.", "The m.a.s.l. of each GPS position was calculated using the GIS program QGIS (https://www.qgis.org/en/site/) and a solid digital terrain model with a resolution of 10 × 10 m (Norwegian Mapping Authority, 2017).", "The altitude information that is the basis of the digital terrain model is primarily height curves and height points from the common map database (FKB).", "The average m.a.s.l. of the main summer grazing area (June–August) with SD, and min and max positions were calculated.", "For climatological calculations, a newly developed gridded data set for Norway (seNorge2) was used (Lussana et al., 2018a and b).", "This is based upon long-term data from all weather stations and makes possible extrapolation of homogenous daily air temperature (2 m above the ground) and precipitation (at 1 × 1 km) for the whole country.", "The method is particularly robust in the summer when the bias and uncertainties are lower than in winter.", "The calculated average elevation of the main summer grazing area, and recorded min and max heights for individual populations were used in the model.", "Data sets for each population were created from grid cells for the calculated heights (±10 m), centered in each of the areas.", "The mean summer (June–August) air temperature and monthly precipitation at the average main grazing area and minimum pasture heights were calculated for the last five summers prior to sampling.", "Long term calculations for the last 60 years (1959–2018) were also carried out.", "Sampling was performed from 2012 to 2014 between August 20-September 30 (median August 31) for reindeer and September 1-December 3 (median September 23) for red deer.", "Faecal samples were collected from the rectum by licensed hunters and sent by mail to the laboratory as soon as possible after hunting.", "Each sample was accompanied by data on the sex, age (calf, yearling [1.5 yr], or adult [≥ 2.5 yr]), date of hunting and geographical location.", "Samples lacking age data or with insufficient quantities of faeces were excluded from the study.", "The number of animals, age distribution and year of sampling for individual populations are shown in Table 1.", "Faecal samples were examined for L1 larvae per gram (LPG; intensity of infection) of Dictyocaulus and Elaphostrongylus, following a slightly modified Baermann technique (Taylor et al., 2007).", "Briefly, 10 g of faeces was weighed and placed in a double gauze cloth, closed with a plastic strip and suspended in a conical glass containing lukewarm water overnight.", "The following day, the faecal parcel was removed and the supernatant aspirated until 5–10 mL liquid remained undisturbed at the bottom the glass.", "This fluid was drawn off into a 15 mL conical centrifuge tube, the glass rinsed, and this fluid also added to the tube.", "This was then centrifuged at 1500 rpm for 3 min and the supernatant aspirated, leaving 1 mL undisturbed at the bottom of the tube.", "A 100 μL subsample was removed from the re-suspended contents of the tube and examined under the microscope at x40-x100magnification.", "Larvae were identified based on larval morphology and the LPG calculated.", "In cases in which no larvae were detected in the first subsample, a second 100 μL subsample was examined from the base of the tube after sedimentation had been allowed to occur.", "If no larvae were observed in this second sample, the result was recorded as no larvae detected.", "L1 of E. rangiferi and E.cervi were identified by measuring body length (ten larvae per animal) and the presence of a dorsal spine on the tail (Lankester and Northcott, 1979; Mason, 1995).", "No other protostrongylid nematodes are known to parasitize reindeer in Europe.", "The protostrongylid lung worm Varestrongylus sagittatus of red deer produces dorsal-spined L1 that can be separated from those of E. cervi on the basis of their shorter body length (Mason, 1995).", "Dictyocaulus L1 were identified on shape, length and the presence of dark brown (chromatin) food granules within their intestinal cells (Taylor et al., 2007).", "The prevalence and intensity data for Elaphostrongylus and Dictyocaulus larvae were analyzed by a multiple regression analysis, using the R-package pscl (Jackman, 2017) and a hurdle model (Zeileis et al., 2008).", "In a hurdle model, the zero-data are modelled by logistic regression, while counts larger than zero are modelled by a truncated count distribution.", "Here, we used a truncated negative binomial distribution.", "The most parsimonious model was selected according to the smallest AICc-value, and we considered an absolute difference in AICc-values less than −2 to be insignificant (Burnham and Anderson, 2002).", "The full model included age-class, sex and geographical area.", "Alternative models replacing geographical area with altitude for each main summer grazing area (mean and standard deviation) were also tested.", "Mean temperature and precipitation for the last five years prior to sampling were not included in the models due to the high correlations between the climatic variables and altitude (see section 3.3).", "Data from reindeer and red deer were analyzed both separately and together.", "Analyses were performed in R, version 3.5.2 (R Core Team, 2018).", "The calculated average altitude of the main grazing area for each population is illustrated in Fig. 3.", "The grazing areas were significantly higher in Snøhetta (No. 2) and Nordfjella (No. 14), compared to the other four reindeer populations (No. 1, 6, 19, 20).", "Grazing areas for the two red deer populations were significantly lower than for the reindeer populations with the lowest being Kvinnherad (No. 25).", "The prevalence of Elaphostrongylus and Dictyocaulus infections in individual populations are shown in Fig. 4.", "The average prevalence of Elaphostrongylus infection (yearlings, adults) for each species was 46% (76/166) in reindeer and 80% (60/75) in red deer.", "The average prevalence of Dictyocaulus infection (calves, yearlings, adults) was 54% both in reindeer (118/218) and red deer (56/104).", "For reindeer, a significantly lower prevalence of infection was found in the two highest (No. 2, 14) compared to the four lowest (No. 1, 6, 19, 20) located areas (p < 0.0001).", "When modelling the prevalence by average altitude of the populations’ main summer pasture area, instead of using geographical area as a factor, the model performed equally well according to the AICc-criteria (ΔAICc = 0.5).", "The general prevalence was significantly higher (p < 0.001) in reindeer adults (68/134; 51%), compared to yearlings (7/32; 22%).", "This was in contrast to red deer in which the prevalence tended to be higher (p = 0.08) in yearlings (28/32; 88%) than adults (32/43; 74%).", "Only one red deer calf and one reindeer calf, both killed in September, shed Elaphostrongylus L1 larvae.", "Two red deer from Ørsta shed small numbers of L1 morphologically compatible with Varestrongylus sagittatus.", "The general intensity of Elaphostrongylus infection in reindeer was low, with median and maximum LPGs of 6 and 77, respectively.", "The general intensity of Elaphostrongylus infection was significantly higher in red deer compared to reindeer (p < 0.001), with median and maximum LPGs of 28 and 820.", "Significantly higher LPGs were found in adult red deer males (p < 0.001), compared to adult females and yearlings.", "Dictyocaulus spp. was common in all populations and age groups.", "The general prevalence and intensity of infection did not differ significantly between reindeer and red deer.", "There was no correlation between prevalence of infection and elevation of the populations’ main summer pasture areas.", "The median LPG was ≥2 and ≤ 40 for all populations and age groups, except for three adult reindeer and one adult red deer that shed >100 LPG.", "In reindeer, the general prevalence was lower in calves and female adults compared to male adults and yearlings (p < 0.001).", "Intensity of Dictyocaulus infection in reindeer was higher in adults compared to calves (p = 0.001).", "In red deer, there were no significant differences in Dictyocaulus infection between age groups or sexes.", "The calculated mean and range of summer temperature and monthly rainfall for each population's main pasture during the five summers prior to sampling, are illustrated in Fig. 5.", "There was a negative linear correlation between summer temperature and pasture altitude (Pearson correlation r = −0.993, p < 0.001).", "Monthly summer precipitation was also (although more weakly) correlated to elevation (r = −0.684, p = 0.06).", "The calculated mean and range of summer temperature for each population's main pasture during the periods 1959–1988 and 1989–2018, are illustrated in Fig. 6.", "For all areas, there was a significant increase in the mean summer temperature of 0.5–0.8 °C for individual populations (average: 0.7 °C) between the two 30-year-periods (p < 0.001).", "Only a tendency towards increased rainfall was observed (p = 0.09) from the first to the second period.", "The annual number of months in each main summer pasture area with a mean temperature ≥12 °C for the period 1989–2018 is shown in Fig. 7.", "A temperature of ≥12 °C for at least two months was reached for all populations in most years at their lowest recorded summer positions.", "The present study demonstrated a marked reduction in prevalence and intensity of Elaphostrongylus infection with increasing altitude and reduced temperature in the main summer grazing areas.", "We consider these findings to be closely related to the temperature-dependent development of L1 larvae to infective L3 larvae in gastropods (Halvorsen and Skorping, 1982).", "Whether differences in gastropod density between the areas could also be a contributing factor is not known (gastropods are common in Norwegian mountain areas).", "According to our calculations, a sufficiently high temperature is not maintained over a period long enough to allow development of L3 in the course of a single summer in the main summer pasture areas of the two highest located reindeer populations.", "While occurring only exceptionally in the other reindeer areas, these conditions are met regularly in red deer areas.", "It may therefore be speculated that while red deer may become infected over their entire main summer grazing areas, reindeer infection mainly occurs at relatively low altitude, in areas with restricted animal residence time and possibility of infection.", "Halvorsen and Skorping (1982) have hypothesized that the parasite may compensate for low temperature by fulfilling its larval development in overwintering gastropods.", "This could be a factor contributing to maintenance of infection in reindeer pastures, especially in the two highest located populations.", "The patent infection identified in single reindeer and red deer calves may reflect ingestion of L3-containing gastropods in early summer, in which larval development had started the previous year.", "A higher Elaphostrongylus infection pressure in red deer compared to reindeer was reflected both through a generally higher intensity of infection and a larger proportion of yearlings infected (reflecting infection as calves) in the red deer populations.", "With reference to earlier studies (Prosl and Kutzer, 1982; Halvorsen et al., 1985), we consider that the intensity of infection identified was low in reindeer and moderate in red deer.", "The higher intensity of infection found in adult male red deer, compared to adult females and yearlings could be a result of increased larval production due to stress-induced immune suppression during the autumn rutting season in adult males (Gaudernack et al., 1984; Halvorsen et al., 1985).", "For reindeer, the prevalence of infection found in yearlings was less than half of that found in adults.", "This was in contrast to red deer in which the prevalence was similar in yearlings and adults.", "These findings indicate a time of infection as calves, yearlings or young adults in the reindeer, and as calves in red deer.", "Consequently, clinical elaphostrongylosis should be a considered diagnosis among calves and young animals in reindeer and among calves in red deer.", "However, our data suggest mild to moderate infections, with presumably few clinical effects at the population level.", "Nevertheless, it should be emphasized that even slight clinical signs occurring in late autumn and winter may be critical, especially in the harsh environment inhabited by wild reindeer.", "An increasing level of clinical significance of Elaphostrongylus infection can be expected in light of climate warming, as illustrated by the 0.7 °C increase in mean temperature from the period 1959–1988 to 1989–2018 found in the present study.", "This increase in clinical significance could be especially valid for reindeer, as suggested by Handeland and Slettbakk (1994).", "The authors speculated that, due to the marginal summer temperatures in reindeer areas, the animals may have developed little specific immunity prior to heavy infections that may occur within a restricted period of time, following hot summers.", "This was considered in contrast to cervids (red deer) living in temperate areas that, due to more favorable condition for development of L3 in gastropods, probably ingest L3 over a longer summer period, and thus develop more immunological protection prior to the main season of infection in late summer.", "Dictyocaulus infection was common in all populations and age groups of reindeer and red deer and precipitation levels seem adequate for larval development in all studied environments.", "The highest prevalence and intensity of Dictyocaulus infection was found in the reindeer population in Snøhetta (Fig. 4 - No. 2, intensity data not shown).", "Dictyocaulus infection (D. eckerti) is also prevalent in the Norwegian muskox population located in the Snøhetta area (Davidson et al., 2014).", "The mechanism of transmission of Dictyocaulus spp. from one year to another is through overwintering larvae in the pasture, or hypobiotic larvae in the lungs of carrier animals (Taylor et al., 2007).", "A low freeze tolerance has been demonstrated for larvae of D. viviparus (Rose, 1956) whereas the freeze tolerance for Dictyocaulus spp. in cervids is unknown.", "However, the winter conditions in the summer grazing areas of the populations examined in this study are harsh, especially for reindeer and we consider overwintering as hypobiotic larvae in the lungs to be the most likely way of inter-annual transmission.", "The interpretation of intensity of Dictyocaulus infection based on LPG is challenging since it may vary throughout summer (Prosl and Kutzer, 1982; Davidson et al., 2014) and due to a lack of good reference values.", "For D. filaria infection in small ruminants, levels between 2 and 100 LPG are considered to reflect moderate and >100 LPG high degrees of infection respectively (DTU Veterinærinstituttet, 2009).", "By these standards, the infections identified in the present study were of moderate and only exceptionally high grade.", "A high clinical significance of Dictyocaulus infection and death among calves has been reported in farmed deer in various countries (Charleston, 1980; Mason, 1995; Corrigall et al., 1980).", "Harsh climatic conditions, poor pasture and poor body condition have been suggested as contributing factors (Corrigall et al., 1980).", "The significance of dictyocaulosis in wild cervid populations is poorly documented.", "However, several reports suggest this infection to be a common cause of death in young wild red deer (Dunn, 1967) and reindeer (Rahko et al., 1992) in Europe, and in caribou and muskox in North America (see Kutz et al., 2012).", "In reindeer herds in northern Norway, outbreaks of dictyocaulosis with or without contribution of opportunistic bacteria (Pasteurella multocida), have caused severe losses of calves in late April and early May (Kummeneje, 1977).", "These outbreaks were presumably caused by hypobiotic larvae, maturing in the lungs in spring.", "Dictyocaulosis in deer is characterized by reduced food intake and rapid loss of body condition without typical respiratory signs and may be challenging to recognize in live animals (Corrigall et al., 1980).", "Based on our findings, we suggest Dictyocaulus infection to be an unrecognized cause of disease in our wild reindeer and red deer populations.", "In conclusion, this study demonstrated that Elaphostrongylus and Dictyocaulus infections are endemic and highly prevalent in Norwegian wild reindeer and red deer populations.", "We consider Dictyocaulus infection to be a potential cause of disease and death in both species, especially among poorly nourished calves in spring.", "For reindeer, we predict an increased significance of Elaphostrongylus infection in the face of climate warming." ]	[ "6 wild reindeer and 2 red deer populations investigated for nematode larvae.", "Elaphostrongylus spp. and Dictyocaulus spp. prevalence varied: 3–89% and 28–80%.", "Elaphostrongylus spp. prevalence decreased with increasing grazing altitude.", "No relationship between altitude and Dictyocaulus spp. prevalence.", "Summer temperature restrictive factor for Elaphostrongylus sp. in reindeer." ]
Synergistic action of dual IGF1/R and MEK inhibition sensitizes childhood acute lymphoblastic leukemia (ALL) cells to cytotoxic agents and involves downregulation of STAT6 and PDAP1	Heterogeneous upregulation of multiple prosurvival pathways underlies resistance to damage-induced apoptosis in acute lymphoblastic leukemia (ALL) cells despite normal p53 responses. Here, we show that the dual combination of insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1/R) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition using AG1024 + U0126 can sensitize apoptosis-resistant ALL cells to ionizing radiation-induced DNA damage irrespective of effect of single pathway inhibition in vitro. This AG1024 + U0126 combination also significantly potentiates the ability of the core chemotherapy compounds vincristine, dexamethasone, and daunorubicin to kill ALL cells in vitro. Evidence of the synergistic action of AG1024 + U0126 in samples with variable basal levels of phosphorylated IGF1/Rβ and ERK1/2 suggested additional targets of this drug combination. Consistent with this, gene expression profiling identified 32 “synergy genes” differentially targeted by IGF1/R + MEK inhibition and, among these, Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) were the most differentially downregulated cluster. Pearson correlation analysesrevealed that STAT6 and PDAP1 display significant expression codependency and a common expression pattern linked with other key “synergy” genes, supporting their predicted role in an STAT6–ERK–nuclear factor kappa beta (NF-κB) network. Knockdown studies revealed that loss of STAT6, but not PDAP1, impinges on the cell cycle, causing reduced numbers of viable cells. In combination with daunorubicin, STAT6 loss has an additive effect on cell killing, whereas PDAP1 loss is synergistic, indicating an important role of PDAP1 in the cellular response to this anthracycline. Inhibition of STAT6 or PDAP1 may therefore represent a potential novel therapeutic strategy for resistant ALL by enhancing sensitivity to chemotherapy.	[ "BIOC", "MEDI" ]	[]	[ "1306", "1307", "1311", "1312", "2720" ]	[ "Patient bone marrow (BM) samples were collected from Birmingham Children's Hospital with ethical approval and written consent (CCLG 08/H0405/22 and 08/H1208/4).", "Leukemic BM mononuclear cells were separated by density centrifugation and frozen in a viable state before use.", "For clinical data, see Supplementary Table E1 (online only, available at www.exphem.org).", "Cells were treated with 5 Gy ionizing radiation (IR) and incubated with U0126 (Promega, WI, USA), AG1024, or LY294002 (Calbiochem, Darmstadt, Germany) at 37°C for 24hours, as described previously [21].", "Apoptosis was assayed using an Annexin V Apoptosis Kit (BD Pharmingen, San Diego, CA) and analyzed using a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA).", "Cells were considered to be apoptotic if they stained positive for both Annexin V and PI (Ann+/PI+).", "The proportion of IR-induced apoptotic cells was determined by subtracting the proportion of apoptotic cells detected in the absence of IR.", "U0126, AG1024, LY294002, vincristine, and daunorubicin were dissolved in dimethylsulfoxide (DMSO) and dexamethasone in 100% ethanol at stock concentrations of 10mmol/L.", "Cells were incubated with U0126, AG1024, vincristine, daunorubicin, or dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) at the indicated doses at 37°C for 72hours and subsequently reacted with Cell TitreGlo reagent according to the manufacturer's instructions (Promega).", "Luminescence was quantified using a Victor Wallac plate reader.", "Weston blotting was performed as described previously [21].", "Antibodies included IGF1Rβ (#3027), phospho-IGF1Rβ (Tyr1131)/insulin receptor β (Tyr1146) (#3021), ERK1/2 (137F5), phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E), PDAP1 (#4300), procaspase 7 (D2Q3L), cleaved caspase 3 (5A1E) (Cell Signaling Technology, Danvers, MA, USA), STAT6 (ab44718) (Abcam, Cambridge, MA, USA), and PARP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).", "Mouse monoclonal antibody against β-actin (AC-74) (Sigma-Aldrich) served as a loading control.", "Cells were untreated, treated with 5 Gy IR, 30 µmol/L AG1024 + 5 Gy IR, 20 µmol/L U0126 + 5 Gy IR, or 30 µmol/L AG1024 + 20 µmol/L U0126 + 5 Gy IR for 6hours in vitro before RNA extraction using a combined TRIzol/chloroform(Invitrogen)/RNeasy column purification (Qiagen) method as described previously [21].", "After first- and second-strand synthesis and in vitro transcription, samples were hybridized to HuGene1.0 ST v1 gene chips (Affymetrix, Santa Clara, CA, USA).", "Probe-level quantile normalization [22] and robust multiarray analysis [23] on the raw .", "CEL files was performed using AltAnalyze [24].", "Differentially expressed genes were identified using limma with a fold change >1.5 and p < 0.01 [25].", "Small interfering RNA (siRNA) silencing was performed in HeLa cells using SiGenome SMARTpools targeting human STAT6 (6778) and human PDAP1 (11333) (Thermo Scientific, Waltham, MA, USA).", "Two daily sequential siRNA treatments were performed using DharmaFECT transfection (Thermo Scientific) according to the manufacturer's instructions and nontargeting pool #2 (scrambled siRNA) served as a control.", "Data from three separate knockdown experiments are shown.", "Treated cells were fixed in 100% ice-cold ethanol before staining with PI (Sigma-Aldrich) and cell cycle profiles assessed using a Coulter Epics XL-MCL flow cytometer (Beckman Coulter).", "Combination indices (CIs) were determined using dose– response curves and Calcusyn software and potentiation effects (PEs) using paired Student t tests of data normalized to untreated cells and with a single-agent effect subtracted.", "Standard deviations are shown.", "Pearson correlation coefficients were determined by comparing normalized LOG2 expression values as described previously [26].", "Network analysis was performed by seeding synergy genes using Ingenuity Systems IPA software (Qiagen) according to the manufacturer's instructions.", "As a consequence of the heterogeneous upregulation of multiple prosurvival signaling pathways underlying defective apoptotic responses in childhood ALL, we reported previously that individual prosurvival pathway inhibitors targeting MEK, IGF1/R, and PI3K induced patient-specific responses to IR-induced DNA damage in vitro [20,21].", "We hypothesized that dual combination of prosurvival pathway inhibitors might sensitize a broader range of leukemias and therefore inform of a more applicable therapeutic approach.", "In this study, we compared dual combinations of the IGF1/R, MEK, and PI3K inhibitors AG1024, U0126, and LY294002, respectively, at the same micromolar doses we reported as single agents in our previous study [21].", "We identified that the specific combination of AG1024 (IGF1/R inhibitor) and U0126 (MEK inhibitor) was consistently active in sensitizing four DNA damage-resistant ALLto IR, whereas dual combinations involving thePI3K inhibitor LY294002 induced more variable responses (Figures 1A and B).", "The combination of AG1024 + U0126 consistently induced the highest level of IR-induced apoptosis, as measured by Annexin and PI staining 24hours after treatment (Figures 1A and B), and this occurred irrespective of the sensitivity of the cells to each single agent [21].", "The drug vehicle DMSO alone had minimal effect on the survival of primary ALL cells at the same doses even after 72hours (Supplementary Figure E1, online only, available at www.exphem.org).", "In two representative samples, western blotting confirmed that AG1024 + U0126 + IR induced caspase- and PARP1-dependent apoptosis to a greater extent than the single most potent inhibitor for each leukaemia.", "(Supplementary Figure E2, online only, available at www.exphem.org).", "Using dose– response curves (Supplementary Figure E3, online only, available at www.exphem.org) and Calcusyn software, we were able to determine CI values for ALL-141, ALL-102, and ALL-106, which revealed strong synergism, synergism, and a near additive effect between AG1024 and U0126, respectively (Figure 1A and Supplementary Table E2, online only, available at www.exphem.org).", "For ALL-111, the dose curves for both U0126 and AG1024 alone were inhibitory.", "We therefore determined the PE that indicated a strongly synergistic effect of the combined AG1024 + U0126 treatment (Figure 1B and Supplementary Figure E3 and Supplementary Table E2, online only, available at www.exphem.org).", "Therefore, whereas in ALL-141, ALL-102, and ALL-111, the combined effect of AG1024 + U0126 was synergistic, in ALL-106, the combination of AG1024 + U0126 was not superior to U0126 alone: both single-agent treatments exhibited efficacy, as did the AG1024 + LY294002 combination.", "Nonetheless, the sensitivity of ALL-106 to AG1024 and U0126 as single agents or in combination shows that a treatment strategy targeting IGF1/R + MEK pathways would sensitize apoptosis-resistant primary ALL cells to IR-induced DNA damage.", "We observed no synergistic effect of the two agents when combined in normal peripheral blood mononuclear cells in the presence of IR-induced DNA damage (Supplementary Figure E4, online only, available at www.exphem.org).", "We next investigated whether this specific combination of IGF1/R + MEK inhibition could sensitize ALL cells to other clinically relevant cytotoxic agents.", "To do so, we tested whether AG1024 + U0126 would sensitize ALL to the core remission-inducing chemotherapy agents dexamethasone, vincristine, and daunorubicin in vitro.", "We evaluated the effect of the lower doses 1 and 10 µmol/L AG1024 + U0126 on chemotherapy-induced killing after 72hours in eight primary ALL samples and observed chemosensitization for all three drugs (Figure 1C).", "In combination with 100nmol/L dexamethasone, synergism was observed with 1 and 10 µmol/L AG1024 + U0126 (PE, paired t tests, p = 0.0084 and p = 0.0166, respectively).", "AG1024 + U0126 also increased ALL sensitivity to 100nmol/L vincristine, with 1 µmol/L displaying an additive effect and 10 µmol/L displaying synergism (PE, paired t test, p = 0.019).", "Finally, AG1024 + U0126 exerted an additive effect with 10nmol/L daunorubicin and, although synergism was observed with 100nmol/L daunorubicin (paired t test, p = 0.01), the killing induced by daunorubicin alone was already very high and the incremental change was small (not shown).", "The combination of IGF1/R + MEK inhibition at micromolar doses is therefore able to sensitize ALL cells, including high-risk and relapse samples, to submicromolar doses of chemotherapy agents in vitro.", "We next set about investigating the possible mode of action for the synergistic effect of AG1024 + U0126 in sensitizing childhood ALL cells.", "To address this, we investigated whether an association existed between the basal levels of the phosphorylated ERK1/2, an indicator of activated MEK pathway, and phosphorylated IGF1/Rβ, an indicator of activated IGF1 and insulin pathways, in 10 primary ALLs that were sensitive to the synergistic effect of 10 µmol/L AG1024 + U0126 after 72hours in vitro compared with each of the single inhibitors in the absence of other cytotoxic agents (PE, paired t test, p = 0.0001) (Figure 2A).", "We did not observe a synergistic effect using 1 µmol/L AG1024 + U0126 (Supplementary Figure E5, online only, available at www.exphem.org).", "These samples (where material was sufficient) revealed highly variable basal levels of phosphorylated ERK1/2 and IGF1/Rβ proteins (Figure 2B), suggesting that the synergistic action of combined AG1024 + U0126 treatment was not wholly dependent on targeting the MEK and IGF1/R pathways.", "Based on the variable basal levels of activated of MEK and IGF1/R pathways, it appears that the synergistic action of AG1024 + U0126 in ALL cells may function in part through additional or off-target activity.", "To explore possible additional mechanisms of this drug combination, we employed gene expression profiling in four ALLs, ALL-102, ALL-141, ALL-150, and ALL-200, which displayed synergistic responses to AG1024 + U0126.", "Samples were treated either with IR to induce DNA damage, with each of the single inhibitors + IR, or AG1024 + U0126 + IR for 6hours.", "To demonstrate that the individual inhibitors were active, we first evaluated the alteration of known target genes by comparing AG1024 + IR with IR alone and U0126 + IR with IR alone.", "This comparison revealed differential expression of known target genes for each of the individual inhibitors, which, as well as MAPK and G-protein-coupled receptor genes, included miR222, SP1, and DUSP6 [27,28] for the MEK inhibitor U0126 and JUN, SPRY, APAF1, and BAK1 [29] for the IGF1/R inhibitor AG1024, as well as TNS3, previously correlated with IGF1 levels [30].", "We subsequently compared gene expression in cells treated with AG1024 + U0126 + IR with cells treated with each inhibitor alone + IR to identify a set of “synergism” genes that were differentially expressed after 6hours of treatment with AG1024 + U0126 (for Venn analysis, see Supplementary Figure E6, online only, available at www.exphem.org).", "Of the 32 differentially expressed genes, six were consistently differentially upregulated and 26 downregulated by AG1024 + U0126 + IR treatment (Figure 2C).", "Because this set of genes could contribute to apoptosis resistance in some ALL, we set about investigating candidate synergy genes further.", "Of the set of synergy genes we identified, STAT6 and PDAP1 were among the most significantly differentially downregulated (Figure 3A) and were validated by independent quantitative reverse transcription polymerase chain reaction (Supplementary Figure E7, online only, available at www.exphem.org).", "These genes were of particular interest because of their possible role in high-risk subtypes of childhood ALL.", "Phosphorylated STAT6 has been reported to be elevated in Ph+ ALL [31] and PDAP1 appears to be involved in PDGF signaling [32,33], a pathway recently implicated in apoptosis-resistant ALL [21] and Ph-like ALL [8–12,18].", "To provide some insight into how these synergy genes might interact to induce chemosensitization when downregulated, we used logarithmic expression data from 20 arrays used in this study (untreated, IR only, AG1024 + IR, U0126 + IR, and AG1024 + U0126 + IR for each of the four ALL samples) to enable Pearson coefficient correlation analyses.", "This identified a marked (r > 0.6) and highly significant expression codependency between STAT6 and PDAP1 (Figure 3B).", "When we compared a possible relationship between STAT6 and PDAP1 and the remaining synergy genes, we observed a striking pattern of linked expression that was common to both STAT6 and PDAP1 (Figure 3C).", "We next performed network analysis using Ingenuity software to determine whether the synergy genes that we identified were predicted or known to interact in common pathways.", "This analysis identified a major network associated with cell death and survival, cellular function and maintenance, and hematological system development and function.", "STAT6 and PDAP1 were associated with this network, of which STAT6 was a major component in addition to ERK and NF-κB.", "In this STAT6–ERK–NF-κB network, STAT6 and PDAP1 appeared to be closely associated (via PDGF), consistent with our codependent expression data (Figure 3D).", "Other components of this network (PRDM8 and MPZL) had also shown linked expression with both STAT6 and PDAP1 using Pearson correlation analysis (see above).", "This STAT6–ERK–NF-κB network could be an important mechanism underpinning apoptosis resistance in ALL cells and warrants further characterization.", "Because our data suggested that STAT6 and PDAP1 might contribute to apoptosis resistance, we hypothesized that loss of these proteins individually might reduce cell viability and/or sensitize cells to chemotherapy.", "To address the cellular impact of loss of STAT6 and PDAP1, we performed siRNA-mediated knockdown in HeLa cells.", "Because of the difficulties in obtaining gene knockdown in primary human ALL cells, we used HeLa cells as an alternative since they enable reproducible and effective protein knockdown.", "Although HeLa cells are of a different tissue origin than ALL, the rationale for choosing this model system to explore the cellular impact of loss of these proteins on chemotherapy sensitization was because they also represent a widely accepted model for studying DNA damage response proteins [34,35].", "Figure 4A shows reduction of each of the proteins after 72hours of siRNA treatment.", "Interestingly, we observed that the loss of PDAP1 appeared to lead to an increase in STAT6 protein expression.", "This apparent connection at the protein level supports the codependent expression correlations that we observed at the mRNA level for these molecules.", "We first investigated the impact of loss of each of the proteins on cell viability and the cell cycle.", "In untreated cells, STAT6 knockdown by siRNA treatment had the greatest impact on the number of viable cells, whereas PDAP1 loss had no impact on the number of viable cells after 1 week of siRNA treatment (Figure 4B).", "When we assessed alterations in the cell cycle, we found that, consistent with the reduction in the number of viable cells and with previous findings [36], STAT6 caused a 20% increase in the proportion of cells in the G1 phase of the cell cycle, indicating an increased number of arrested cells and a reduction in cycling.", "In contrast, PDAP1 loss had no impact on the cell cycle, which is consistent with the absence of effect of PDAP1 loss on viable cell number (Figure 4C).", "We next investigated the ability of loss of each of these proteins to sensitize cells to core chemotherapy agents used to induce remission in ALL patients.", "When we combined protein knockdown with increasing doses of the anthracycline daunorubicin in vitro, the impact on the total number of viable cells caused by knockdown of STAT6 had an additive effect (Figure 4D), whereas, strikingly, PDAP1 knockdown was synergistic after 72hours (Figure 4D).", "In contrast, when STAT6 was silenced, the impact on cell cycle arrest (Figure 4D) reduced the sensitivity of HeLa cells to increasing doses of vincristine, the mechanism of which is dependent on cell proliferation.", "PDAP1 knockdown had no effect at all on vincristine-induced cell killing after 72hours of treatment (Supplementary Figure E8, online only, available at www.exphem.org).", "The HeLa cells displayed no evidence of sensitivity to dexamethasone with or without knockdown (data not shown).", "These data support the notion that the inhibition of pathways involving STAT6 and PDAP1 contributes to the potentiating effect of combined AG1024 + U0126 treatment on DNA-damaging agents such as IR and daunorubicin.", "The inability of loss of the single genes selected for further exploration in this study to sensitize HeLa cells to vincristine or dexamethasone suggests that it is likely that alternative genes or a combination of genes are implicated in sensitization observed in both HeLa and leukemia cells.", "This is supported by the microarray data.", "However, with respect to daunorubicin, our data from HeLa cells indicate that STAT6, and in particular PDAP1, appear to function in the cellular response to daunorubicin and warrant further investigation in leukemia cells.", "In summary, we have identified a specific combination of prosurvival signaling pathway inhibitors, AG1024 + U0126, which was consistently able to sensitize apoptosis-resistant primary ALL cells to DNA-damaging agents.", "This dual combination targets a specific set of “synergy” genes that includes STAT6 and PDAP1, which are predicted to function in an STAT6–ERK–NF-κB regulatory network.", "Childhood ALL is genetically heterogeneous and deregulation of different prosurvival signaling pathways can contribute to apoptosis resistance.", "In this study, we sought to determine whether the inhibition of a specific combination of prosurvival pathways could sensitize ALL irrespective of response to single pathway inhibition and if this might inform of a more applicable uniform treatment approach for ALL patients.", "We have shown that the combined inhibition of the IGF1/R and MEK pathways using AG1024 + U0126 can sensitize ALL cells in a synergistic manner with IR-induced DNA damage and can also potentiate the effects of core chemotherapy agents in vitro.", "Gene expression profiling revealed a set of synergy genes that included STAT6 and PDAP1, which are predicted to function in a STAT6–ERK–NF-κB regulatory network.", "JAK/STAT signaling is implicated in many cancers [36].", "STAT6 hyperactivation has been described in a several lymphoid malignancies and, recently, STAT6-activating mutations have been identified in a range of lymphomas with a frequency of up to 40% [36–39].", "In ALL, phosphorylated STAT6 levels are elevated in Ph+ disease [31] and, given the similarities, it is interesting to speculate that STAT6 activity might also be upregulated in the high-risk Ph-like ALL subtype.", "STAT6 signaling has been implicated specifically in treatment resistance and progression in several malignancies, including the response of chronic lymphocytic leukemia cells to B-cell receptor-mediated treatment and radioresistance in inflammatory breast cancer cells [40–43].", "There is mounting evidence, therefore, that STAT6 signaling plays a relevant role in the pathophysiology and clinical responses of lymphoid and other malignancies.", "These data support our conclusions that STAT6 is likely to be important in the cellular response to DNA damage and could contribute to apoptosis resistance in ALL.", "Although the function is largely unknown, PDAP1 was originally identified as a PDGF-interacting protein [32].", "Indeed, PDAP1 was shown recently to be an effector of PDGR signaling in glioma cells and was associated with proliferation and disease progression, highlighting PDAP1 as a potential therapeutic target [33].", "PDGF signaling is a pathway that we recently implicated in apoptosis resistance in childhood ALL [21] and, furthermore, PDGFRB translocations are a recurrent feature of high-risk ALL [8–12,18].", "Interestingly, cells with NF1 inactivation and consequential hyperactivation of the RAS–MAPK–ERK signaling pathway also display overexpression of PDGFRs, which contributes to RAS-induced proliferation [44,45] and supports a role for PDAP1 in a putative STAT6–ERK–NF-κB regulatory network.", "In our study, we showed that loss of PDAP1 synergized significantly with daunorubicin to induce killing in HeLa cells.", "We suggest that the role of PDAP1 in the cellular response to DNA-damaging agents should be investigated further in leukemia cells because PDAP1 might represent an interesting novel therapeutic target for chemosensitization.", "In summary, through dual IGF1/R and MEK inhibition, we have identified a group of genes that appear to contribute to impaired apoptotic responses to DNA damage and, when targeted, can sensitize ALL cells to chemotherapy agents.", "In particular, we have demonstrated that STAT6 and PDAP1, via a putative STAT6–ERK–NF-κB network, may represent useful molecular targets for treatment-resistant ALL, particularly in the absence of clinically available IGF1/R inhibitors.", "It has already been demonstrated that JAK inhibitors such as ruxolitinib or leflunomide, which abrogate JAK3/STAT6 tyrosine phosphorylation, could represent useful treatment approaches for some ALL.", "A novel therapeutic approach for ALL could also potentially include STAT6 inhibition.", "STAT6 small-molecule inhibitors are currently under development for the treatment of asthma and could also be evaluated in the context of ALL.", "Overall, it will be important to delineate STAT6/PDAP1 signaling precisely in ALL and other malignancies to elucidate the role of these molecules in treatment resistance." ]	[ "Dual insulin-like growth factor 1 receptor (IGF1/R) + mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition synergistically sensitize apoptosis-resistant acute lymphoblastic leukemia (ALL) cells.", "Dual IGF1/R + MEK inhibition potentiates chemotherapy-induced killing of ALL cells.", "Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) are downregulated differentially by this synergistic action.", "STAT6 and PDAP1 are predicted to act in a putative STAT6–ERK–nuclear factor kappa beta (NF-κB) survival network.", "Targeting this network may be useful for sensitizing ALL to chemotherapy agents." ]
Effect of aerobic exercise on hippocampal volume in humans: A systematic review and meta-analysis	Hippocampal volume increase in response to aerobic exercise has been consistently observed in animal models. However, the evidence from human studies is equivocal. We undertook a systematic review to identify all controlled trials examining the effect of aerobic exercise on the hippocampal volumes in humans, and applied meta-analytic techniques to determine if aerobic exercise resulted in volumetric increases. We also sought to establish how volume changes differed in relation to unilateral measures of left/right hippocampal volume, and across the lifespan. A systematic search identified 4398 articles, of which 14 were eligible for inclusion in the primary analysis. A random-effects meta-analysis showed no significant effect of aerobic exercise on total hippocampal volume across the 737 participants. However, aerobic exercise had significant positive effects on left hippocampal volume in comparison to control conditions. Post-hoc analyses indicated effects were driven through exercise preventing the volumetric decreases which occur over time. These results provide meta-analytic evidence for exercise-induced volumetric retention in the left hippocampus. Aerobic exercise interventions may be useful for preventing age-related hippocampal deterioration and maintaining neuronal health.	[ "NEUR" ]	[ "Hippocampus", "Neurodegeneration", "Neurogenesis", "Physical activity", "Plasticity", "Resistance" ]	[ "2805", "2808" ]	[ "The hippocampus is one of the major brain sites of neuroplasticity, and has been identified as a brain region sensitive to the effects of physical activity, and in particular aerobic exercise interventions (Cotman et al., 2007).", "The impact of aerobic exercise on hippocampal size and function has been extensively studied in rodents (Van Praag et al., 2005).", "These animal studies have shown that voluntary exercise is associated with hippocampal neurogenesis (Van Praag, 2008) and prevents age-related decline in cell-proliferation in this brain structure.", "Changes in hippocampal structure may be mediated by trophic factors, such as insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF) (Ferris et al., 2007; Vaynman et al., 2004).", "Angiogenesis may also contribute, with evidence that dentate gyrus cerebral blood volume (CBV) is increased after exercise in rodents (Swain et al., 2003; Van Praag et al., 2005).", "In addition, dentate gyrus CBV was significantly associated with increased oxygen uptake (V02) following aerobic exercise in healthy human volunteers (r = 0.66, p < 0.05) (Pereira et al., 2007).", "Such changes have also been linked to cognitive benefits in both animal and human studies (Adlard et al., 2005; Kimhy et al., 2015).", "Whilst there is clear evidence in animal studies that exercise training is associated with increased hippocampal neurogenesis, the evidence from human studies is equivocal.", "Along with a recent meta-analysis showing that physical activity is correlated with greater white matter volume (Sexton et al., 2016), there are also several studies reporting cross-sectional relationships between physical activity levels and hippocampal volumes (Erickson et al., 2010).", "However, determining causality from such correlational analyses is not possible.", "Some randomized controlled trials (RCTs) have reported exercise-induced increases in hippocampal volumes.", "For instance, Pajonk et al. (2010) and Erickson et al. (2011a) demonstrated that exercise increased hippocampal volumes among people with schizophrenia and older adults respectively.", "A previous review of exercise-induced changes in brain regions implicated in the default mode network also presented some preliminary evidence that exercise increased right hippocampal volume (Li et al., 2016).", "However, these findings were limited to four studies, with a combined total of 132 participants receiving exercise interventions.", "Since this review, a number of large controlled studies have been published (e.g. Jonasson et al., 2016; Maass et al., 2015; Lin et al., 2015; Rosano et al., 2016; Malchow et al., 2016; Thomas et al., 2016) – but again with equivocal findings.", "We conducted a systematic review and meta-analysis to determine the impact of aerobic exercise interventions on hippocampal volumes in human subjects.", "We included controlled studies conducted in human subjects, regardless of age or clinical status.", "We also conducted a range of subgroup analyses to explore effects of exercise in unilateral measures of left/right hippocampal volume, and establish effects on the hippocampus in clinical and non-clinical populations.", "Additionally, we employed meta-regression techniques to examine how exercise effects differed with age.", "This systematic review was prospectively registered with the PROSPERO database (#CRD42014009686) and was conducted in accordance with the MOOSE guidelines (Stroup et al., 2000) and in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standard (Moher et al., 2009).", "Two independent authors (SR, JF) searched Medline, PsycARTICLES, AMED, Embase and CINAHL from database inception to August 14th, 2017 for controlled intervention studies.", "Key words used were “exercise” AND “hippocampus” OR “hippocampal volume” in the title, abstract or index term fields.", "Manual searches were also conducted using the reference lists from recovered articles.", "After the removal of duplicates, both reviewers screened the titles and abstracts of all potentially eligible articles.", "Both authors applied the eligibility criteria, and a list of full text articles was developed through consensus.", "The two reviewers then considered the full texts of these articles and the final list of included articles was reached through consensus.", "A third reviewer (BS) was available for mediation throughout this process, however this was not required due to consistent agreement between reviewers.", "Inclusion in this review was restricted to studies of adults aged at least 18 years.", "Studies were included regardless of clinical diagnosis at the time of assessment.", "All populations were used in main analysis, with subgroup analyses used to explore how the effects of exercise on hippocampal volume differ with respect to clinical status.", "Exercise interventions were defined as physical activity that is planned, structured, repetitive and purposive in the sense that improvement or maintenance of physical fitness or health as an objective (Caspersen et al., 1985).", "We restricted this review to aerobic exercise interventions only and excluded other physical activity interventions such as resistance training, as these alternative methods of exercise have different neurological actions to aerobic training (Cassilhas et al., 2012).", "Studies were considered eligible if they examined the effects of a structured, aerobic exercise intervention on hippocampal volumes in comparison to an inactive control condition, or a time-and-attention matched comparator of minimal activity (such as stretching or balance training), of a similar duration.", "Studies which compared two forms of aerobic exercise, without a non-aerobic exercise comparator, were unable to be included.", "The primary outcome measure was absolute hippocampal volume assessed via structural magnetic resonance imaging (MRI) and estimated using either manual tracing or automated methods.", "We included both bilateral and unilateral (i.e. left and right) measures of the hippocampus.", "The method of assessment was recorded.", "When more than three studies were identified recruiting participants with similar diagnoses (e.g. mental disorders), subgroup analyses were conducted.", "We excluded studies with: (a) insufficient data for extraction of primary outcome data; (b) restriction to children and/or adolescents, (c) animal studies and (d) non-exercise interventions.", "In the case of multiple publications from the same study, only the most relevant paper or article was included.", "There were no language or time restrictions placed upon identified articles.", "The quality of included studies was assessed using the Cochrane's Collaboration risk of bias tool (Higgins, 2011).", "This tool was applied to rank each study for ‘Low’, ‘High’ or ‘Unclear’ risk of bias for six aspects of trial design that could introduce different sources of bias (sequence generation, allocation sequence concealment, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, other sources).", "Random-effects meta-analyses were conducted using Comprehensive Meta-Analysis software (Version 2).", "Intervention effect sizes (differences between intervention and control groups) for the primary outcome measure (hippocampal volume) at post-treatment, were calculated using Hedges' g statistic, along with 95% confidence intervals (CIs) around the estimated effect-size.", "Intervention effect sizes were also calculated to determine effects of exercise on hippocampal volume in (i) studies using automated vs. manual segmentation methods, and (ii) unilateral measures of left and right hippocampal volume.", "No study reported a pre and post-test correlation, therefore we assumed a conservative correlation of 0.7 for the primary outcome.", "Effect sizes were categorized as small (0.2), medium (0.5), or large (0.8 or greater.", "Statistical heterogeneity was quantified using the I2 statistic: I2 of more than 75% is considered to indicate considerable heterogeneity, I2 of 50%–75% is considered to indicate substantial heterogeneity, and an I2 of less than 40% is considered to indicate limited heterogeneity.", "Publication bias was tested using the Egger's regression method and Begg & Mazumdar test, with a p-value <0.05 suggesting the presence of bias (Begg and Mazumdar, 1994; Egger et al., 1997).", "Where significant bias was detected, a Duval and Tweedie (2000) trim-and-fill analysis was applied.", "In addition, a funnel plot was created, in which the study-specific effect estimates are displayed in relation to the standard error in order to assess the potential presence of publication bias.", "Subgroup analyses were conducted to examine the effects of exercise on bilateral and unilateral hippocampal volume for any population studied by > 2 trials.", "Furthermore, meta-regression analyses were used to explore the relationship between mean sample age (years) and intervention effect size.", "A total of 4398 records were identified, reduced to 2260 after duplicates were removed.", "After screening and the application of the eligibility criteria, 14 eligible studies were identified and included in the primary meta-analysis (hippocampal volume).", "Fig. 1 presents the flow of studies through the review.", "Table 1 summarizes the characteristics of included trials, including participant characteristics, summary of the intervention, and control group protocol.", "Fourteen trials were included, with eligible outcome data from 737 participants.", "Participants mean age in the included trials ranged from ranged from 24 to 76 years (median = 66 years).", "Four studies recruited participants with a DSM diagnosis of schizophrenia or other psychotic disorders (Lin et al., 2015; Malchow et al., 2016; Pajonk et al., 2010; Scheewe et al., 2013), six recruited healthy older adults (Burzynska et al., 2017; Erickson et al., 2011a; Kleemeyer et al., 2016; Maass et al., 2015; Niemann et al., 2014b; Rosano et al., 2016), whilst individual studies examined the effects of exercise of hippocampal volumes in people with depression (Krogh et al., 2014), mild cognitive impairment (Brinke et al., 2014), probable Alzheimer's disease (Morris et al., 2017), and healthy young-middle aged adults (Thomas et al., 2016).", "Study participants were recruited from the general community (Burzynska et al., 2017; Erickson et al., 2011a; Jonasson et al., 2016; Kleemeyer et al., 2016; Krogh et al., 2014; Maass et al., 2015; Niemann et al., 2014b; Rosano et al., 2016; Thomas et al., 2016), university medical clinics (Brinke et al., 2014), community or outpatient treatment or referred from clinical settings (Lin et al., 2015; Malchow et al., 2016; Morris et al., 2017; Pajonk et al., 2010; Scheewe et al., 2013).", "Risk of bias assessments found that studies varied in their quality and only three studies met the Cochrane standard for Low Risk of bias on all criteria (Burzynska et al., 2017; Krogh et al., 2014; Lin et al., 2015).", "The results of bias assessments for each study are displayed in Supplement 1.", "Four of the studies utilised a stationary cycling intervention (Thomas et al., 2016; Krogh et al., 2014; Pajonk et al., 2010; Malchow et al., 2016), five studies investigated walking-based interventions (Burzynska et al., 2017; Niemann et al., 2014a,b; Erickson et al., 2011a,b; Ten Brinke et al., 2015; Rosano et al., 2016), four combined multiple modalities of aerobic exercise (Lin et al., 2015; Jonasson et al., 2016; Morris et al., 2017; Scheewe et al., 2013), and one used treadmill running (Maass et al., 2015).", "The length of the interventions ranged from three to 24 months with a range of 2–5 sessions per week (see Table 1).", "All studies used supervised exercise sessions.", "Results from all meta-analyses are presented in Table 2.", "Eligible outcome data was available from 14 studies investigating the effect of aerobic exercise on hippocampal volume.", "Pooled effects across 737 participants (387 in exercise interventions and 350 controls) showed a non-significant effect of exercise on total hippocampal volume (Hedges g = 0.120, 95% CI: -0.02 to 0.26, p = 0.082, Fig. 2).", "There was some no evidence of heterogeneity (Q = 14.05, p = 0.371, I2 = 7.45%) and indication of publication bias (Kendall tau = 0.451, p = 0.014, Eggers regression = 0.771, p = 0.13).", "The trim-and-fill analysis identified one outlier studies introducing publication bias, further reducing the non-significant effect (Adjusted g = 0.104, 95% CI = -0.05 to 0.26).", "Sensitivity analyses confirmed that null effects of exercise on hippocampal volume were found from studies using automated segmentation (N = 7, n = 407, g = 0.167, 95% C.I. = -0.01 to 0.34, p = 0.06) and manual segmentation (N = 6, n = 254, g = 0.118, 95% C.I. = -0.13 to 36, p = 0.34), with no significant differences between these two methods (Q = 0.113, p = 0.74).", "Meta-analyses were also performed for the nine studies (total n = 501) that reported changes in left and right hippocampal volumes separately (Fig. 3).", "This showed no significant effect of exercise on right hippocampal volumes (g = 0.164, 95% CI = -0.01 to 0.34, p = 0.065).", "However, aerobic exercise had significant positive effects on left hippocampal volume in comparison to control conditions (g = 0.265, 95% CI = 0.09 to 0.44, p = 0.003) and there was no indication of heterogeneity (I2 = 0%) or publication bias influencing these results (Kendall tau = 0.19, p = 0.23, Eggers regression = 0.23, p = 0.40).", "In order to explore if the positive effects of exercise were due to increasing hippocampal volume, rather than attenuating volume loss in comparison to control conditions) post-hoc analyses were performed to pool the mean changes in left hippocampal volume reported in the exercise conditions.", "Raw change data was available for 8 studies (total n = 256) no significant increase in left hippocampal volume following the exercise interventions (g = 0.049, 95% C.I. = -0.06 to 0.16, p = 0.384), thus indicating the positive effects observed in the primary analysis are due to exercise attenuating age-related neurodegeneration (rather than exercise significantly increasing hippocampal volume).", "Data was pooled from six studies to determine the effect of aerobic exercise on hippocampal volume in healthy older adults (n = 390).", "Aerobic exercise did not significantly increase total hippocampal volume compared to control conditions (g = 0.146, 95% CI: -0.011 to 0.303, p = 0.07).", "Five studies (total n = 332) reported effects of exercise on left/right hippocampus separately.", "There was a statistically significant positive effect of exercise in left hippocampal volume in healthy older adults (g = 0.355, 95% CI = 0.14 to 0.57, p = 0.001), with no indication of heterogeneity or publication bias influencing these results.", "There was also a significant effect of exercise on right hippocampal volume in healthy older adults (g = 0.237, 95% CI = -0.02 to 0.45, p = 0.032), again with no indication of heterogeneity or publication bias.", "Post-hoc analyses examining raw volumetric changes in exercise conditions alone found that exercise in older adults did not significantly increase volume in the left (N = 4, n = 168, g = 0.04, p = 0.70) or right hippocampus (g = 0.034, p = 0.71), again indicating that positive effects observed from exercise are due to slowing down or preventing the volume loss occurring over time among the older adults in control conditions.", "Four studies examined the effect of aerobic exercise on hippocampal volume in people with schizophrenia or first episode psychosis (n = 107).", "Aerobic exercise did not significantly increase total hippocampal volume compared to control conditions (g = 0.149, 95% CI: -0.31 to 0.60, p = 0.53, Table 2).", "Among the two studies which reported effects on left/right hippocampus separately, there was no evidence of effects in either region (both p > 0.1).", "There was also no evidence of heterogeneity or publication bias influencing these results.", "Data in other populations was insufficient for pooled meta-analyses, and so results from individual trials are summarised below.", "Individual trials which examined effects of aerobic exercise in patients with depression (Krogh et al., 2014), mild cognitive impairment (Brinke et al., 2014) and probable Alzheimer's disease (Morris et al., 2017) all found no significant effects on total or left/right hippocampal volumes.", "One study examining the effects of exercise in young-to-middle-aged adults found no change in total hippocampal volume but did find a significant increase in anterior hippocampal volume following 6 weeks of aerobic exercise (Thomas et al., 2016).", "Meta-regression analyses were performed to examine the relationship between mean sample age and effects of exercise on hippocampal volume.", "No statistically significant associations of effects of exercise with sample age were found for total, right or left hippocampal volume (all p > 0.05).", "This meta-analysis examined the effects of exercise on hippocampal volumes in clinical and non-clinical populations.", "Fourteen controlled trials comparing aerobic exercise with various control conditions were eligible for inclusion, with a total of 737 participants.", "Random effects meta-analyses found that aerobic exercise had no effect on total hippocampal volumes (Fig. 2), with no statistical heterogeneity between studies.", "This finding is consistent with that of a previous review which also found no effect of aerobic exercise on total hippocampal volume (Li et al., 2016), although in an analysis that included fewer studies of a smaller number of participants (Five studies, 184 participants).", "The current meta-analysis revealed a significant effect of aerobic exercise on left hippocampal volumes (g = 0.265, 95% CI = 0.1 to 0.44, p = 0.003).", "Interestingly, Li et al. (2016) reported significant effects of exercise in the right hippocampus, but no statistically significant changes in the left hippocampus.", "The current analyses included more than twice as many controlled trials, comprising a total of nine studies (including five newer studies which were not available for inclusion in the earlier review).", "Moreover, the earlier meta-analysis examined post-intervention differences in hippocampal volume and therefore could not account for baseline volumes of the hippocampus or the mean change from pre to post test.", "Given that some of the controlled trials were not randomized and included relatively small numbers of participants, relying solely on posttest changes does not take into account group differences at baseline, which may have impacted the findings.", "Furthermore, there was no evidence of heterogeneity or publication bias influencing our results.", "Nonetheless, the findings should be interpreted with some caution due to the fact that all but two studies had potentially moderate/high risk of bias.", "The same observation of exercise having significantly positive effects on left hippocampal volume was also observed for the subgroup of studies conducted in healthy older adults (g = 0.36, p = 0.001).", "The demonstrated potential for exercise to positively affect hippocampal volume in healthy older adults has important implications, since neuronal mass decreases with increasing age in this area in almost all people during healthy ageing (Raz et al., 2005), and is associated with cognitive decline (Mosconi et al., 2008).", "In the ageing population, the potential benefits of any intervention which attenuates cognitive decline would have a substantial positive impact on public health.", "Indeed, our post-hoc analyses indicated that the positive effects of exercise on hippocampal volume were observed as a result of exercise promoting retention of hippocampal volume (while volume loss occurred in control conditions), rather than actually increasing neuronal mass per-say.", "However, given the limited number of studies in this analysis, caution is required when interpreting this result.", "No significant effects from exercise were observed in people with schizophrenia, although only two studies could be included in the subgroup analysis examining left hippocampal volume change from exercise in this population (total n = 69).", "Similarly, we did not find any significant effects from exercise on left, right or total hippocampal volume in people with depression (Krogh et al., 2014), mild cognitive impairment (Brinke et al., 2014) or probable Alzheimer's disease (Morris et al., 2017).", "However, meta-analyses were not possible for these samples, given there was only data from single studies for each patient group.", "Considering also that all of these studies had small sample sizes, it is equally likely that the non-significant effects observed in these clinical populations are due to underpowered studies.", "Thus, the possible benefits of exercise for hippocampal volume in schizophrenia, depression or cognitive impairment cannot yet be ruled out on the basis of our null findings.", "The neurochemical mechanisms through which exercise promotes retention of hippocampal volume in aging humans has not been fully established.", "However, a comprehensive narrative review examining the neurological effects of exercise across the lifespan (Voss et al., 2011) found evidence from animal models supporting the theory that aerobic exercise acts by preventing the usual decrease in neurogenesis associated with aging, thus resulting in greater retention of neural matter – particularly in the hippocampus.", "Whereas angiogenesis (i.e. increase in brain vasculature) may also be induced by aerobic exercise, this is not observed in animal models using older rodents, and increases in cognitive performance occur independently of vascular changes (Van Praag et al., 2005).", "Voss et al. (2011), concluded that the primary candidate for stimulating neurogenesis in relation to exercise is BDNF.", "The findings of the current review are consistent with this suggestion, as BDNF is found in high concentrations within the hippocampus (Murer et al., 2001) and recent meta-analytic evidence has demonstrated this is upregulated by regular exercise in humans (Szuhany et al., 2015).", "Another factor that may contribute to the more consistent effects of exercise on hippocampal volumes observed among older adults is the likelihood that there may have been greater consistency of non-intervention specific physical activity among older adults.", "As one of the most plastic regions of brain, the hippocampus is more likely to reflect the impact of a wide range of non-specific behavioral effects that are more difficult to constrain in human subjects than in laboratory animals.", "This would in turn reduce the capacity to determine exercise-specific hippocampal volume changes in human subjects.", "The effects of exercise on hippocampal volume in humans have also been linked with improvements cardiorespiratory fitness.", "Cross-sectional studies have previously reported a link between fitness and brain volumes (Erickson et al., 2014), and several of the RCTs also observed a positive association of volumetric changes in the hippocampus with exercise-induced fitness improvement (Erickson et al., 2011b; Kleemeyer et al., 2016; Pajonk et al., 2010).", "Again, the mechanistic link between these two variables is unclear.", "Nonetheless, these findings hold important implications from an implementation perspective, as while it may be difficult to develop exercise programs specifically for improving brain volume, focusing on fitness improvement appears to be one method for designing exercise interventions to confer neurobiological benefits.", "Indeed, previous research has shown that increases in cardiorespiratory fitness are also positively associated with the improvements in cognitive performance which occur from exercise, in both clinical and non-clinical populations (Firth et al., 2017; Hötting and Röder, 2013).", "Considering this alongside the broader benefits for metabolic risk (Kodama et al., 2009), physical capacities (Lord et al., 2003), and mental health (such as reducing depression) (Schuch et al., 2016; Sui et al., 2009), prescribing aerobic exercise with an emphasis on cardiorespiratory fitness enhancement appears to be a suitable intervention for promoting healthy aging, in order to maintain both physical and neurological functioning into old age.", "There are limitations to this review, which are largely reflected by limitations in the primary studies.", "One limitation of this review that we examined changes only in hippocampal volume, without analyzing other brain regions.", "Although the hippocampus has long been recognized as a particularly sensitive region for detecting exercise-induced alterations in both animal and human studies (Hillman et al., 2008; Molteni et al., 2002), other areas are also important to examine for future research (see Table 1).", "For instance, Malchow et al. (2016) showed that aerobic exercise significantly increased brain volume in various areas of the temporal gyri in people with schizophrenia, independently of hippocampal volume change.", "Additionally, there is growing evidence that exercise may alter structure and connectivity in white matter regions (Riggs et al., 2016; Voss et al., 2013) and the basal ganglia - changes which are further related to the cognitive and functional improvements which occur from aerobic exercise (Niemann et al., 2014a).", "Additionally, although we found an overall effect of aerobic exercise on left hippocampal volume, the intensity, frequency and length of exercise programs differed considerably across the studies, and was not reported in sufficient detail to perform comparative subgroup meta-analyses; thus making it difficult to determine which intervention factors are most important for influencing hippocampal volume.", "Perhaps most importantly, the exercise interventions were all relatively short (mostly <12 months) and longer, adequately powered RCTs are required to consider if exercise can influence hippocampal volume over the longer term.", "Future research which compares different exercise modalities, along with varying the frequency and intensity of aerobic training, could provide new insights into optimal program design.", "However, it is important that a ‘fair’ comparator is given when comparing exercise to a control condition, since active controls in other studies have demonstrated considerable improvements in primary outcomes (Stubbs et al., 2016).", "Furthermore, the neurobiological benefits of non-traditional approaches towards aerobic fitness improvement should also be established.", "For instance, ‘high intensity interval training’ (HITT) has recently been shown to produce similar cardiovascular benefits to long-duration endurance training in short periods of times (Wisløff et al., 2009), although the effects on brain health have yet to be established.", "One further consideration is that evidence is currently lacking with regards to a direct link between exercise-induced increases in brain volume and actual cognitive benefits in terms of task performance.", "Assuming that greater brain volume is directly associated with better neural functioning and thus cognitive task performance is reductive, and does not follow from the available evidence (Eyler et al., 2011).", "Indeed, one of the studies in this review even found a negative relationship; with exercise-induced increases in the left hippocampus significantly associated with worse cognitive performance after a 6-month intervention (Ten Brinke et al., 2015).", "Thus, although exercise significantly increased some areas of hippocampal volume and cognition in humans, it cannot be concluded that structural increases are causative or necessary for performance increases (Hillman et al., 2008).", "This is also true for white matter regional increases, as although cardiorespiratory fitness is linked to both improved white matter integrity and cognitive performance, there is no independent link between these variables (Voss et al., 2013).", "In conclusion, this meta-analysis found no effects of exercise on total hippocampal volume, but did find that exercise interventions retained left hippocampal volume significantly more than control conditions.", "As these positive effects were also observed among the subgroup of studies of healthy older adults, the findings hold promising implications for using exercise to attenuate age-related neurological decline.", "Currently, the overall quality of the evidence is compromised by the fact that 10 of the 12 studies included some risk of bias, therefore more high-quality RCTs are now required.", "In additional to RCTs, a prospective meta-analysis examining how changes in physical activity and fitness predict hippocampal retention/deterioration across the lifespan would provide novel insights into longer-term neural effects of exercise, while also reducing the impact of methodological heterogeneity often found across exercise RCTs.", "Further research is also required to determine effects in younger people (Riggs et al., 2016), and establish the neurobiological mechanisms through which exercise exerts these effects, in order to design optimal exercise programs for producing neurocognitive enhancements.", "However, the functional relevance of structural improvements has also yet to be ascertained.", "Nonetheless, the link between cardiorespiratory fitness with both structural and performance increases indicates this as a suitable target for aerobic training programs to improve brain health.", "All authors declare no conflict of interests." ]	[]
MIDAS: Open-source framework for distributed online analysis of data streams	Data streams are pervasive but implementing online analysis of streaming data is often nontrivial as data streams can have different, domain-specific formats. Regardless of the stream, the analysis task is essentially the same: features are extracted from the stream, e.g., to employ machine learning and data mining methods. We present the Modular Integrated Distributed Analysis System (MIDAS) for constructing distributed online stream processing systems for heterogeneous data. The MIDAS framework makes it possible to process raw data streams, extract features, perform machine learning and make the results available through an HTTP API for easy integration with various applications. MIDAS is agnostic with regard to the type of data stream and is suitable for multiple domains.	[ "COMP" ]	[ "Data streams", "Distributed systems", "Machine learning", "Online analysis" ]	[ "1706", "1712" ]	[ "Devices in the Internet of Things (IoT) are found in various fields, e.g., in healthcare [1] or in environmental and agricultural applications [2].", "IoT is also relevant in Human–Computer Interaction (HCI) (e.g., biosignals for controlling user interfaces).", "Many IoT data streams are time series signals with a volume ranging from one channel sampled at 1 Hz (e.g., a temperature sensor) to 16 channels of electroencephalographic (EEG) data sampled at 500 Hz.", "Time series must be processed sequentially sample-by-sample in contrast to batch processing of data, where the processing order of data items is less important.", "Regardless of the source of data streams, potentially important information can be extracted from them.", "Data streams in different domains have varying properties, but the data processing task is essentially identical: features of interest are extracted from the streams and used in decision making.", "There is hence a need for stream processing systems for online extraction and analysis of streaming signals, that can handle various data formats and high data rates.", "Since a typical task is to simultaneously collect data from multiple sensors this suggests a distributed system for balancing the computational load.", "Current stream processing systems are either domain-specific (e.g., Brain–Computer Interface (BCI) frameworks such as BCI2000 [3], OpenViBE [4] or BCILAB [5]) or automation systems (e.g., QIVICON (https://www.qivicon.com/) or EPICS (http://www.aps.anl.gov/epics/)).", "Such systems are monolithic and optimised for processing a small number of parallel data streams arriving at a high rate in a particular domain.", "There are also generic data processing frameworks for handling huge amounts of incoming data at a high rate, e.g., Twitter data streams, at massive scales (e.g., Storm (http://storm.apache.org/), Samza (http://samza.apache.org/) and Spark Streaming (https://spark.apache.org/streaming/)).", "Finally, there are dedicated IoT platforms (e.g., General Electric Predix (http://https://www.predix.com/), PTC Thingworx (https://www.thingworx.com/) and Amazon AWS IoT (https://aws.amazon.com/iot/) or Kaa (https://www.kaaproject.org/)).", "Although the distributed data processing systems and the IoT systems are highly capable they are in many cases unnecessary complex to set up for smaller-scale analyses with few data streams, e.g., for prototyping.", "The focus of this paper is the cross-platform midas (Modular Integrated Distributed Analysis System) framework, primarily engineered to handle high-velocity time series originating from wearables and IoT sensors for use in HCI applications.", "The philosophy of the midas framework is to help researchers create and manage setups with multimodal signal sources, enabling them to focus on the signal processing and machine learning aspects of the system.", "The key design consideration was to create a flexible stream processing system with a distributed architecture for load balancing that is easy to set up and which scales to different types of hardware, such as laptops, desktops and single-board computers.", "midas was engineered with the following design goals in mind, common to systems for online analysis of data streams regardless of the domain.", "The system is data agnostic, i.e., different types of data streams can be analysed since streams may provide complementary information.", "The system is modular and is composed of small autonomous, interconnected units, making it simple to add and remove data streams and analysis components, speeding up the workflow.", "The distributed architecture ensures scalability and distribution of computational load.", "Accessibility is provided using an API built on top of standard protocols, e.g., clients to the system can use HTTP.", "midas is written in Python and is lightweight, e.g., it can be installed in seconds.", "midas does not aim to compete with domain-specific or dedicated high-performing stream processing systems.", "midas aims to be a free, open-source, lightweight alternative to the other frameworks for creating IoT systems, e.g., setting up real-time analysis pipelines for multimodal time-series data for prototyping or research.", "Handling data streams essentially consists of (i) data input, (ii) data processing (feature extraction) and (iii) serving data to clients.", "Offloading these stages on multiple instances benefits the efficiency and flexibility of the system.", "This naturally suggests a three-layered design for the high-level architecture of the system (Fig. 1(a)); the input, processing and client layers.", "The flow of information is from producers on the left to consumers on the right.", "midas comprises the middle processing layer connecting producers and clients.", "midas consists of nodes and dispatchers.", "The nodes are the processing units for the data streams and contain the signal processing, feature extraction and machine learning functionalities.", "Nodes process incoming data into metrics i.e., quantities derived from one or several data streams.", "Data streams can be processed continuously or the node can produce results when requested by a client.", "Automatic broadcasting of results is also supported.", "The dispatcher coordinates the communication between clients and nodes.", "The input layer consists of sensors and streamers.", "Sensors acquire signals and transmit them using an often vendor-specific protocol over, e.g., Bluetooth, USB, or TCP/IP.", "Signals must be decoupled from vendor-specific protocols before entering midas so generic analysis methods can be applied to the data.", "The Lab Streaming Layer (LSL, https://github.com/sccn/labstreaminglayer ) is a networked cross-platform protocol for transmission of time series.", "The LSL allows time synchronisation and discovery of the streams and is the primary data input format from sensors to midas.", "The nodes receive incoming data streams and perform high-level operations on the data.", "A node is the main processing unit in midas and the most complex component with multiple internal units.", "All nodes share the architecture of Fig. 1(b).", "Nodes can be of two main types, depending on their inputs:", "The units inside the node are next described in greater detail.", "The receiver receives incoming data.", "For primary nodes, the data is typically an LSL stream, whereas secondary nodes request data from primary nodes through the dispatcher.", "The incoming signal is stored in a data container.", "The broker receives messages from clients and performs load-balancing and routes incoming requests to an available responder allowing for concurrency.", "A responder receives incoming requests and performs different tasks depending on the type of the message.", "The messaging protocol is described in the midas API (https://github.com/bwrc/midas/wiki/API).", "Each node has a UDP beacon broadcasting status information (IP address, port etc.) for initiating communication with the dispatcher.", "The dispatcher listens for node beacons and uses the information in the messages to automatically add, remove and reconnect nodes.", "The analysis functions represent the functional part of the node, e.g., preprocessing, feature extraction and classification.", "The analysis functions are task-specific and must be implemented by the user.", "The key design philosophy behind midas is to enable the development of analysis methods with minimal effort.", "The only requirement is that the user must follow the defined format for how data is passed to the analysis function, and it is therefore easy to use existing analysis functions in midas.", "Analysis functions have three modes of operation: (i) metric, (ii) process and (iii) publish.", "These are described in more detail below.", "Metric functions are triggered by a request–reply pattern initiated by the client.", "Analysis functions receive the input data as a function parameter and return a value passed back to the client.", "The API supports passing arguments to metric functions.", "Process functions run continuously and typically read from primary data buffers, process the data and push processed values into secondary data buffers.", "Process functions are optimal for continuous computation of a specific variable, e.g., continuously extracting features from incoming data streams.", "This can be useful if computations are slow, in which case the node may return the last computed metric to the client reducing wait-time.", "Publishing functions are similar to process functions and broadcast messages using ZeroMQ (http://zeromq.org/).", "These functions can also act as “watchdogs”, publishing messages if certain conditions are met, e.g., if a metric exceeds a set threshold.", "The broadcasting is realised using a publisher–subscriber model, described next.", "The publisher–subscriber messaging model in midas can broadcast a message, e.g., if the heart rate of the subject exceeds 150 beats per minute (the contingent case) or broadcast the average heart rate every five seconds (the continuous case).", "Clients can then receive data without requests but this requires client support for ZeroMQ.", "All messages published by nodes are routed to clients through the dispatcher.", "A distributed system is defined as a collection of multiple independent computers that appears as one from the perspective of the user [6].", "midas follows a client–server architecture in which the dispatcher handles the bi-directional messaging between nodes and clients.", "The role of the dispatcher in midas is to simplify the communication patterns between components in the system, such as node–node or node–client messaging.", "Hence, although midas is distributed it appears to client applications as one system, since all communication between clients and nodes goes through one (or more) dispatcher.", "Clients only need to communicate with a dispatcher, which simplifies the configuration process since clients only need to be set up with the dispatcher address and can ignore the full network topology.", "Multiple dispatchers can be used concurrently without inconsistency problems since a message from a client to a node and back is routed through the same dispatcher.", "The added benefit of a multi-dispatcher topology is increased robustness, load balancing and redundancy against a single-point-of-failure.", "Communication between clients and nodes takes place through a request–reply messaging pattern over HTTP initiated by the client.", "The communication between the nodes and a dispatcher is realised using an internal messaging protocolimplemented on top of ZeroMQ.", "These network communication protocols allow a distributed architecture where all midas components (nodes, clients and dispatchers), can be run on different computers.", "The discovery of the system topology in midas is automatic and based on the node beacons.", "Nodes can be added and removed during run-time without reconfiguring the system.", "Automated discovery also adds reliability as the connection to a node in an offline state can be automatically restored once the node becomes online.", "The dispatcher can be configured to only communicate with a subset of nodes, which can be used to partition nodes into multiple midas networks, each with their own dispatchers.", "Redundant and load-balanced system topologies where several nodes perform identical tasks can be built using midas.", "A client may then request a metric from one of several nodes yielding the same result.", "Running multiple dispatchers further increases the reliability, redundancy and balances the load.", "Synchronisation of data streams is especially important for analyses utilising shared information between different data streams.", "One way of accounting for network-related latencies is the round-trip time (RTT) metric describing the network latency between the components and the time required to process the request.", "In the LSL protocol each sample is time-stamped at the source and the LSL can constantly measure the RTT between the streamer and the receiving node.", "Network latencies calculated from the RTT can then be used to synchronise data streams from different streamers.", "midas provides methods for determining the RTT between nodes and the dispatcher.", "The client layer interfaces with midas using an HTTP API, making it easy and flexible to request data from the midas network.", "Both requests and responses are in the JSON format.", "The performance of stream processing systems, such as midas, is in general difficult to evaluate since performance is affected by factors such as network topology, configuration of individual components and the type and amount of data being processed.", "These factors all contribute to the overall latency of the system.", "The latency of each request can roughly be attributed to the time for routing the request and reply between the client and the target node, and to the processing time of the node.", "To evaluate the performance of midas under normal operation conditions, we investigated the extreme case where a single node receives requests from multiple clients: 1, 5, 25, 125, 250 and 500.", "We limit our investigation to the network latency of a “dummy” node with ten responders and no processing capabilities.", "The performance test was performed using (i) one dispatcher and (ii) two dispatchers.", "The node and the dispatchers use ten responders and threads for incoming queries, respectively.", "Each client contacts the node in 100 ms intervals over a time period of 200 s.", "The distribution of round-trip-times (RTTs) was calculated from the last 100 requests.", "The network used for testing consisted of four computers (one for the node, two for the dispatchers and one for the clients) connected using wired gigabit Ethernet.", "The testing setup allowed determination of the network latency which can be considered additive to the actual computational load of the nodes.", "The results are displayed in Fig. 2.", "The median RTT using a single dispatcher is around 3 ms when the number of clients is 250.", "At 500 clients with one dispatcher, the median RTT and standard deviation increases.", "Adding a second dispatcher balances the load and the median RTTs remain stable.", "midas has been used in several projects for integrating online signal analysis into different applications.", "midas has been used to process EEG signals1 (refer to [8] for a demonstration of this project predating midas) and is a supported input protocol for eye data in an eye-tracking application [9].", "midas was used to develop a prototype showcasing adaptation of an information seeking system based on psychophysiological metrics.2", "midas was discussed in a review of psychophysiological methods for HCI [10, Section 3.12].", "Section 3.12 in that paper was written by the present authors and discusses solutions for online signal processing.", "A midas tutorial on online analysis of psychophysiological signals was held in conjunction with the 4th International Workshop on Symbiotic Interaction.3", "In 2015 midas was demonstrated at a booth in the Upgraded Life Festival in Helsinki.", "The Finnish Institute of Occupational Health participates in the Finnish Defense Force’s Research Programme 2017–2020.", "The plan is to monitor physiological activity online using midas on wearable computers.", "We encourage users of midas to share their processing nodes by issuing pull requests to the repository https://github.com/bwrc/midas-nodes, so users working on similar research tasks can benefit from the open source nature of this project.", "midas is a modular and generic framework for online distributed analysis of data streams, agnostic with regard to the type of data streams.", "The system is suitable for multiple domains, i.e., midas has a high generic applicability.", "This, together with the lightweight and modular nature of midas allows rapid development of distributed applications.", "midas provides a high-level abstraction allowing the user to focus on implementing analysis functions without having to consider, e.g., communication patterns and data handling.", "midas is well suited for research use, e.g., in human–computer interaction." ]	[]
Photoelectrocatalytic Reduction of CO2 to Paraffin Using p-n Heterojunctions	Nowadays, photoelectrocatalytic (PEC) reduction of CO2 represents a very promising solution for storing solar energy in value-added chemicals, but so far it has been hampered by the lack of highly efficient catalyst of photocathode. Enlightened by the Calvin cycle of plants, here we show that a series of three-dimensional C/N-doped heterojunctions of Znx:Coy@Cu are successfully fabricated and applied as photocathodes in the PEC reduction of CO2 to generate paraffin product. These materials integrate semiconductors of p-type Co3O4 and n-type ZnO on Cu foam to construct fine heterojunctions with multiple active sites, which result in excellent C-C coupling control in reduction of CO2. The best catalyst of Zn0.2:Co1@Cu yields paraffin at a rate of 325 μg·h−1 under −0.4 V versus saturated calomel electrode without H2 release. The apparent quantum efficiency of PEC cell is up to 1.95%.	[ "MULT" ]	[ "Catalysis", "Electrochemical Materials Science", "Materials Design" ]	[ "1000" ]	[ "Solar energy as a clean, cheap, and sustainable energy source remains the final hope to human beings.", "Photosynthesis in plants and algae can efficiently utilize sunlight to convert CO2 and water to various organic compounds and O2 that feed organisms to complete the natural carbon cycle in the planet (Govindjee and Krogmann, 2004; Nürnberg et al., 2018).", "The photoelectrocatalytic (PEC) reduction of CO2 can be termed as artificial photosynthesis, which mimics natural photosynthesis and efficiently converts CO2 and H2O into hydrocarbons and O2, tackling both energy and global environmental problems (Halmann, 1978; Wang et al., 2019).", "Recently, semiconductors are commonly utilized as catalysts in PEC reduction of CO2, because they combine the advantages of both photocatalysis and electrocatalysis (EC) to promote the separation of photogenerated electron-holes leading to high solar conversion efficiency (Chang et al., 2019).", "However, their product distribution is usually limited to C1 and C2 compounds (CO, HCOOH, CH3OH, and C2H5OH, etc.) (Barton et al., 2008; Cardoso et al., 2018; Schreier et al., 2015; Shan et al., 2019).", "In contrast, the production of multi-carbon chemicals is much sought after as potential sustainable fuels, which represents a promising path toward establishing a carbon-neutral cycle (Loiudice et al., 2016; Zhuang et al., 2018).", "Unfortunately, it is difficult to generate multi-carbon chemicals using direct CO2 reduction owing to the low activity of the present catalysts in C-C coupling.", "This is currently the major scientific challenge in sustainable energy research.", "Based on the knowledge of Calvin cycle (Bassham et al., 1954; Mao et al., 2004), we speculate that the photocathode of three-dimensional (3D) semiconductor heterojunction with multiple active sites is favorable for C-C coupling process in CO2 reduction.", "Recently, our groups introduced that constructing suitable semiconductor heterojunctions is a valuable strategy to improve their PEC performances (Xu et al., 2018) by enhancing the efficiency of separation of photogenerated electron-holes.", "Moreover, nitrogen-modified semiconductors show favorable performance in the absorption and activation of CO2 (Jia et al., 2017).", "In addition, metal oxides have been explored as efficient PEC catalysts.", "Among them, Co3O4 is an important p-type semiconductor with attractive photoelectric properties (Long et al., 2006; Tang et al., 2016); ZnO has been recognized as an excellent material for photocatalytic reactions (Liu et al., 2016; Yu et al., 2015), and Cu is confirmed in previous work as a unique metal that retains the significant faradaic yields of hydrocarbons and oxygenates (Lee et al., 2018; Lum and Ager, 2018; Shibata et al., 2008).", "As mentioned above, we choose Zn/Co-based zeolitic imidazolate frameworks (Znx/Coy-ZIFs) as the substrates that were in situ assembled on Cu foam and led to Znx:Coy@Cu through a calcined process.", "Herein, we report that well-designed 3D C/N-doped heterojunctions of Znx:Coy@Cu in situ integrated semiconductors of p-type Co3O4 with n-type ZnO on Cu foam are successfully used as photocathode in PEC cell for efficient solar-driven CO2 reduction.", "Notably, visible paraffin is produced through excellent C-C coupling control in our PEC cell of Znx:Coy@Cu \| KHCO3 \| BiVO4.", "We reason that the possible mechanism of generating paraffin product arises from these ideal heterojunctions that ameliorate their abilities of harvesting solar light and enhance the separation efficiency of photogenerated electron-holes; the multiple active sites of catalyst result in a cooperative effect to realize high efficiency of C-C coupling and suppress H2 release.", "This is an important scientific discovery because this is the first tangible evidence uncovering chain propagation during PEC reduction of CO2 over semiconductor photocathode.", "The fabrication procedures of Znx:Coy@Cu are illustrated in Figure 1A, and the experimental details are summarized in the Transparent Methods.", "The precursors of Znx/Coy-ZIFs are synthesized based on reported method (Banerjee et al., 2008; Wu et al., 2014); their morphology shows a regular dodecahedron structure (Figure 1B).", "In this work, the samples are prepared with different molar ratios of Zn(NO3)2·6H2O to Co(NO3)2·6H2O, and the actual ratios are determined by inductively coupled plasma-optical emission spectroscopy and listed in Table S1.", "We take the Zn0.2:Co1@Cu photocathode as the model catalyst for analyzing the morphology and structure.", "Scanning electron microscopy reveals an approximate diameter of 400-nm shrink hollow dodecahedron structure with sharp edges and rough surfaces (Figure 1C and Table S2).", "The X-ray diffraction patterns demonstrate spinel structure for ZnxCo3-xO4 (Figure 1G).", "High-resolution transmission electron microscopic (HRTEM) analysis indicates that the clear lattice fringes are related to the plane (220) of Co3O4 (2.86 Å), the plane (002) of ZnO (2.59 Å), and the plane (002) of CuO (2.52 Å) (Figure 1F).", "In addition, the rough surfaces of Zn0.2:Co1@Cu reveal abundant defects (Figure 1E), which are composed of missing link/cluster in crystal as well as residues of N for replacing O in metal oxide phases (Liu et al., 2019).", "The elemental mapping (Figure 1D) images illustrate that the Zn, Co, Cu, C, N, and O elements are uniformly distributed in the dodecahedral structure of Zn0.2:Co1@Cu, in which Cu is doped in dodecahedron due to the dissolution of a fraction of Cu foam.", "Thus multiple active sites would be composed of multicomponent metal and defects and activate CO2 molecules in reaction.", "Figures S1–S4 summarize the morphology and structures of heterojunctions Znx:Coy@Cu, which confirm that various molar ratios of Zn to Co give rise to different crystal phases: Zn0:Co1@Cu to cubic Co3O4, Zn1:Co1@Cu to hexagonal ZnO, and cubic Co3O4, Zn1:Co0@Cu to hexagonal ZnO.", "The photocurrent densities of representative four photocathodes show excellent characteristic curves of photoelectron response (Figure 2A), in which photocathode of Zn0.2:Co1@Cu gives the highest photocurrent.", "The linear sweep voltammetry curves (Figure 2B) show that the current density under PEC condition is much higher than that under argon and EC condition.", "Furthermore, the cyclic voltammetry curves demonstrate that the CO2 reduction peak of photocathode Zn0.2:Co1@Cu is approximately at −0.4 V versus saturated calomel electrode (SCE) (Figure S5).", "We investigate the performance of CO2 reduction by using PEC cells of Znx:Coy@Cu \|KHCO3\|BiVO4.", "Under potential of −0.4 V versus SCE, liquid products such as methanol (MeOH), ethanol (EtOH), and acetone can be detected in almost all PEC cells (Figure S13).", "The PEC cell of Zn0.2:Co1@Cu \|KHCO3\|BiVO4 gains the topmost yield of paraffin at a rate of 325 μg h−1 and releases O2 at a rate of 41 μmol h−1 (Figure 2C).", "The apparent quantum efficiency value of Zn0.2:Co1@Cu reaches 1.95%, which is about 9 times larger than that of the Zn0:Co1@Cu if its apparent faradaic efficiency equals 100% (Figure 2D).", "In addition, we test the activity of catalyst Zn0.2:Co1@Cu under different potentials from −0.2 to −1.3 V versus SCE (Figure S6).", "The paraffin products are generated under all potentials due to good C-C coupling control.", "Notably, this type of PEC system can tolerate voltages of up to −3.3 V (−1.0 versus SCE) to yield paraffin and release O2 at a rate of 181 μmol h−1 without H2 emission (Figure S7 and Video S1), which has possible potential application in industry.", "To further understand the behavior of this effective PEC cell, its electrochemical impedance spectroscopy is obtained under EC condition (Figure 2E).", "The second arc radius of Zn0.2:Co1@Cu is smaller than others, which suggests a faster interfacial charge transfer from the cathode to electrolyte and favors the formation of active hydrogen atoms reducing CO2 to paraffin.", "Besides, Mott-Schottky (M-S) relationships of Znx:Coy@Cu are gained to illustrate their semiconductor properties and carrier concentration (Nq) (Figures 2F and S9).", "The slopes of the M-S curves present both positive and negative values for the Znx:Coy@Cu cathode, implying the successful formation of p-n heterojunction of n-type ZnO and p-type Co3O4 on Cu foam (Cardon and Gomes, 1978).", "Furthermore, the Nq values of all cathodes can be calculated by the slopes of M-S curves, in which a lower slope of M-S curve reflects a higher Nq (Luo et al., 2013).", "Therefore, the Nq of the Zn0.2:Co1@Cu is higher than that of others (Figure S9D); this is the main reason why Zn0.2:Co1@Cu shows high photocurrent.", "Theoretically, the inner electric field will be built in the interfaces of p-n heterojunctions (Zhang et al., 2010).", "Specifically, charge carriers inside the Znx:Coy@Cu are diffused and drifted between p-type Co3O4 (2.76 eV) and n-type ZnO (3.24 eV) (Mohamed Reda et al., 2017; Tak and Yong, 2008), thus forming a depletion layer at the interface.", "In this depletion layer a built-in electric field is formed due to the formation of positive and negative charges at the n- and p-sides, respectively.", "When the p-n heterojunction of Znx:Coy@Cu is irradiated, it can enhance the concentration of carriers and tolerance for high external voltage, resulting in high efficiency of photogenerated electron-hole separation and excellent performance in CO2 reduction.", "UV-visible absorption spectra of Znx:Coy@Cu catalysts reveal good absorption near UV to visible light (300–1,000 nm) (Figure S10).", "Therefore the heterojunctions not only ameliorate the ability of harvesting solar light but also obviously enhance the separation efficiency of photogenerated electron-holes.", "To clarify the structure of paraffin product (Figure 3C), the 1H NMR, 13C NMR, and infrared spectra (Figures 3A, 3B, and S11B) are obtained.", "The typical peaks confirm a mixture of long-chain alkane compounds.", "The corresponding visible paraffin product is shown in Figure 3B; when the electrolyte was standing for several days, the transparent visible paraffin-like product floated on the surface of electrolyte (Figure S12).", "To trace the carbon source, 13CO2 labeling experiments are also carried out by using Zn0.2:Co1@Cu as photocathode.", "The distribution of molecular weight of the 13C labeling products is carefully examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).", "We can find that these paraffin products are a series of long-chain oxygen-containing hydrocarbons and their molecular weight increases from 875 to 3,434 (Figure 3D).", "As shown in Figure 3E, we speculate that these paraffin products might have the following structural formula: [13CH2OH(13CH2)55(CH2)7][(13CH2)4(CH2)]3CH2OH for 1208; [13CH2OH(13CH2)54(CH2)7][(13CH2)4(CH2)]3 [13CHOH] CH2OH for 1225, and [13CH2OH(13CH2)54(CH2)](13CH2)20 (CHOH)2 CH2OH for 1247.", "Therefore, we can see that the long chain grows step by step with a unit of C5 (74 (13CH2)4(CH2) or 75 (13CH2)5), which is like a unit of C6 glucose in the Calvin cycle of plant cell.", "Obviously, Figure 3F further illustrates that the peak with a molecular weight of 1,283 in the case of using 13CO2 as the carbon source is very strong; this means the PEC cell converts CO2 and H2O to paraffin products.", "The common experiment of CO2 reduction in PEC cell of Zn0.2:Co1@Cu\|KHCO3\|BiVO4 gives similar long-carbon-chain compounds: [CH3(CH2)51CH2OH][(CH2)20]x (1039, 1320, 1601, 1882, x = 1, 2, 3, 4) and [CH2OH(CH2)80CH2OH] for 1183 (Figure S11A).", "These phenomena could be attributed to the proper 3D dodecahedra structure of heterojunction with multiple active sites favoring C-C coupling.", "The C54H110 (801) long-chain hydrocarbon is a basic unit that could be generated by the control of dodecahedral cage and multiple active sites for C-C coupling.", "For a deeper understanding of the relationship between the activity and the structural feature of catalysts, the fine valence states of Co, Zn, and Cu are revealed by X-ray absorption edge structure analyses.", "The Co K-edge X-ray absorption near-edge structure (XANES) spectra of Znx:Coy@Cu exhibit similar features, indicating that Co3O4 phase is formed in each sample (Figure 4A).", "In detail, the illustration II in Figure 4A shows that the average valence state of Co elements in Zn0.2:Co1@Cu is higher than that in others.", "The octahedral coordination has a lower intensity at peak I than the tetrahedron one, as well as a higher intensity at peak III in Figure 4A.", "These imply that some Co2+ cations in the lattice of crystal Co3O4 are replaced by Zn2+ cations (Rong et al., 2015) (Figure S14).", "The Zn K-edge XANES spectrum of Zn0.2:Co1@Cu appears as an absorption edge energy at 9,668.9 eV that is typical characteristics of Zn2+.", "The two small shoulders at 9,664.8 and 9,674.4 eV indicate that some tetrahedra of Zn-N are successfully retained in this catalyst compared with the curve of ZIF-8.", "To further investigate the structure, Fourier transforms of Co, Zn, and Cu extended X-ray absorption fine structure (EXAFS) spectra of the Zn0.2:Co1@Cu catalyst are showed in Figure 4B.", "It is noted that the second peak of Zn K-edge has a slight shift to 3 Å and a new peak appears around 5 Å compared with ZnO, which suggests a structural distort compared with other catalysts.", "The Cu K-edge gives rise to a very intense peak at about 2.5 Å, which is similar to the observed peak in the Co K-edge; this means that amounts of Co2+ are substituted by Cu2+ in the Co3O4 structure.", "As strong evidences, the wavelet transform plots (Figure 4C) show the maximum intensity related to Zn-Zn bonding at ∼7 Å−1 in Zn foil and ZnO.", "The signal of Zn-Zn in Zn0.2:Co1@Cu shifts to ∼6 Å−1, which explains the formation of Zn-Co bond.", "Besides, the new weak signal at ∼8 Å−1 is related to the peak of 5 Å in the Zn EXAFS spectrum; it could be attributed to the nanostructure commutative replacement in the lattices of ZnO and Co3O4 phases, which is well consistent with the HRTEM analysis.", "Hence, it is concluded that some Co2+ cations in the tetrahedral units of spinal Co3O4 have been successfully replaced by Zn2+ and Cu2+ in Zn0.2:Co1@Cu heterojunction.", "A dodecahedron structure related to the above results and elemental mapping images are illustrated in Figure 4D.", "In the (100) plane of Zn0.2:Co1@Cu, five CO2 molecules could be captured by Co atoms and connected each other in the CO2 reduction process as well as in plant cell, where the Zn2+ and Cu2+ dispersed well in samples resulting in a cooperative effect.", "According to the experimental results and characterizations, a possible mechanism for the PEC reduction of CO2 to paraffin product is proposed and illustrated in Figure 5.", "Protons could move to the Znx:Coy@Cu photocathode under a low-bias potential and converted to active hydrogen atoms by high-energy photoelectrons in semiconductors.", "On the other hand, hydroxyl groups (OH−) transfer to the photoanode of BiVO4 releasing O2 (Seabold and Choi, 2012).", "When light irradiation is applied to this PEC cell, photogenerated electrons (e−) and holes (h+) can be generated in Znx:Coy@Cu and quickly separated by the built-in electric field resulting in higher mobilities of charge carriers.", "Simultaneously, the electrons could be transferred from the conduction band (CB) of Co3O4 to the CB of the n-type ZnO and the holes are captured by electrons from the circuit or OH−.", "Hence, the high concentration of photoelectrons was captured by protons in the surfaces to form abundant active hydrogen atoms that could reduce multiple CO2 molecules into paraffin as the Calvin cycle in plant.", "In this process, the H2 release is suppressed due to the rapid rate of CO2 reduction at multiple active sites of metals and nitrogen.", "In summary, for the first time we demonstrate a strategy to construct C/N-doped Znx:Coy@Cu heterojunctions as photocathodes, which mimic the Calvin cycle of plants and achieve multiple C-C couplings in our PEC cell to generate the paraffin product.", "At the optimal photocathode of Zn0.2:Co1@Cu, some Co2+ cations in the tetrahedral units of spinal Co3O4 have been successfully replaced by Zn2+ and Cu2+ and form favorable heterojunctions, in which CO2 molecules are adsorbed and activated by multiple active sites of metals and nitrogen and reduced to paraffin product by active hydrogen atoms.", "Although there are still many challenges in applying our system to industrial paraffin product, an understanding of the mechanism could provide different perspectives for CO2 reduction.", "All methods can be found in the accompanying Transparent Methods supplemental file." ]	[ "Heterojunctions of Znx:Coy@Cu are applied in photoelectrocatalytic reduction of CO2", "Multiple active sites result in excellent C-C coupling like natural photosynthesis", "Photoelectrocatalytic system can tolerate the higher voltage without H2 emission", "Paraffin product is reported for the first time in CO2 reduction" ]
Extruded meat analogues based on yellow, heterotrophically cultivated Auxenochlorella protothecoides microalgae	Alternatives to animal proteins with similar texture, appearance and taste are demanded by an increasing group of consumers. Plant-based meat analogues produced by high moisture extrusion cooking can provide relevant solutions. Here, microalgae combined to soy concentrates were shown to create fibrillary textured extrudates. The incorporation of spray-dried microalgae biomass in up to 50% affected the formation of fibers, which could be balanced by reducing moisture levels. The elevated fat content of microalgae biomass led to lubrication effects, while probably undisrupted microalgae cells acted as passive fillers and limited the access of intracellular proteins. Both effects may have reduced texturing but increased tenderness in comparison to pure soy based extrudates. By using heterotrophically cultivated Auxenochlorella protothecoides with a light-yellow coloration, a consumer-adverse visual appearance could be omitted. Microalgae integration improved the extrudate's nutritional profile by incorporating vitamins B and E, where over 95% was retained in the final product. Industrial relevance: The meat analogue industry strives to be more than an alternative for vegetarian and vegan customers. Large initial public offerings of relevant players underline the current economic and industrial interest. Besides animal wellbeing, meat analogues are praised mainly for their reduced ecological and environmental impact. Yet, most of the products on the market are based on environmentally questionable resources, such as soy. Hence, this study with focus on microalgae as a protein alternative to the ubiquitously applied soy protein concentrate is relevant to the evolving industry and research. In this manuscript, a fibrillary textured plant-based product using microalgae is shown without an adverse color.	[ "AGRI", "CHEM", "ENGI" ]	[ "Alternative proteins", "Auxenochlorella protothecoides", "Heterotrophic microalgae", "High moisture extrusion cooking", "Nutritional profile", "Plant-based meat analogues" ]	[ "1106", "1600", "2209" ]	[ "In the upcoming years, population growth and the subsequent increase in food demand will affect food markets worldwide (Godfray et al., 2010).", "Meanwhile, there is a steadily growing number of consumers in favor of reducing the consumption of animal-derived products due to concerns for the animals' well-being, dietary restrictions as vegan, vegetarian or flexitarian, health or environmental issues (Chaudhary, Gustafson, & Mathys, 2018; Chen, Chaudhary, & Mathys, 2019).", "Meat analogues are a class of food products that contain no animal-derived protein but attempt to incorporate meat-like characteristics (Smetana, Mathys, Knoch, & Heinz, 2015; Smetana, Schmitt, & Mathys, 2019).", "Different techniques to structure plant based proteins are available with extrusion most often applied (Dekkers, Boom, & van der Goot, 2018).", "Extrusion comprises a continuous, thermomechanical processing unit mainly to transform and structure raw materials.", "Depending on the moisture content it is differentiated between low moisture extrusion with moisture content below 35% and high moisture extrusion cooking (HMEC) with moisture content above 50%.", "Especially HMEC offers the possibility to create fibrous structured meat analogues that are ready to eat while mimicking appearance, texture, and flavor of meat (Bouvier & Campanella, 2014; Cheftel, Kitagawa, & Quéguiner, 1992; Egbert & Borders, 2006; Mezzenga, Schurtenberger, Burbidge, & Michel, 2005).", "It has been argued that extrusion is limited to form fibrous structures on microscale in comparison to animal derived meat fibers i.e. in chicken muscle with a fiber diameter of about 50 μm (Dekkers et al., 2018; Smith & Fletcher, 1988).", "HMEC is performed in twin screw extruders comprising two intermeshing screws that are encased by a row of barrel segments.", "Powders of protein-rich raw material are fed into the first barrel while water is injected into the following barrel.", "The material is hydrated, conveyed, and intermeshed by typically co-rotating screws.", "Therefore, raw materials are exposed to mechanical and thermal energy, whereby the proteins undergo endothermic physicochemical transformations.", "The mechanical energy input is largely dependent on the screw design and on the raw material's properties.", "Thermal energy is introduced by barrel heating and/or injection of live steam.", "Cooking temperatures around 130 °C to 180 °C and holding times up to 150 s are required to melt the proteins (Cheftel et al., 1992).", "To produce meat analogues using HMEC, a long cooling die channel is necessary as it allows the protein melt to slowly cool down without expanding.", "By cooling down, the protein molecules and small aggregates align and solidify in a three dimensional cross-linked network with a layered and fibrillary texture (Akdogan, 1996, 1999; Cheftel et al., 1992; Wild, 2017).", "A multitude of plant ingredients is used to produce meat analogues with soy protein being the most frequently used ingredient due to its functional properties rather than nutritional values (Malav, Talukder, Gokulakrishnan, & Chand, 2015; P. Singh, Kumar, Sabapathy, & Bawa, 2008).", "Soy protein concentrates, produced from soybean flour or defatted flakes, are rich in proteins (~70%) but with few flavor components as a result of refining processes.", "However, soy-derived products have been used, alone or mixed with other materials, to improve the characteristics of final HMEC products (Cheftel et al., 1992; Fang, Zhang, & Wei, 2014; Lin, Huff, & Hsieh, 2000, 2002; Liu & Hsieh, 2008; Wu et al., 2018; Yao, Liu, & Hsieh, 2004).", "Recently, microalgae have been used as raw material for HMEC together with soy protein concentrates (Grahl et al., 2018).", "Microalgae contain valuable nutritional compounds that provide health benefits (Caporgno & Mathys, 2018) and, when cultivated heterotrophically, they were calculated to yield more environmentally sustainable meat analogues than other protein sources (Smetana, Sandmann, Rohn, Pleissner, & Heinz, 2017).", "Microalgae can accumulate >50% of protein in their dry matter (Becker, 2007), they grow faster than terrestrial plants, and reach higher protein productivities per area than other crops, such as soybean, pulse legumes or wheat (Bleakley & Hayes, 2017).", "Additionally, microalgae can be incorporated in different food categories given their functional properties, such as gelation, water and fat absorption capacity, emulsification capacity, and foaming capacity (Buchmann et al., 2019; Caporgno & Mathys, 2018; Chronakis & Madsen, 2011).", "In spite of the nutritional and environmental benefits, the reported high moisture extrusion of microalgae and soy protein concentrate mixtures revealed some drawbacks (Grahl et al., 2018).", "Incorporating microalgae beyond 50% dry weight strongly affected the distinctive structure of pure soy-based extrudates, which are characterized by fibrillar, elastic, firm, and layered properties.", "Moreover, the pigments of photoautotrophic cultivated microalga Arthrospira platensis (Spirulina) resulted in a dark green colored product, which is one of the strongest barriers for using microalgae as a food ingredient (Becker, 2007).", "Yet, under controlled cultivation conditions, some microalgae species can synthesize and accumulate different pigments, such as carotenoids, resulting in a light yellow biomass (Caporgno, Haberkorn, Böcker, & Mathys, 2019; Hu, Nagarajan, Zhang, Chang, & Lee, 2018).", "In this study, we aimed to evaluate the possibility of producing meat analogues from different formulations of soy protein concentrate and a commercial heterotrophic microalga that produces yellow pigments.", "We focused on the characteristics of the final products, such as texture, appearance, and nutritional benefits.", "Raw material consisted of two commercially available products: a microalgae powder and a soy protein concentrate.", "The microalgae powder (MA) is commercialized in Switzerland as Golden Chlorella (Alver AG, Chardonne, Switzerland; lot code: C2B07-C318234).", "The MA is composed of dried Auxenochlorella protothecoides - strain KT158710.1, which was genetically verified.", "Total DNA was isolated from 114 g of the algae powder using the QIAamp Fast DNA Stool Mini Kit following suppliers instructions.", "The total DNA was quantified using PicoGreen® and normalized to 1 ng/μl before PCR.", "The PCR product was purified and Sanger sequenced at Microsynth AG (Balgach, Switzerland) using the PCR primers (Hadi et al., 2016) as well as two internal primers (Internal forward primer: 5′-TGCAACGTGTGACTTGTTTGA-3′; Internal reverse primer: 5′-GGAGAAGAGGGAAGGTCGAC-3′) (White, Bruns, Lee, & Taylor, 1990).", "Sanger sequencing results were assembled and manually edited and the consensus sequence was blasted against the NCBI nc database for species identification.", "The MA was composed of 63% proteins, 19% carbohydrates, 11% of lipids, 4% ash, and 3% water.", "The soy protein concentrate (SP), commercially named Alpha® 8 IP, manufactured by DUPONT™ DANISCO® (Copenhagen, Denmark), was composed of 67.5% proteins, 19.6% carbohydrates (of which 18% was dietary fibers), 1.6% lipids, 7% ash, and 4% water.", "Preliminary kneading measurements were performed for formulations containing MA and SP in different proportions and with increasing moisture content.", "The torque of a measuring kneader was recorded to assess the effect of alterations in the formulation.", "For such, an IKA-Visc MKD 0.6 kneader (IKA Werke GmbH & Co.", "KG, Staufen, Germany, product number 1.319527) was equipped with two intermeshing duplex kneading blades.", "The kneading blades, 9.5 cm long and 5.7 cm in diameter, were set to rotate at 30 rpm.", "The cooling jacket was tempered at 20 °C.", "Measurements began by mixing the dry powder for 1 min before adding tap water during 10 s to a final wet weight of 200 g. Torque was recorded over 16 min.", "All measurements were carried out in triplicates.", "Formulations containing pre-disrupted microalgae were prepared in order to assess the effect of microalgae disruption on torque measurements.", "Microalgae disruption was performed in a M-110EH Pilot Scale Microfluidizer (Microfluidics Corp., Westwood MA, USA; product number 2005013).", "A suspension containing 10% MADW was processed with this high-pressure homogenizer at 1′000 bar and was passed through the system three times.", "Light microscopy was used to confirm cell disruption (Leica DM6, Leica Microsystems AG, Heerbrugg, Switzerland).", "Dry extrusion was performed as pre-trials to evaluate both the extrudability of MA and the behavior of microalgae cells when processed under elevated shear forces, temperature, and pressure.", "Pre-mixtures containing the desired proportion of MA and SP, with a moisture content between 32% and 35%, were fed to a 20DN co-rotating intermeshing twin-screw extruder (Brabender GmbH & Co.", "KG, Duisburg, Germany, product number 177501).", "The screws had a diameter of 20 mm, an L/D ratio of 25:1, and a screw speed that was set between 60 and 190 rpm.", "The barrel possessed four temperature-controlled segments, which were set to 80 °C, 110 °C, 130 °C, and 100 °C, respectively.", "The dosage was varied between 0.3 and 0.5 kg/h when optimizing processing conditions to reach stable product properties.", "A tap water cooled stainless steel pipe, with a diameter of 0.8 cm and a length of 18.3 cm, was attached to the twin-screw extruder to act as a cooling die in order to reduce product expansion and to induce possible fiber formation.", "Pressure, product temperature, screw speed, torque, product throughput, and residence time were monitored.", "A BuEx2-32/44 co-rotating intermeshing twin-screw extruder composed of eleven segmented barrels (Buhler AG, Uzwil, Switzerland; product number 78745/1) was used for the trials.", "The screws had a diameter of 30.5 mm, an L/D ratio of 44:1, and a screw speed that was constantly kept at 375 rpm.", "In the first three barrels, forward conveying and mixing elements were installed.", "In barrels four to seven, screw segments consisted of kneading, shearing, backward, and forward conveying elements.", "In the last barrel, only forward conveying screws were applied.", "The temperature profile throughout the eleven barrels was set as follows: 20 °C, 20 °C, 20 °C, 70 °C, 90 °C, 90 °C, 130 °C, 140 °C, 140 °C, 140 °C, and 140 °C.", "A stainless steel cooling die, with internal dimensions of 128 × 6 × 1 cm3 (L × W × H), was attached to the end of the extruder.", "Cooling was conducted using a circulating water cooler (Variocool VC 1200, Lauda Dr. R. Wobser GmbH & Co.", "KG, Lauda-Königshofen, Germany) at 56 ± 2 °C to reach a product temperature below 100 °C at the die outlet.", "The cooling die consisted of six elements each equipped with temperature and pressure sensors.", "Different from the dry extruder, the HMEC extruder had two separate dry feeders in the first barrel, for MA and SP, and a piston dosing water pump that feeds tap water at 70 °C into the second barrel.", "The product throughput was held constant around 430 g/min.", "The extrudates were vacuum packed right after production and were stored at 7 °C until texture characterization.", "Different parameters were monitored during the extrusion process: product temperature, pressure, torque, product throughput, and residence time.", "Temperature dependence of cp was based on the raw material components and the numerical relations based on Singh and Heldman (2014).", "The heating and cooling portions of the extrusion process were considered for the STE calculation.", "Additionally, the maximum temperature holding section was considered to operate under adiabatic conditions.", "Texture evaluation was done using cutting strength and double compression analyses in a Z010 Zwick Universal testing machine (ZwickRoell GmbH & Co.", "KG, Ulm, Germany) with the software testXpert V10.11 (ZwickRoell).", "Commercially available reference products to the meat analogues were also evaluated, such as chicken breasts (Migros Optigal, Altigon, Switzerland; Ross broilers; bird age at slaughter 35 days) and tofu (Bio Tofu, Frostag Food-Centrum AG, Landquart, Switzerland).", "Both products were vacuum packed and boiled in water for 15 min.", "Prior to the analysis, all samples were cut into equally sized portions to prevent that the products' dimensions could affect the result.", "All samples were also allowed to reach room temperature prior to testing.", "Cutting strength was measured with a Warner Bratzler shear blade.", "Pieces of the product with dimensions of 6 × 2.8 × 1 cm3 (L × W × H) were cut with a cutting speed of 50 mm/min over a total distance of 40 mm.", "Cuts were performed transversally (FT) and longitudinally (FL) to the flow direction in the extruder.", "Thus, longitudinal (FL) cuts were performed in the same direction of the fibers in the extrudates.", "The peak forces for FT and FL were measured five times in all extrudates and reference products.", "The double compression analysis, also known as the texture profile analysis (TPA), was performed using one cubic centimeter pieces of each sample.", "TPA was evaluated compressing the samples transversally (FT) and longitudinally (FL) to the flow direction in the extruder with a compression speed set to 60 mm/min and a decompression speed of 180 mm/min.", "The flat compression probe had a diameter of 23 mm.", "Both compressions were performed until reaching 50% of original sample height.", "Analyses of all samples were repeated five times.", "To quantify the vitamins and carotenoids present in the samples, a high moisture extrudate with 50% MADW, as well as the microalgae and soy protein concentrates, were analyzed by Eurofins Scientific AG (Schönenwerd, Switzerland) according to the following protocols: thiamine (vitamin B1) was quantified according to BS EN 14122-2014; riboflavin (vitamin B2) according to EN 14152:2006 (with modifications); niacin (vitamin B3) according to EN 15652:2009; pyridoxine (vitamin B6) according to EN 14164:2014; alpha-tocopherol (vitamin E) according to EN 12822:2014; lutein and zeaxanthin according to Roche Index n° 2264; and beta-carotene according to EN 12823-2:2000.", "HMEC-samples were prepared for scanning electron microscopy by drying the extrudates at 37 °C and then fracturing them with a blade.", "Pieces with a fresh surface were glued to aluminium stubs with Leit-C conductive carbon cement (Plano GmbH, Wetzlar, Germany) and coated with 4 nm of Pt/Pd.", "Dry extrusion samples could be fractured directly with a blade without prior drying.", "For a comparison with the microalgae biomass, images from the initial MA powder were also taken.", "For such, the powder was fixed on stubs containing double adhesive carbon-pads and then cut with a sharp blade to observe cross fractions of the cells and agglomerates.", "In addition, samples of the extrudates containing microalgae were observed with a light microscope (Leica DM6, Leica Microsystems AG, Germany) in order to observe the presence of intact cells.", "Such samples were prepared by soaking a piece of the extrudate in deionized water and breaking the fibrillar structure, which allows an aliquot of the aqueous phase to be observed under the microscope.", "Shear and heat transfer during extrusion determine the aligned meat-like fibrous structure in meat analogues.", "The energy input from shear and heat transfer depends on the extruder's setup, its processing parameters, and the properties unique to each raw material being used.", "The specific thermal and mechanical energy (STE and SME) inputs are used to characterize the overall energy input per extruded product.", "Moreover, the SME is directly proportional to the motor torque (Akdogan, 1996) and essential to optimize and scale up the extrusion process (Bouvier & Campanella, 2014).", "As an indication on how the SME may respond to the incorporation of MA in an extrusion process, the torque of a measuring kneader was recorded for different proportions of MA and SP at 55 and 70% moisture contents (Fig. 1).", "At 55% and 70% moisture content, increasing the MA fraction resulted in a decrease of the torque (Fig. 1).", "The least texturized formulation contained 50% microalgae and 70% moisture and could be described as a liquid, sticky paste.", "These results suggest that the formulations containing high amounts of microalgae would need lower moisture contents to balance the loss in cohesion and structure.", "Additionally, two formulations were tested with pre-disrupted MA.", "In downstream processing of microalgae, it is well known that cell disruption leads to a release of intracellular contents, including proteins and polysaccharides, which increases the suspension's viscosity (Lee, Lewis, & Ashman, 2012).", "Thus, the formulations with disrupted MA required a higher torque than that by formulations with non-disrupted MA at similar moisture contents (Fig. 1).", "Even though these trials were carried out at 20 °C, whereas in dry and high moisture extrusion processes temperatures reach 140 °C, the results obtained indicate how the extrusion process must be adapted with increases in MA content.", "Dry extrusion trials were carried out on a lab-scale extruder with soy protein concentrate (SP) and increasing amounts of microalgae per dry weight (MADW): 0% MADW, 14% MADW, 29% MADW, and 44% MADW.", "Operating parameters, such as feeding rate, screw speed, and temperature profile, were modified during the trial to obtain a clearly visible fibrous texture.", "Moisture content was set to 35% but had to be reduced to 33% for the highest MA content (44%) in order to obtain some texture.", "Unfortunately, a constant process could not be established.", "Fluctuations in pressure (10 to 43 bar) and torque (44 to 77 Nm) occurred without changes in the formulation.", "The heterogeneity of the formulations, especially in terms of moisture, resulted in bubble formation and expansion, which could explain the observed fluctuations in pressure and torque during the experiments.", "Fluctuations in torque are a sensitive indicator of an erratic process during extrusions (Bouvier & Campanella, 2014) and resulted in unstable products.", "Consequently, SME values could not be calculated, which is why results are limited to macro- and microscopic characteristics of the products.", "Several extrudates were produced with different MA contents (Fig. 2).", "Extrudates produced purely from SP showed a clear fibrous texture (Fig. 2a), while increasing amounts of MA led to the loss of such fibrous texture.", "With the formulation that contained 44% MA, the moisture content was decreased from 35% to 33% to induce fibrillary structures.", "However, additional changes in operating parameters, such as feed rate, screw speed, and barrel temperature, did not improve the characteristics of the final product, which had the appearance of a warm, doughy paste without cohesion (Fig. 2d).", "To better visualize textural changes and to investigate if microalgae cells remained intact after the dry extrusion process, samples were observed by scanning electron microscopy (SEM).", "The initial spray-dried MA powder presented large, even spheres with a slightly uneven surface (Fig. 2e).", "Cell agglomerates showed diameters between 40 and 70 μm, whereas fresh Chlorella microalgae samples showed diameters ranging between 3 and 6 μm.", "Fibrillar structure (arrows) and spherical particles (dashed line) were observed in the 29% MADW extrudate (Fig. 2h).", "The spherical particles (Fig. 2f-h) were much smaller than the spheres from the dried microalgae (Fig. 2e).", "These results indicate that the operational conditions during the dry extrusion process were harsh enough to disintegrate microalgae cell agglomerates, yet the shear forces did not seem to disrupt MA cells.", "Unbroken A. protothecoides cells from the dry extrusion highlights its mechanical resistance, which could hamper the extrusion process as such resistance prevents the release of intracellular components.", "Yet, formation of a fibrous texture from a protein-based raw material involves swelling, dissolving, and unfolding of protein (Wild, 2017).", "Thus, protein must be available in the aqueous phase and should not be enclosed by a robust cell structure in order to enable fiber formation.", "Mimicking meat-like texture and flavor is a distinctive goal of meat analogues (Egbert & Borders, 2006).", "Thus, HMEC was used in an attempt to produce a final extrudate with a striated, layered structure.", "We obtained extrudates with a clearly visible fibrous texture, even in formulations containing MA (Fig. 3).", "However, the fibrillary structure of the extrudates decreased with increasing amounts of MA.", "Therefore, the moisture level was reduced with increasing amounts of MA to maintain the fibrous texture.", "Increasing MA content affected not only the fibrillary structure, but also the product's stickiness.", "Elevated stickiness negatively affects the subsequent processes, such as cutting and packaging, and also required reduced moisture levels with increasing MA contents.", "Extrudates obtained with 50% MADW at 55% moisture showed very low internal cohesion due to diminished protein cross-linking (Fig. 3c).", "These extrudates almost crumbled, but could be slightly improved by decreasing the moisture content to 50%.", "Similarly, Grahl et al. (2018) reported a lack of fibrillary structure and firmness of extrudates when increasing the fraction of another microalgae, Arthrospira platensis (Spirulina).", "The authors also observed changes in the color of the final product when A. platensis was used: they obtained dark-green to black extrudates, which was attributed to the dark color of the microalgae used.", "In contrast, the heterotrophically cultivated A. protothecoides introduced a yellowish color to the extrudates (Fig. 3d), which is more likely to be accepted by consumers.", "Apart from the visual (qualitative) evaluation, the extrudates were also quantitatively assessed to determine how the different proportions of MA affected texture in comparison to boiled chicken breast and tofu.", "Cutting strength, which yields information about the tenderness of the product, was evaluated with the Warner-Bratzler method, while the texture profile was evaluated with a double compression test.", "For the compression test, sample hardness, represented by the maximum compression force, was extracted.", "Both tests were performed across (FT) and along (FL) the flow direction in the extruder, which is also the fibers' orientation.", "For all extrudates, the cutting and compressing force decreased with increasing MA contents or with increasing moisture (Fig. 4).", "These results indicate that the extrudates became more tender and softer when MA was used in the formulation.", "They underline the visual evaluation which identified a crumbling tendency with increasing MA.", "Crumbling is related to brittle failure whereas tenderness assumes yielding and plastic deformation, both requiring less fibrous structured materials with reduced elastic behavior.", "Cutting strength decreased with increasing MA contents in both cutting directions (Fig. 4 top).", "The highest cutting force was observed for the soy extrudate with 60% moisture content across the direction of the fibers (FT = 49.2 ± 2.9 N), similar to the values obtained for chicken breasts (FT = 55.2 ± 25.1 N).", "Chicken breast values varied greatly due to the dissimilarity in the samples, such as the presence of tendons and ligaments (Smith & Fletcher, 1988).", "It has to be noted that tenderness values are furthermore strongly dependent on the preparation method, which was shown for chicken breast meat but is also applicable to mechanical testing of meat analogues (Lyon & Lyon, 1990).", "In all extrudates, differences in cutting strength between cutting across and along the fibers' direction varied with MA and moisture contents.", "A greater cutting strength was needed with lower MA and moisture contents, which was in agreement with the visually perceived amount of fiber formation.", "The more pronounced structure of an extrudate corresponded to a higher fiber formation, which correlated with a greater difference between cutting strength across and along fiber directions.", "This was exemplified by the comparative chicken and tofu samples.", "Tofu possessed a uniform structure in all directions, and no difference was found between cutting across and along the fibers' direction.", "Contrarily, in chicken breasts, the cutting strength differed between both directions due to the anisotropic mechanical properties of fibers.", "In the double compression assay (Fig. 4 bottom), similar trends were observed.", "The test recorded the force profile during a double compression with the peak force indicating the sample's hardness.", "Peak force decreased with increasing MA content but increased with decreasing moisture content, similar to the results obtained from the cutting strength test.", "In the double compression test, tofu also showed the lowest resistance with 5.7 ± 1.0 N and 5.6 ± 1.6 N across and along the flow direction, respectively.", "The highest values were observed for the pure soy extrudates with 60% moisture content (FT = 102.0 ± 12.7 N).", "However, in contrast to the cutting strength results, chicken was much softer (FT = 22.7 ± 3.2 N).", "The combination with MA reduced hardness, yielding extrudates that showed comparable hardness values as the chicken breast, i.e. extrudates with 50% MADW and 55% moisture content had a hardness across the fiber direction of FT = 20.4 ± 3.9 N which is similar to the chicken breast with FT = 22.7 ± 3.2 N.", "Such results suggest that extrudates containing high MA contents showed greater resemblance to the texture of chicken (in terms of hardness) than that by extrudates without MA.", "The incorporation of MA reduced the formation of a fibrous structure, which was achieved with pure SP extrudates, leading to a reduction in cutting strength and in its resemblance to chicken breasts.", "On the other hand, MA introduced softness to the structure, which pure SP extrudates lacked.", "Contrary to the dry extrusion experiments, the stability of the operational parameters during high moisture extrusion allowed to determine SME values, which is an important parameter to optimize and enhance the process.", "SME ranged from 60 to 160 kJ/kg depending only on the proportions of the raw materials used as the extruder setup and the operational parameters, such as screw speed and barrel temperatures, were kept constant (Fig. 5).", "Similar to what was observed in the torque measuring kneader (Fig. 1), increasing the moisture content while maintaining the MA content constant decreased the SME values (Fig. 5).", "Likewise, the SME decreased with increasing MA content at a constant moisture level (Fig. 5).", "The results are similar to those reported for a typical high moisture extrusion process, in which levels of SME in the range of 200 kJ/kg or lower are needed to generate a fibrous texture from a protein-based raw material (Bouvier & Campanella, 2014).", "The overall energy input is required to create a melt, which can be aligned in flow and cross-linked upon cooling to obtain a wet textured protein structure.", "In this study, specific thermal energy (STE) inputs between 500 and 560 kJ/kg were applied to heat up the ingredients to 140 °C and cool them down below 100 °C in the cooling die.", "This range was calculated assuming that the mechanical energy input did not contribute to the increase in temperature.", "Consequently, the reported STE values indicate maximum values.", "Moreover, the holding section at the maximum temperature was considered to be adiabatic.", "In non-adiabatic conditions, the thermal energy input during the holding section must be included for the STE calculation.", "Likewise, one must consider that the present study was done on a pilot scale extruder, which requires extensive heating to reach processing temperatures around 140 °C.", "On an industrial scale, relatively more heat dissipation is created by the mechanical energy input, which demands a lower input of external heat transfer, while the ratio between SME and STE is expected to differ (Bouvier & Campanella, 2014).", "As both SP and MA have a similar composition in terms of protein and carbohydrates, extrudates with a comparable structure could have been expected.", "Yet, the incorporation of MA caused changes in the texture formation, which have been shown visually, in cutting and compression tests, and in alterations of the processing conditions (i.e. SME values).", "We suggest two explanations for such results.", "The first one refers to the differences in the composition of SP and MA.", "Although protein and carbohydrate contents were much alike (67.5% and 63% protein and 19.6% and 19% carbohydrates for SP and MA, respectively), the fat content differed largely between SP (1.6%) and MA (11%).", "The greater fat content probably resulted in lubricating effects that decreased shear during extrusion and, thereby, prevented the formation of a firm cross-linked texture (Akdogan, 1999).", "Lubrication leads to less friction between particles and between material and screw surfaces, which results in less overall friction.", "Thus, a lower SME would be required to extrude the product, which was an observed effect of incorporating MA to the raw material.", "If no additional STE is provided by injecting steam or if an altered screw configuration is used to increase SME, the overall energy input will be lowered by lubrication, which could have affected fiber formation.", "The second explanation is based on the assumption that MA cells were not disrupted by the HMEC process.", "Images from scanning electron microscopy were obtained from different extrudates and from the MA powder initially used to verify this assumption (Fig. 6).", "The images show evident changes in the microstructure from formulations that only contain SP (Fig. 6a) and those containing SP and MA, where less pronounced fibrillary structure in SP and MA formulations was clearly visible (Fig. 6b).", "Pictures of the two raw materials stress differences in their initial state (Fig. 6c–d).", "The intial MA powder was fractured with a blade for SEM imaging and hollow cavities could be identified in the spray-dried agglomerates (Fig. 6d–e), which probably originated from vaporization during spray-drying (L. P. Lin, 1985).", "Additionally, small round-shaped cavities were observed in the extrudate containing MA (6b) and in the cross-section of the spray-dried MA agglomerates (6e).", "This suggests to be remainings of intact microalgae cells.", "Therefore, an extrudate combining MA and SP was soaked in water and the aqueous phase was investigated in light microscopy, in which we were able to confirm the presence of intact microalgae cells in the extrudate with 50% MADW (Fig. 6f–g).", "Intact MA cells could have had implications in two ways.", "First, they could be considered as insoluble material acting as fillers in the molten material.", "When they exceed a critical volume fraction, they could negatively affect the flow properties in the extruder and the final structure of the extrudates (Bouvier & Campanella, 2014).", "This critical volume fraction is not easy to quantify in the system used in this study, but there was a decrease in fibrous structure from formulations containing over 30% MA.", "Secondly, intact MA cells would have limited the availability of functional intracellular proteins preventing them to form fibers.", "Reduced accessibility of internal compounds from dried microalgae cells has been previously shown (Mussgnug, Klassen, Schlüter, & Kruse, 2010; Safi et al., 2013).", "The incorporation of dried heterotrophic MA biomass has led to a reduced fiber formation but also to a higher tenderness than that of pure SP extrudates.", "Changes in structure formation were probably due to a greater fat content and to the presence of intact microalgae cells that acted as passive filling material and prevented the access of intracellular water soluble proteins.", "The extrudates with combined soy and microalgae could have their fibrous texture improved by reducing moisture content levels.", "However, as previously discussed, changes in the process, such as screw profile, speed, and temperature profile, might be required in order to obtain a more fibrillary structure for extrudates with high MA contents.", "Based on our results, we suggest MA cells be treated prior to the extrusion process in order to disrupt the cells.", "Moreover, processing fresh instead of dried biomass could result in higher disruption rates during extrusion and in a reduced energy consumption, as drying and pre-disrupting microalgae biomass could be omitted.", "Microalgae are known for its high nutritional quality, especially in terms of its protein and omega-3 fatty acid content, but also in its vitamin levels (Caporgno & Mathys, 2018).", "From a macronutrient point of view, minor changes were induced by the microalgae incorporation.", "The extrudate's fat content increased from 1.6% for the soy based product to 6.3% with 50% microalgae incorporation.", "The level of proteins and carbohydrates stayed almost constant with 67.5% and 19.6% versus 65.25% and 19.3%, respectively.", "Regarding vitamins, the microalgae incorporation had a larger effect.", "The heterotrophic A. protothecoides used was especially high in vitamins B and E (Fig. 7).", "In case of thiamine B1 and riboflavin B2, 100 g of the raw material powder exceeded the recommended daily intake (RDI) levels with 1.7 ± 0.3 mg > 1.1 mg RDIB1 and 5.3 ± 0.8 mg > 1.4 mg RDIB2, respectively (FDA - Food and Drug Administration, 2016).", "The soy concentrate, on the other hand, did not contain any relevant levels of vitamins B or E. Thus, the combination of MA and SP raw materials resulted in enhanced vitamin levels.", "Such elevated levels could even be retained in the high-pressure, high-temperature and time conditions of the extruder (Fig. 7).", "Other authors have shown comparable retention levels for riboflavin and thiamine in extrusion cooking (Beetner, Tsao, Frey, & Harper, 1974).", "Moreover, A. protothecoides is also known for being rich in lutein and zeaxanthin, which are carotenoids associated to health benefits (Hu et al., 2018).", "No recommended daily intake level could yet be defined for the two structurally and physiologically similar xanthophylls.", "However, protective effects could be observed against eye diseases with daily intake levels of 10 mg lutein and 1 mg zeaxanthin (Dawczynski et al., 2013).", "Thus, lutein and zeaxanthin concentrations of 0.6 ± 0.1 and 0.3 ± 0.1 mg/100 g found in the extrudate with 50% MADW are encouraging results, especially when considering that nearly no losses could be detected due to the extrusion processing.", "Consequently, the incorporation of MA led to an enhanced nutritional profile of the final product.", "HMEC is applicable to create ready to eat meat analogues that mimic meat characteristics, satisfying consumers seeking fibrous food products based on non-animal proteins.", "Incorporating heterotrophic microalgae in the production of meat analogues resulted in a final product with benefits from a nutritional point of view, while maintaining an attractive color.", "The combination of soy protein with microalgae influenced the fibrous structure formation, which is probably due to the highly resistant microalgal cell wall and microalgae's elevated fat content.", "Reducing moisture content simultaneously could counterbalance the impeded fiber formation.", "Based on fiber level evaluation and mechanical texture analysis, extrudates with promising properties could be obtained with 30% microalgae incorporation at a moisture level of 60%.", "The presented results have shown that the strong coloration of meat analogues containing microalgae can be overcome by applying heterotrophic microalgae." ]	[ "Bright yellow fibrillary meat analogues were produced using heterotrophic microalgae.", "Moisture reduction was required to develop fibers with increasing microalgae content.", "Microalgae cells remained visible after the extrusion processing.", "Enhanced nutritional value of extrudates as a result of microalgae incorporation." ]
Controlled semantic cognition relies upon dynamic and flexible interactions between the executive ‘semantic control’ and hub-and-spoke ‘semantic representation’ systems	Built upon a wealth of neuroimaging, neurostimulation, and neuropsychology data, a recent proposal set forth a framework termed controlled semantic cognition (CSC) to account for how the brain underpins the ability to flexibly use semantic knowledge (Lambon Ralph et al., 2017; Nature Reviews Neuroscience). In CSC, the ‘semantic control’ system, underpinned predominantly by the prefrontal cortex, dynamically monitors and modulates the ‘semantic representation’ system that consists of a ‘hub’ (anterior temporal lobe, ATL) and multiple ‘spokes’ (modality-specific areas). CSC predicts that unfamiliar and exacting semantic tasks should intensify communication between the ‘control’ and ‘representation’ systems, relative to familiar and less taxing tasks. In the present study, we used functional magnetic resonance imaging (fMRI) to test this hypothesis. Participants paired unrelated concepts by canonical colours (a less accustomed task – e.g., pairing ketchup with fire-extinguishers due to both being red) or paired well-related concepts by semantic relationship (a typical task – e.g., ketchup is related to mustard). We found the ‘control’ system was more engaged by atypical than typical pairing. While both tasks activated the ATL ‘hub’ colour pairing additionally involved occipitotemporal ‘spoke’ regions abutting areas of hue perception. Furthermore, we uncovered a gradient along the ventral temporal cortex, transitioning from the caudal ‘spoke’ zones preferring canonical colour processing to the rostral ‘hub’ zones preferring semantic relationship. Functional connectivity also differed between the tasks: Compared with semantic pairing, colour pairing relied more upon the inferior frontal gyrus, a key node of the control system, driving enhanced connectivity with occipitotemporal ‘spoke’. Together, our findings characterise the interaction within the neural architecture of semantic cognition – the control system dynamically heightens its connectivity with relevant components of the representation system, in response to different semantic contents and difficulty levels.	[ "NEUR", "PSYC" ]	[ "Anterior temporal lobe", "Connectivity", "Hub-and-spoke", "Inferior frontal gyrus", "Semantic cognition" ]	[ "2805", "3205", "3206" ]	[ "Based on an abundance of data from patients and healthy individuals, investigated using behavioural assessments, neuroimaging, and brain stimulation, Lambon Ralph, Jefferies, Patterson, & Rogers (2017) enunciated a detailed framework termed controlled semantic cognition (CSC).", "In CSC, representations of semantic knowledge are underpinned by a distributed system that involves both a pan-modality hub and multiple modality-specific spokes.", "In addition to semantic representations, there is a ‘semantic control’ system that manages how the hub and functionally diverse spokes split division of labour.", "Neuroimaging data have identified key regions of CSC: Performing semantic tasks activates polymodal regions generally believed to be the hub, such as the ventrolateral parts of anterior temporal lobe (ATL), as well as regions supporting executive control, such as the inferior frontal gyrus (IFG) and posterior temporoparietal regions (Binder, Desai, Graves, & Conant, 2009; Lambon Ralph et al., 2017).", "Furthermore, the CSC framework makes specific predictions that, when the usage of semantic knowledge is customary and well-practised, the semantic representation system needs little input from the ‘control’ mechanisms to output a correct response (e.g., associating ducklings with eggs); by contrast, input from the ‘control’ system would ramp up when a context accentuates atypical usage of semantic information (e.g., associating ducklings with dandelion due to both being yellow) or when precise scrutiny of semantic attributes is necessary.", "At the neural level, it remains to be tested how different components of CSC join forces dynamically for different tasks.", "The principal target of this study was, therefore, to understand the flexible division of labour and functional connectivity among the hub, spoke, and executive areas in semantic cognition.", "Investigations into the white-matter connectivity of the temporal lobe have identified the anatomical ‘infrastructures’ underpinning communication between the semantic control and representation systems.", "Using probabilistic tractography, Binney, Parker, and Lambon Ralph (2012) found that convergence of sensory information in the temporal lobe is a graded process occurring along both its coronal and sagittal axes.", "Along the coronal axis, different gyri of the temporal lobe connect laterally to each other, making the inferior temporal gyrus (ITG) the midpoint that receives information both from ventromedial and dorsolateral sources.", "Along the sagittal axis, both short- and long-range longitudinal tracts course through the temporal lobe, resulting in increasing information convergence towards the rostral temporal areas (as also found in non-human primates: Moran, Mufson, & Mesulam, 1987).", "In addition to this graded connectivity structure within the temporal lobe, white matter tracts also extend to regions outside the temporal lobe, such as prefrontal and parietal regions that are crucial for executive function (Fedorenko, Duncan, & Kanwisher, 2013).", "This includes the uncinate fasciculus that links the prefrontal cortex (particularly pars orbitalis) to the ATL, as well as the inferior fronto-occipital fasciculus that links the prefrontal cortex (particularly pars opercularis and triangularis) to ventral occipitotemporal areas (Bajada et al., 2017; Bajada, Lambon Ralph, & Cloutman, 2015).", "Such structural-anatomical findings align with the workflow that CSC proposes to explain how semantic cognition is implemented by the brain (Lambon Ralph et al., 2017): information from modality-specific areas (spokes) merge at the ATL (hub), where polymodal, generalisable semantic concepts are crafted by melding componential information coded in unimodal spoke areas (Lambon Ralph, Sage, Jones, & Mayberry, 2010; Rogers et al., 2004).", "There are also abundant neural tracts linking this hub-and-spokes structure to the IFG, permitting the prefrontal system to regulate.", "It is crucial to note that, although the tractography evidence shows the physiological ‘hardware’ that semantic processing rests upon, it does not explain how information processing is conveyed in this structure and how it is modulated by tasks.", "Our primary goal was to understand the flexible interplay between hub, spoke and executive regions under different contexts.", "We narrowed down this broad aim to test a specific prediction of CSC that a less-practised, atypical context that requires precise scrutiny of semantic attributes would elicit greater prefrontal-executive regulation to the hub-and-spoke system, compared to a well-practised, familiar context.", "This was achieved by first characterising the potential ‘hub’ and ‘spoke’ sections in the ventral temporal cortex (VTC), using a novel approach termed vectors of region-of-interest (Konkle & Caramazza, 2013) which mapped the evolution of functional responses along the VTC.", "We next utilised psycho–physiological interaction analyses (PPI; Friston et al., 1997) and dynamic causal modelling (DCM; Friston, Harrison, & Penny, 2003; Stephan et al., 2010) to explore how the communication between the prefrontal ‘control’ region and ‘hub-and-spoke’ structure varied between tasks.", "A secondary goal was to investigate how visual cortices respond to sensory stimulation (visually-presented colour patches) versus conceptual simulation of visual knowledge (colour-related concepts).", "We identified the occipitotemporal clusters that exhibited enhanced connectivity with the IFG (connectivity-defined clusters) and tested whether these connectivity-based clusters overlapped with those activated by perceiving sensory hue or retrieving colour knowledge.", "This was achieved by conducting a novel analysis of receiver operating characteristic (ROC) on the spatial distribution of sensory-, concept-, and connectivity-demarcated occipital voxels.", "All results were then integrated, enabling us to examine how they fit with CSC.", "Eighteen right-handed, native English-speaking volunteers (14 females, 23 ± 3 years) gave informed consent before participating in the study.", "All had normal colour vision, assessed using a colour-blindness test (Ishihara, 1960), completed Magnetic resonance imaging safety screening before participation, and had no neurological or psychiatric conditions.", "This study was reviewed and approved by the local research ethics committee.", "Participants completed five functional scans.", "In Scans 1–3 (main tasks), they did (i) a colour knowledge task that required pairing semantically unrelated objects based on canonical colour, (ii) a semantic-associative that required pairing items based on conceptual relationship, (iii) a non-conceptual control task of comparing visual configurations.", "The contrast of colour versus semantic task allowed us to examine whether task contexts alter the interaction between the control and representational systems, as CSC would envisage.", "In the colour knowledge task, participants saw a triad of words in each trial, one above the centre and the other two below, equidistant from the midline.", "The three words were not related to one another; each word referred to an object that was associated with a canonical colour (e.g., duckling, dandelion, and plum).", "Participants judged which one of the two bottom objects has a typical colour more similar to that of the top object.", "In the semantic knowledge task, participants also saw a triad of words (e.g., ostrich, eggshell, and cheese).", "They judged which of the two bottom words was semantically more associated with the top word.", "In the non-conceptual condition, we used a well-established control task (Visser, Jefferies, Embleton, & Lambon Ralph, 2012; Visser & Lambon Ralph, 2011) in which participants saw a triad of scrambled visual patterns.", "They judged which one of the two bottom patterns was the left-right mirror inverse of the top item.", "We tested two different modes of semantic operation – while keeping the association strength of probe (the top word) with foil (the non-target bottom word) identical in both tasks, linking a target to a probe item in the colour task is a semantically arbitrary process that requires deliberately pairing of two words that bear minimal prior relationship, whereas linking a target to a probe in the semantic task is based on pre-existing and well-learnt knowledge.", "Thus, the two tasks differ on the automaticity of semantic association.", "To achieve this, we used the same probes and foils for the colour and semantic tasks, while they differed in the target item.", "For example, a ‘probe-target-foil’ triad in the colour task was ‘mustard, smiley, hawk’ and its counterpart in the semantic task was ‘mustard, ketchup, hawk’.", "With careful selection we ensured that (i) in the colour task, both the target and foil were semantically unrelated to the probe while all three words were strongly associated with a typical colour; and (ii) in the semantic task, neither of the two option words were associated with a colour similar to the probe's colour and only the target was semantically related to the probe.", "Our selection of stimuli was facilitated using latent semantic analysis (LSA), a computational technique that calculates the associative strength between word meanings from a large text corpora (Hoffman, Rogers, & Lambon Ralph, 2011; Landauer & Dumais, 1997).", "While selecting words for each triad of stimuli, we used LSA to extract the pairwise similarity of ‘probe and colour target’ (e.g., duckling – dandelion), ‘probe and foil’ (e.g., duckling – plum), and ‘probe and semantic target’ (e.g., duckling – goose).", "With these corpus statistics, we examined (i) whether the semantic targets were more associated with probes compared to foils and colour targets, and (ii) whether foils and colour targets did not differ in associative strength to probes.", "Results of these statistical tests supported the adequacy of our stimuli set: The strength of connection for ‘probes and semantic targets’ was significantly greater, compared to that of ‘probes and foils’ and ‘probes and colour targets’ (both ps < 10−16), while no difference existed for ‘probe and foil’ versus ‘probe and colour target’ (p > .59).", "In addition, we also controlled for word length (number of letters) and lexical frequency (based on the British National Corpus) to ensure no systematic difference between conditions.", "The lexical stimuli set are provided in Supplemental Information (SI).", "Taken together, with rigorous selection and control we ensured that pairing semantic targets with probes would be easier compared to pairing colour targets with probes.1", "Stimuli were presented in a block design, controlled using E-Prime (Psychology Software Tools).", "A functional scan of the main tasks contained six blocks of 19 sec for each of the three tasks and six 19-sec resting periods, giving 456 sec in duration.", "The order in which task conditions and stimuli sets were presented was fully counterbalanced across our sample of 18 participants so that each condition and stimuli was equally likely to appear in every possible position of the sequences, with stimuli randomly drawn from a designated stimuli-set for a given scan and shuffled across blocks.", "A task block contained five trials.", "Each trial began with a fixation display prompting task demand (colour, association, or inverse), presented for 800 msec.", "Subsequently, a triad of stimuli (words or scrambled patterns) was displayed for 3 sec during which participants had to indicate their choice by pressing one of the two designated buttons on a MR-compatible response pad.", "All visual stimuli were black and displayed on a mid-grey background, presented via a mirror mounted on the head coil.", "The target options were equally likely to appear on the left and right side.", "Prior to entering the scanner, participants completed two practise blocks for each task.", "In Scans 4–5, participants did the Farnsworth-Munsell 100 Hue Task (Hsu, Kraemer, Oliver, Schlichting, & Thompson-Schill, 2011; Simmons et al., 2007) that localised brain regions underlying colour perception.", "In each trial participants saw an annulus stimulus presented centrally.", "The annulus comprised five ‘wedges’ and could be coloured or greyscale (lightness matched to its counterpart coloured stimulus; presented in separate blocks of trials).", "The five component wedges were varied in lightness, arranged either in an orderly sequence (from lightest to darkest; 50% of the trials) or disorderly (50%).", "Among the sequentially-ordered stimuli, the wedges were equally likely to move from lightest to darkest clockwise and anti-clockwise.", "A localiser scan contained 10 blocks of 15 sec for each of the coloured and greyscale conditions, with a 10-sec resting period between blocks, giving 500 sec in duration.", "The coloured and greyscale blocks were randomly interleaved within a scan and counterbalanced across participants.", "Each block contained 5 trials.", "At the beginning of a trial, a fixation display was shown (500 msec), followed by the annulus stimuli (2.5 sec).", "Participants were asked to judge if the lightness of component wedges was arranged sequentially or not by pressing a designated button.", "They performed the localiser task after they had completed the main tasks.", "All scans were acquired using a 3T Phillips Achieva scanner equipped with a 32-channel head coil and a SENSE factor of 2.5.", "A dual gradient-echo EPI sequence was used to maximise signal-to-noise ratio in the ventral ATLs (Halai, Welbourne, Embleton, & Parkes, 2014).", "Using this technique, each scan consisted of two images acquired simultaneously with different echo times: a short echo optimised to obtain maximum signal from the ventral ATLs and a long echo optimised for whole-brain coverage.", "The sequence included 31 slices covering the whole brain with repetition time (TR) = 2.8 sec, short/long echo times (TE) = 12/35 msec, flip angle = 85°, field of view (FOV) = 240 × 240 mm, resolution matrix = 80 × 80, slice thickness = 4 mm (no interleaving gap), and in-plane resolution = 3 × 3 mm.", "To reduce ghosting artefacts in the temporal lobes, all functional scans were acquired using a tilted angle, upward 45° off the AC-PC line.", "Functional scans of the main tasks were collected over three runs; each run was 456-sec long during which 163 dynamic scans were acquired (alongside 2 dummy scans, discarded).", "Functional scans of the localiser task were collected over two runs; each run was 500-sec long during which 179 dynamic scans were acquired (alongside 2 dummy scans).", "To tackle field-inhomogeneity, a B0 field-map was acquired using identical parameters to the functional scans except for the following: TR = 599 msec, short/long TEs = 5.19/6.65 msec.", "Total B0 scan time was 1.6 min.", "A high-resolution T1-weighted structural scan was acquired for spatial normalisation; this included 260 slices covering the whole brain with TR = 8.4 msec, TE = 3.9 msec, flip angle = 8°, FOV = 240 × 191 mm, resolution matrix = 256 × 163, and voxel size = .9 × 1.7 × .9 mm.", "The structural scan took 8.19 min.", "Analysis was carried out using SPM8 (Wellcome Department of Imaging Neuroscience, London).", "The functional images from the short and long echoes were integrated using a customised procedure of linear summation (Halai et al., 2014; Poser, Versluis, Hoogduin, & Norris, 2006).", "The combined images were realigned using rigid body transformation (correction for motion-induced artefacts) and un-warped using B0 field map (correction for field-inhomogeneity).", "The averaged functional images were co-registered to each individual participant's T1 structural scan.", "Spatial normalisation into the MNI space was performed using the standardised DARTEL protocol by group-wise registration of individual's grey and white matter into a template brain created from the group average (Ashburner, 2007).", "This optimises inter-participant alignment, allowing more precise localisation.", "The functional images were then resampled to a 3 × 3 × 3 mm voxel size.", "Voxel-smoothing was applied using an 8-mm Gaussian FWHM kernel, in accordance with the default setting of SPM.", "Contrasts of interest were estimated using general linear models convolving a box-car function of all experimental conditions with a canonical haemodynamic response function (main tasks: the colour, semantic, and control conditions; localiser: the coloured and greyscale conditions), with resting periods modelled implicitly.", "Motion parameters and reaction times were entered into the model as parametric covariates of non-interest, which accounted for brain activities driven by head movements and task difficulty/effort.", "Low frequency drifts were removed using a high-pass filter of 128 sec.", "We performed a vectors-of-ROI analysis (Konkle & Caramazza, 2013) to explore the evolution of preferential response for different tasks.", "As illustrated in Fig. 1, the procedure of constructing a vector contained the following steps: (i) Along the ventral pathway, we specified a medial and a lateral sector based on the demarcation of anatomical template, and for each sector we secured a series of anchor points on a cortical path of interest; (ii) we fitted a spline across these anchor points using linear approximation of piecewise function; (iii) we created a series of spherical ROIs, evenly distributed along the spline; (iv) we computed and extracted the response strength for all contrasts of interest in each ROI (β weights for ‘colour vs. control’, ‘semantic vs. control’, and ‘colour vs. semantic’).", "Specifically, based on the sulcal/gyral definitions given in the Wake Forest University Pickatlas toolbox (Maldjian, Laurienti, Kraft, & Burdette, 2003) we first segregated the ventral occipital and temporal cortices into two compartments: one sector included the lateral occipital complex concatenated with the inferior temporal gyrus, and the other sector comprised the lingual and fusiform gyri.", "Subsequently, we partitioned each sector into five segments of approximately equated length (on the y-axis) and set the centroid of each segment as the ‘anchor points’.", "Using a function of piecewise linear approximation, we fitted a ‘spline’ across the five anchor points for each sector.", "On each spline, the piecewise distance between each pair of anchors was divided into three equal pieces, giving two intermediate points between anchors.", "Finally, a series of 13 non-overlapping spherical ROIs (radius = 3 mm) was created, centred at the five anchors and eight intermediate points.", "The two vectors of ROI spanned from the occipital to the temporopolar cortex (range on the y-axis: −78 to 5).", "Great care was taken to adjust the coordinates so that on the coronal and axial planes the ROIs of the two vectors were matched on the y-axis and z-axis, with them only differing on the sagittal plane (x-axis).", "For each contrast of interest, we extracted β weights for each ROI sphere from the GLM results for further statistical analysis.", "We investigated whole-brain functional connectivity to the left ventral ATL and IFG using PPI analyses.", "We first extracted the time-series of estimated neural signal from a seed region (the left ATL/IFG) and constructed a GLM model containing three factors: a psychological variable (contrast of interest), a physiological variable (seed activity), and a psychophysiological variable (the interaction term of the former two).", "For each participant and each run of main tasks, BOLD activity was extracted from a sphere (radius = 6 mm) centred on the local maxima of ATL and IFG activation identified by the relevant contrasts in GLM and converted into neural time-series using the standard deconvolution algorithm of SPM8.", "We then performed a whole-brain search to identify voxels whose activity could be explained by the PPI factor.", "We conducted two PPIs: (i) colour vs. semantic, with ATL seed, (ii) colour vs. semantic, with IFG seed.", "DCM analysis was performed using DCM10 included in SPM8.", "As the considerations for constructing the DCM were based on the results of other analyses, to improve comprehensibility we report the details about rationales, model configuration, and selection of nodes in the Results section 3.6 after we have reported the results that DCM relied upon.", "Here we outline key information about our DCM: We tested two models; the ‘top-down’ model assumes that performing atypical pairing based on colour knowledge would enhance modulatory signals flowing ‘downstream’ from the prefrontal cortex to colour-related clusters of ventral occipitotemporal cortex.", "The contrasting ‘bottom-up’ model has otherwise identical structure and nodes, but instead assumes that the colour task would reinforce feed-forward information moving from occipitotemporal to prefrontal cortex.", "For each participant and each of the five target nodes (the left IFG, ventromedial ATL, ventrolateral ATL, occipital ‘concept’ area, and occipital ‘percept’ area), we extracted the BOLD time-series.", "The target nodes were localised based on individual's maximal response closest to the peak activation point identified in group analysis and defined as spherical ROIs of 6-mm radius; BOLD series were converted into neural activity using the first eigenvector extracted by the default algorithm of SPM8.", "For both the ‘top-down’ and ‘bottom-up’ model, the five nodes were all set to be bi-directionally connected with one another.", "Each model tests 20 ‘endogenous’ parameters that reflect baseline connectivity in the absence of experimental perturbation, as well as five parameters that reflect changes due to different experimental contexts.", "Subsequently, we examined the explanatory power of each model by performing fixed-effect (FFX) Bayesian selection, comparing the top-down model against the bottom-up model, using the algorithm implemented in SPM8 (Stephan, Penny, Daunizeau, Moran, & Friston, 2009).", "The FFX analysis assumes that the optimal model would be identical across individuals.", "It computes ‘model posterior probabilities’, which gauge each model's ability to interpret the causative strength of synaptic connections among neuronal populations (nodes) and their susceptibility to contextual modulation.", "This allowed us to assess the probability that one model offers a better description for a given data-set than another model.", "However, because the FFX analysis might be vulnerable to outliers, we also implemented a random effect (RFX) analysis, which took into consideration the heterogeneity of model structure across individuals.", "It uses hierarchical Bayesian modelling that calculates parameters of a Dirichlet distribution which describes the probabilities of both models considered.", "These probabilities define a multinomial distribution over model space, enabling the computation of the posterior probability (the likelihood of being true) of each model given the data of all subjects and the models considered.", "The results of RFX analysis are reported in terms of ‘model exceedance probabilities’, indicating if one model is more probable to hold true than the other model.", "The ROC analysis was performed based on the results of the initial whole-brain GLM and PPI analyses.", "The rationales and procedures of the ROC analysis are described in detail in Results section 3.7.", "Here we only provide key information: We first identified all active voxels showing sensitivity to colour perception (localiser: coloured > greyscale), colour concept (main tasks: colour > semantic), and colour-related co-variation with the IFG (PPI: colour > semantic) in the left occipital lobe mask, threshold at p < .01.", "This liberal threshold was purposefully used to allow scrutiny of the full breadth of different activity levels, from weakly to strongly responsive.", "All identified voxels were ranked based on their t value (activation strength) into a percentile.", "We then examined the location of each voxel, starting from the most active voxel to the least.", "For each type of colour processing (concept, percept, and PPI), three ROC curves for each functionally-defined target ROIs (concept-specific, percept-specific, and PPI) were created.", "The computation of each ROC function began with the voxel most active for a certain effect, and finished with the least active.", "After constructing the ROCs, we calculated values of area under the curve (AUC) to quantify the likelihood of voxels appearing in a certain region.", "Accuracy was high for the three main tasks.", "As expected, there was a main effect of task (F2, 34 = 12.27, p < .001, ηp2 = .42), with arbitrary pairing by canonical colour (86%) being less accurate than semantic pairing by meaning (94%) and the control task (91%).", "This effect was also evident in RTs (F2, 34 = 16.70, p < .001, ηp2 = .49), with slower RTs for the colour (1569 msec) than semantic (1387 msec) and control (1354 msec) tasks.", "These results are consistent with our prediction that arbitrary pairing would be more effortful than the other tasks.", "In the localiser experiment, accuracy and RTs did not significantly differ between coloured (83%; 1123 msec) and greyscale (82%; 1144 msec) stimuli (both ps > .24).", "Note that in subsequent fMRI analysis, a parametric modulator of reaction time was included to account for and rule out its potential influences.", "The whole-brain interrogation was stringently thresholded at p < .0001 for voxel intensity and p < .05 (FWE-corrected for multiple comparisons) for clusters.", "Relative to the control task, both colour and semantic decisions elicited greater activation in a strongly left-lateralised distributed network well-established in previous inquiries of semantic memory, including the IFG and a vast swathe of the temporal cortex, encompassing superior to inferior subregions (see Fig. 2).", "Critically, both colour and semantic tasks triggered robust activation in the ATL (the anterior fusiform gyrus, FG), an area reliably identified as the key substrate of semantic ‘hub’2 (Binney, Embleton, Jefferies, Parker, & Lambon Ralph, 2010; Mion et al., 2010; Shimotake et al., 2014; Visser, Embleton, Jefferies, Parker, & Lambon Ralph, 2010).", "Compared to semantic pairing (colour > semantic), colour pairing enhanced activation in the prefrontal ‘executive control’ regions, including the IFG and its neighbouring middle frontal gyrus, as well as the intraparietal sulcus.", "This dovetails with the prediction of CSC that tasks demanding atypical and precise processing necessitate more input from the ‘control’ network, hence heightened activity in the frontoparietal network.", "The reverse contrast (semantic > colour) revealed greater activation in the dorsolateral aspect of the ATL.", "The ‘hub-and-spokes’ view postulates that concepts, particularly those about perceptual features, engage the ATL hub plus modality-specific ‘spoke’ cortices.", "Thus, we specifically focused on the visual cortices, examining the extent to which colour knowledge involved the visual regions and comparing it to colour perception.", "We independently identified occipital voxels sensitive to colour percept (chromatic > greyscale, localiser task) and colour concept (colour > semantic, main task), thresholded at p < .005 for each voxel and further limited by a cluster constraint of contiguously extending at least 270 mm3.", "Consistent with the ‘hub-and-spoke’ view and embodied cognition (for review, see Barsalou, 2008; Martin, 2015), we found percept and concept engage adjacent sections of the visual cortex (Fig. 3A).", "Interestingly, while percept and concept voxels adjoin and partially overlap, we noticed that percept tends to be situated more medially and posteriorly whereas concept tends to be located more laterally and anteriorly.", "This separation is more manifest when rendered on the cortical surface with a liberal threshold (Fig. 3B).", "To systematically delineate the spatial layout of percept vs. concept voxels, we extracted the normalised activation strength (Z-value) of left-hemisphere voxels as a functional of position and represented content, with best-fitting polynomial functions representing the trend of this clearly separable cortical distribution.", "As illustrated in Fig. 3C, percept and concept voxels were distributed in a graded fashion, with a lateral and anterior shift into conceptual processing.", "This is consistent with a representational gradient, arguing against clear-cut modular separation.", "It is noteworthy that, while we were able to detect the effect of colour knowledge > semantic association in broad expanses of prefrontal ‘control’ areas with stringent criteria (voxel: p < .0001, cluster: p < .05 FWE-corrected; see Fig. 2), we had to apply liberal thresholds3 to detect the same effect in the visual cortex (voxel: p < .005, cluster: at least 270 mm3).", "This points to a need to recognise that whilst modality-specific embodied representations contribute as building blocks for concepts (especially during the initial acquisition of a novel concept), they have to be considered together with higher-level regions as constituents of a greater functional neural architecture.", "To explore how neural responses unfold along the caudal ‘spoke’ and rostral ‘hub’ zones of ventral-temporal pathway, we performed a vectors-of-ROI procedure (Konkle & Caramazza, 2013).", "Our paradigm provided optimal circumstances for studying this transition: the control task demanded pairing stimuli using perceptual conformation; the semantic task demanded pairing concepts using meaningful relationship; whilst the colour knowledge task straddled between perception and semantics (pairing concepts using their known perceptual features stored in semantic memory).", "We created a series of parallel, non-overlapping spherical ROIs along the FG and ITG, spanning ventral occipital and temporal cortices (from y = −78 to +5), and extracted β-weight for each contrast of interest.", "Along both the vectors of FG (Fig. 4A) and ITG (Fig. 4B) we observed clear gradients evolving from perception to semantics.", "Both gyri shifted from being more active for visual configuration processing (control task) to conceptual knowledge (the colour and semantic tasks) as neural processing proceeded anteriorly.", "Critically, closer scrutiny of Fig. 3B and C revealed two functional distinctions between the FG and ITG vectors: (i) the ‘tipping point’ that switched from perceptual to conceptual processing was evidently more posterior in the ITG than FG vector; and (ii) the two vectors also differed in their response profiles in the most rostral poles of ROIs; anterior FG was equally responsive to the colour and semantic knowledge tasks, whereas anterior ITG was apparently more active for the semantic task than colour task.", "Formal statistical analysis fully corroborated these visual inspections (Fig. 4C).", "We obtained a three-way interaction, indicating that the evolution of neural responses across the ROIs significantly differed between contrasts and vectors (F24, 408 = 10.03, p < .001, ηp2 > .37).", "For colour vs. control, the difference of FG vs. ITG activations significantly interacted with anterior-to-posterior positions (F12, 204 = 15.21, p < .001, ηp2 > .47; Fig. 4C left).", "The two vectors significantly differed in the posterior-to-middle segments (y = −63 to −39), with ITG regions exhibiting greater response to colour knowledge than did FG regions (all ps < .01, indicated by the golden rectangle).", "However, in more rostral and most caudal cortices, the two vectors did not differ in response profile (all ps > .1).", "For semantic vs. control, the two vectors' response also significantly interacted with positions (F12, 204 = 12.54, p < .001, ηp2 > .42; Fig. 4C middle): Compared to the FG vector, the ITG vector displayed greater response to the semantic task in the posterior-to-middle sections (y = −63 to −39) and, crucially, in the most rostral ROI, situated in the temporal pole (y = +5; all ps < .05; note this rostral, temporopolar section showed no between-gyri difference for colour vs. control while exhibiting a significant difference for semantic vs. control).", "Finally, in the direct contrast of colour vs. semantic decisions, the difference between the two vectors was evident in the most posterior and anterior extremes (interaction: F12, 204 = 6.64, p < .001, ηp2 > .28; Fig. 4C right): in the caudal segment (y = −71 to −63), the ITG was more active for the colour knowledge task, whereas in the rostral segment (y > −8), the ITG was more active for the semantic association task, relative to the fusiform ROIs (all ps < .05).", "To investigate how performing different tasks altered connectivity to the ‘semantic control’ and ‘semantic representation’ systems, we conducted PPI, thresholded at p < .005 for voxel and further constrained cluster size (contiguous extension ≥ 270 mm3).", "We know that, compared to pairing using well-learnt semantics, deliberate pairing by colour was behaviourally more effortful and elicited greater activation of the prefrontal cortex.", "Thus, we tested a key prediction of CSC that the more demanding colour task might augment communication between the prefrontal ‘control’ system and the ventral-temporal ‘representation’ regions, relative to the semantic task.", "We localised the seeds at the ATL ‘hub’ (anterior FG) and the prefrontal ‘control’ region (the IFG) using peaks of the initial GLM analyses, and compared the contextual connectivity of colour knowledge > semantic association.", "For the ATL seed, PPI showed that a cluster of the orbitofrontal cortex was more connected with the ‘hub’ during the colour than semantic task (Fig. 5); this orbitofrontal cluster is evidently more ventral than the IFG seed, which is situated at the dorsal part of pars triangularis.", "For the IFG seed, PPI revealed two major groups of brain regions showing enhanced connectivity to the IFG: (i) Widely distributed areas of ‘control’ network, including an extensive stretch of left prefrontal ‘executive-control’ regions, spreading dorsally (to the middle frontal gyrus) and ventrally (to pars orbitalis), as well as the intraparietal sulcus and the posterior middle/superior temporal gyrus.", "(ii) Most intriguingly, the left visual ‘spoke’ cortex was also linked to the IFG, peaking at the lateral occipital area and middle fusiform gyrus (Fig. 5).", "The pattern of connectivity to the IFG revealed key features of the neural architecture for colour knowledge: To access specifics of remembered colour attributes, the IFG becomes more tightly connected with other nodes of the frontoparietal ‘control’ network.", "Furthermore, its connectivity with the occipitotemporal ‘spoke’ cortex is also reinforced.", "This particular channel of connectivity might provide a crucial avenue for the prefrontal control centre to retrieve the embodied representations of colour stored in the occipitotemporal regions.", "In the reverse test (‘semantic > colour’) with the IFG seed, we found various nodes of the default mode network (DMN; Anticevic et al., 2012; Spreng & Grady, 2010) were more connected to the seed, including the angular gyrus, cuneus, and medial prefrontal cortices (Fig. 5).", "Further analysis showed that this stronger IFG-DMN connectivity during the semantic condition was driven by greater deactivation of the DMN during the more demanding colour task (for details see SI), consistent with its propensity to deactivate during cognitively taxing situations (Humphreys, Hoffman, Visser, Binney, & Lambon Ralph, 2015).", "The aim of DCM is to make directional inferences about the impact that one brain region exerts over another and how this is affected by tasks.", "We used DCM to adjudicate two hypotheses that could potentially explain our PPI results.", "The top-down hypothesis posits that the PPI connectivity reflects the prefrontal ‘control’ regions modulating the occipitotemporal ‘spoke’ regions via giving feedback signal.", "This might be due to imagery during the colour task (e.g., conjuring up mental pictures of object colours, a common strategy for the task).", "By contrast, the bottom-up view assumes that activities of the ‘spoke’ areas arise early and precede subsequent higher-level processing that leads to semantic decisions; thus, it would be the ‘spoke’ feeding forward to the ‘control’ region.", "We selected the nodes of DCM network based on the following empirical evidence of our prior GLM, PPI, and vectors-of-ROI analyses, and localised the nodes based on GLM outcomes.", "Both colour knowledge and associative semantics drove robust activation of the anterior FG (the semantic ‘hub’), peaking at medial-ventral parts of the ATL, as the GLM and ROI-vectors analyses both showed;", "The colour knowledge task induced greater activation of the left IFG (control centre) and lateral occipitotemporal regions (modality-specific ‘spoke’) relative to associative semantics, as indicated by the GLM and ROI results;", "The associative semantic task induced greater activation of the anterior ITG (lateral-ventral parts of the ATL) relative to colour knowledge, as revealed by the ROI-vectors analysis (the GLM results similarly revealed the anterior ITG, when thresholded more liberally);", "The occipital concept-related (colour knowledge > associative semantics) clusters were adjacent to the percept-related (colour images > greyscale) clusters, with a trend shifting laterally and anteriorly for concept processing, as shown by the GLM analysis;", "The colour knowledge task enhanced functional connectivity between the ‘control centre’ IFG and the visual ‘spoke’ clusters (the lateral occipital cortex and posterior FG), as revealed by the PPI analysis.", "Based on the considerations and evidence laid out above, we constructed the DCM models using the regions that showed significant effects in the relevant contrasts of interest.", "The models consisted of bilateral, intrinsic connections (thin black arrows) between the IFG, the ventral ATL (anterior FG/hub), the lateral ATL (anterior ITG), the lateral occipital colour-concept cluster, and the medial occipital percept cluster (Fig. 6A).", "We performed both fixed-effect (FFX) and random-effect (RFX) Bayesian model selections (Penny, Stephan, Mechelli, & Friston, 2004) to verify which model maximised explanatory accuracy and minimised model complexity.", "In the top-down model (Fig. 6A left), the triggering input entered the model through the IFG and ventral ATL due to their established significance for semantic cognition.", "Five modulatory parameters (thick red arrows) were specified to examine whether inter-node communication was altered by task: (i) the mutual influence between ventral ATL hub and IFG control centre, (ii) the mutual influence between ventral ATL hub and its nearby lateral ATL; and (iii) we assumed that the connectivity between IFG and occipital ‘colour-concept’ area (i.e., the PPI effect) is driven by top-down messages from the IFG to downstream visual cortex.", "The bottom-up model (Fig. 6B right) was otherwise identical to the top-down model but differed in two aspects: first, the input entered the model via the medial occipital percept node; second, the connectivity identified by PPI was driven by the lateral occipital ‘colour-concept’ node triggering the IFG.", "Both the FFX and RFX Bayesian selection methods showed that the top-down model overwhelmingly outperformed the other model (Fig. 6A middle).", "We subsequently tested endogenous parameters (baseline strength of connection, without task-related modulation) of this winning top-down model and the results showed that all links were significantly excitatory (relative to zero, all ps < 1 × 10−10).", "Crucially, different task conditions significantly modulated effective connectivity (F8, 136 = 23.38, p < .001, ηp2 = .57; see Fig. 6B).", "Closer inspection of the results revealed two distinct patterns of contextually-driven connectivity.", "Performing the associative-semantics task significantly strengthened the modulatory impact from the ventral ATL hub to IFG (simple main effect: F2, 34 = 30.85, p < .001) and from the hub to lateral ATL (F2, 34 = 40.39, p < .001), as well as the reciprocal effect from lateral ATL to the hub (F2, 34 = 12.29, p < .001).", "By contrast, the more effortful colour knowledge task relied on the IFG – performing this task significantly enhanced the impact that the IFG wielded on the occipital ‘colour-concept’ cluster, relative to other tasks (F2, 34 = 3.86, p = .03).", "The modulatory impacts that the IFG exerted on the ventral ATL hub did not differ between tasks (F2, 34 = 1.09, p = .34, n.s.), although a planned t-test revealed that the modulation is significantly greater than zero for the colour task (p = .02).", "Within the ventral-posterior occipitotemporal cortices, we observed that voxels sensitive to colour perception (localiser: coloured > greyscale), colour concept (main tasks: colour knowledge > associative semantics), and showing co-variance with the IFG (PPI effect: colour knowledge > associative semantics) occupied neighbouring and partially overlapped cortical zones.", "When rendered on the cortex (Fig. 7A), the spatial layout of the three types of colour-related voxels exhibited an evident continuum: colour perception was coded in posterior sections, colour knowledge occupied intermediate zones, and concept-related communication with the IFG occurred in anterior patches of visual cortex.", "To characterise and quantify how the locations of the three types of colour processing were spatially organised in relation to each other, we performed a ROC analysis that has been exploited to tackle related issues and is able to circumvent the drawbacks of the conventional ROI approach (for precedent use of the ROC, see Konkle & Caramazza, 2013).", "The typical ROI analysis tests whether an independently defined ROI (e.g., colour-concept area) is engaged in a cognitive process of interest (e.g., hue perception or dialogue with the IFG), by averaging activation of all voxels within the ROI.", "This conventional approach fails to capture more nuanced variations in activity strength between voxels (due to the averaging process), and is validated using liberal statistical threshold (usually α = .05) to arbitrate if an effect is present within the ROI.", "By contrast, the ROC bypasses these issues by (i) considering the activation strength of each individual voxel and (ii) examining each voxel based on its percentile rank, covering the full breadth of intensity.", "We first identified all active voxels showing sensitivity to colour perception, colour concept and enhanced coupling with the IFG (i.e., the PPI effect of colour concept) in the left occipital lobe,4 threshold at p < .01.", "We deliberately adopted this liberal threshold to include voxels so as to gain insight about variation across the full breadth of activation level, from weakly to strongly responsive.", "All identified voxels were ranked based on their t value into a percentile (separately for the three types of colour processing).", "We then examined the location of each voxel, starting from the most to least active percentile of voxels, and computed the proportions of voxels that fell in a target zone (e.g., concept area) vs. non-target zones (e.g., percept and PPI areas).", "Note that this examination protocol was free from the constraints of spatial contiguity or anatomical location, enabling us to inspect which zone of the occipital cortex an individual active voxel was most/least likely to fall into.", "This procedure gave the ROC curves (with increasing numbers of voxels scrutinised, the curve plotting the proportion of target zone filled vs. non-target zone filled) and allowed computation of area under the curve (AUC).", "The ROC curves and AUC values were computed for each colour processing (concept, percept, and PPI) and each functionally-identified ROI as the target zone.", "If the ROC curve of a certain processing is situated well above/below the diagonal line (chance, AUC = .5), it means that the likelihood that activated voxels fall into the target zone is higher/lower than chance.", "If the ROC courses along the chance line with little deviation, it means that active voxels are equally likely to appear inside or outside this target zone.", "Results of the ROC analysis complement the caudal-to-rostral gradient found in our ROI-vector analysis and further demonstrate the subtlety of representational transition.", "Most critically (see Fig. 7B), we found that voxels sensitive to colour concept (colour > semantic) were significantly more likely to fall into the intermediate concept-specific zone (AUC = .61; compared to chance, Z = 3.66, p < .001) and less likely to appear in the posterior percept-specific zone (AUC = .17; Z = 36.66, p < 1 × 10−10), with the likelihood of appearing in the most anterior IFG-driven zone equal to chance (AUC = .49; Z = 1.0, p = .32, n.s.).", "The AUC values of each curve also significantly differed from one another (all Zs > 3.79, ps < .0002; see Fig. 7B inset box).", "This pattern suggests a gradient structure at a micro within-occipital scale: Colour knowledge is represented in adjoining cortical zones anterior and lateral to those for colour percept.", "While a minimal number of voxels respond commonly to both concept and percept (as previous studies have demonstrated), the ROC showed that the great majority of activated voxels by concept tend not to fall into the perceptual zone.5", "Furthermore, as illustrated in Fig. 7B, a proportion of concept-sensitive voxels appeared in the anterior zones where communication with the IFG occurred, sprinkled alongside the connectivity-sensitive voxels.", "The majority of concept-sensitive voxels are located in the intermediate area, sandwiched between percept- and IFG-driven zones.", "Together, the spatial layout of voxels constitutes a graded pattern, from sensory colour processing in the posterior section, through to conceptual colour processing that shifts anteriorly and laterally, to clusters that intensify connectivity with the IFG at the most anterior end of this gradation.", "We tested a key prediction of the CSC theory that the frontoparietal ‘control’ machinery regulates the hub-and-spoke ‘representation’ system depending on semantic contents and task characteristics.", "Specifically, a semantic task that requires pairing semantic concepts in an atypical/infrequent manner (or one that requires high degree of semantic specificity) would need greater executive control, constraining how neural signals propagate within the hub-and-spoke system.", "Compared to a typical associative-semantic task, we found that atypical pairing using specific canonical colour enhanced activity of the prefrontal and parietal ‘control’ regions, replicating previous work using similar designs to the present study (e.g., Thompson-Schill, D'Esposito, Aguirre, & Farah, 1997).", "The prefrontal activation might reflect its role in sustaining a task-defined attention-set and coordinating other brain regions to associate two ostensibly unrelated words using arbitrary rules requested by the task.", "Both tasks robustly activated the anterior sections of the FG, which has previously been proposed to be the centre–point of a graded representational ‘hub’ for semantic processing (Lambon Ralph et al., 2017).", "In addition, there was a gradient along the inferior temporal gyrus, with its caudal sector favouring the colour task (perhaps reflecting embodied colour simulation) and rostral sector preferring the typical association task.", "Furthermore, the PPI and DCM analyses revealed distinct patterns of connectivity for different tasks: Compared to the less effortful semantic task, PPI detected stronger coupling between the IFG (a key node of the ‘control’ network) and the occipitotemporal ‘spoke’ area (which adjoins the regions for colour perception, see below).", "DCM further revealed that this coupling reflects directional modulation from the IFG to the occipitotemporal cortex, implying strategic use of colour imagery during the task.", "Thus, while the two tasks engage largely overlapping prefrontal and ventral temporal cortices, the key distinction of their neural architecture lies in greater involvement of the frontoparietal control system during colour knowledge and the connectivity whereby the IFG attains embodied-visual information from the colour-related cortex.", "The absence of a direct IFG-ATL linkage in the PPI results is somewhat surprising – we speculate that this might be due to a lack of statistical power to detect the subtle PPI effect.", "Specifically, largely overlapping regions of the IFG and ATL were recruited by both the colour and semantic tasks.", "When the strong main effect of task-driven activation was partialled out, we might not have sufficient power to detect the more elusive PPI-related changes in connectivity (especially when the PPI-driven area might be overlapped with task-driven zones of ATL and IFG).", "All in all, our results highlight the complementary roles of the control and representation systems in supporting flexible use of semantic knowledge in a wide variety of contexts.", "Our findings lend support to a key tenet of the CSC theory – semantic cognition is subserved by two functionally and anatomically distinct yet interacting machineries.", "First, a ‘hub-and-spoke’ system is in charge of representing semantic content: knowledge about perceptual features (e.g., ketchup is red) relies on modality-specific cortices (spoke) while a coherent concept (e.g., ketchup as fusion of multiple perceptual and functional traits) is built upon a ‘coalition’ of a polymodal hub (the anterior FG/vATL) and various modality-specific spokes (Pobric, Jefferies, & Lambon Ralph, 2010; Rogers et al., 2004).", "Second, an executively-related machinery for semantic control is implemented primarily by the left frontoparietal network (Badre, Poldrack, Paré-Blagoev, Insler, & Wagner, 2005; Jefferies & Lambon Ralph, 2006).", "This network constantly interacts with the hub-and-spokes structure to promote efficacious selection of task-relevant knowledge from a multitude of information in the representational database.", "These two neural systems can be selectively lesioned, causing double dissociation: Whereas ATL atrophy leads to semantic dementia (erosion of semantic memory with preserved capability of semantic control), frontoparietal stroke leads to semantic aphasia (inability to select appropriate pieces of semantic information, exacerbated by infrequent or unfamiliar meaning, while semantic memory per se is intact; for example, see Thompson et al., 2018; Thompson et al., 2017).", "It is noteworthy that we chose to focus on the IFG in the present study owing to its established role in selecting context-relevant semantic information, particularly under unfamiliar or ambiguous situations.", "Apart from the IFG, however, the intraparietal sulcus (IPS) and temporoparietal junction have been known to underpin semantic control (e.g., Davey et al., 2016; Whitney, Kirk, O'Sullivan, Lambon Ralph, & Jefferies, 2011; for review, see; Noonan, Jefferies, Visser, & Lambon Ralph, 2013).", "In fact, our data also revealed these regions: in the GLM contrast between tasks (Fig. 2), the more demanding colour task induced greater activation of both the IFG and IPS; in the PPI results (Fig. 5), the colour task drove the IFG to become more connected with the IPS and a superior temporoparietal cluster.", "These are compatible with the literature and support the notion that regions other than the IFG are involved in semantic control.", "Together, there appears to be a triangulation (polymodal hub, unimodal spokes, and executive mechanism) of neurocomputation underling semantic cognition.", "Our findings highlight the importance of connectivity in understanding semantic processing and the potential jeopardy of focusing solely on modality-specific regions, echoing recent proposals (Binney et al., 2012; Chen, Lambon Ralph, & Rogers, 2017; Mahon, 2015a, 2015b, 2015c).", "Indeed, complete models of semantic cognition need to be able to surmount two challenges: (i) they need to expound how functionally distinct and often anatomically remote modules cooperate to engender semantic knowledge, transcending beyond the regional function of each component; and (ii) they also need to take the intrinsic wiring of neural tracts into consideration when explaining how cortical response and connectivity are couched in and constrained by neurophysiological infrastructure.", "Using a series of novel analyses to address these issues, we unravelled the functional interactions among constituent components of the semantic network.", "Furthermore, we revealed the representational gradients of cortical activity, both at an intra-lobar scale (within-occipital) and an inter-lobar scale (encompassing the entire VTC, spanning across the occipital and temporal lobes), and how neural connectivity alters its dynamics in response to different contexts.", "Utilising PPI and DCM connectivity analyses, combined with ROI-vectors, we mapped the semantic networks of the brain.", "It is important to note that the present results fit closely with the pattern of intra- and inter-lobar white-matter connections in both human and non-human primates (Binney et al., 2012; Jung, Cloutman, Binney, & Lambon Ralph, 2016; Moran et al., 1987).", "The gradual transition of ROI-vectors, shifting anteriorly from perceptual to conceptual processing, meshes with the pattern of long-range fasciculi and short-range U-fibres of VTC.", "Across the caudal to rostral VTC, multiple long-range fasciculi (including inferior/middle longitudinal, uncinate fasciculi, and anterior commissure) all terminate in the anterior temporal region.", "Across medial to lateral VTC, multiple short-range fibres connect neighbouring gyri (Bajada et al., 2017; Binney et al., 2012; Papinutto et al., 2016).", "These short- and long-range connections provide the neural scaffolding, wherein information converges at the anterior temporal region and amalgamates both within and across sensory modalities (Binney et al., 2012).", "With this graded and converging structure, neural processing shifts gradually from perceptual computations for a single modality towards aggregate computations for poly-modalities and higher-level semantics.", "There are also inter-lobar connections, such as the inferior fronto-occipital fasciculus (IFOF) that links the IFG to occipitotemporal regions, as well as the uncinate fasciculus (UF) that links the IFG to ATL.", "The functional network that we identified in the present study fits with the tractography evidence, suggesting that the IFOF and UF might serves as conduits allowing the prefrontal cortex to exert top-down modulation on posterior regions (Almairac, Herbet, Moritz-Gasser, de Champfleur, & Duffau, 2015).", "An anatomical connectivity ‘triangle’ is completed by the inferior longitudinal fasciculus which directly connects the ATL with occipitotemporal and more posterior striate and extrastriate cortices (Binney et al., 2012).", "The gradient within the visual cortex for ‘concept vs. percept’ dovetails with previous work that colour knowledge activates regions in proximity to regions for hue perception (Hsu, Frankland, et al. 2012; Rugg & Thompson-Schill, 2013; Simmons et al., 2007).", "Critically, while concept and percept occupy partially overlapping regions, concept tends to be more anterior and lateral.", "This pattern offers clues about the dissociation of patients and healthy individuals' data.", "Specifically, in healthy participants retrieval of colour knowledge induces occipitotemporal activity adjacent to regions of colour perception.", "However, in neurological patients there is double dissociation between perceptual and conceptual deficits: some patients lose their ability to perceive and recognise colours but retain the knowledge about canonical object colour when probed using imagery tasks (Shuren, Brott, Schefft, & Houston, 1996), whereas other patients lose colour knowledge while keeping normal colour perception (Miceli et al., 2001; Stasenko, Garcea, & Mahon, 2013).", "This dissociation fits our ROC analysis for voxel distribution that, despite them being represented in adjoining regions, the core substrates of percept and concept might still be separable.", "Our results speak directly to a spectrum of views on the nature of semantic representations, spanning from strong ‘embodied’ theories (concepts are rooted in sensorimotor activation, simulating original experiences) through to symbolic accounts which postulate that concepts reflect ‘amodal’ representations (Fodor, 1983; Mahon, 2015b; Meteyard, Cuadrado, Bahrami, & Vigliocco, 2012).", "A key advantage of the ‘embodied’ approaches is that the source of information from which concepts can be learnt is apparent.", "However, this view struggles to tackle the fact that a conglomeration of perceptual features, by themselves, is not sufficient for creating coherent, generalisable concepts (Lambon Ralph et al., 2010), thus eliciting critiques (Dove, 2016; Leshinskaya & Caramazza, 2016).", "In light of the criticisms, treatments have proposed that the neural substrates of object knowledge entails both the perceptual cortex that codes embodied-experiential attributes, plus a polymodal region on which information from different perceptual modules converges (Lambon Ralph et al., 2017; Mahon, 2015c; Rogers et al., 2004).", "Echoing these proposals, our connectivity analyses provide clear evidence that the coordinated interplay among unimodal (spoke), polymodal (hub), and executive regions underpins the retrieval of task-relevant semantic representations.", "The involvement of visual cortices in colour knowledge concurs with previous studies (e.g., Simmons et al., 2007) and argues against a sharp separation between percept and concept.", "Critically though, the involvement of the polymodal ATL and the executive-related IFG, underlines the need for an additional contribution from a transmodal level so that the semantic system is able to establish accurate links between perceptual attributes (canonical colour) and their multimodal properties (object identity).", "These results also fit with accruing evidence that the ATL, particularly its ventral sector, serves the need to represent both concrete and abstract knowledge irrespective of input modality (Lambon Ralph et al., 2017; Peelen & Caramazza, 2012; Visser & Lambon Ralph, 2011) and contributes directly to colour knowledge (Adlam et al., 2006; Chiou & Lambon Ralph, 2016; Chiou, Sowman, Etchell, & Rich, 2014; Ikeda, Patterson, Graham, Ralph, & Hodges, 2006; Rogers, Patterson, & Graham, 2007), establishing the status of the ventral ATL as the centre–point of a graded, transmodal hub.", "As we demonstrated in the present work, the polymodal hub (ATL), unimodal spoke (occipitotemporal cortex in the case of colour knowledge), and executive system (IFG) work as a flexible, task-related coalition to represent different forms of semantic knowledge." ]	[]
Nucleic acid separations using superficially porous silica particles	Ion pair reverse-phase liquid chromatography has been widely employed for nucleic acid separations. A wide range of alternative stationary phases have been utilised in conjunction with ion pair reverse-phase chromatography, including totally porous particles, non-porous particles, macroporous particles and monolithic stationary phases. In this study we have utilised superficially porous silica particles in conjunction with ion pair reverse-phase liquid chromatography for the analysis of nucleic acids. We have investigated a range of different pore-sizes and phases for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA. The pore size of the superficially porous silica particles was shown to significantly affect the resolution of the nucleic acids. Optimum separations of small oligonucleotides such as those generated in RNase mapping experiments were obtained with 80 Å pore sizes and can readily be interfaced with mass spectrometry analysis. Improved resolution of larger oligonucleotides (>19 mers) was observed with pore sizes of 150 Å. The optimum resolution for larger dsDNA/RNA molecules was achieved using superficially porous silica particles with pore sizes of 400 Å. Furthermore, we have utilised 150 Å pore size solid-core particles to separate typical impurities of a fully phosphorothioated oligonucleotide, which are often generated in the synthesis of this important class of therapeutic oligonucleotide.	[ "BIOC", "CHEM" ]	[ "Ion pair reverse-phase chromatography", "Nucleic acids", "Oligonucleotides", "RNA" ]	[ "1303", "1602", "1605" ]	[ "Ion pair reverse-phase chromatography (IP RP HPLC) has been widely employed for nucleic acid separations [1–3].", "A wide range of reverse-phase stationary phases have been utilised for IP RP HPLC including traditional totally porous C18 particles (silica and polymeric stationary phases) [1–5].", "However, slow mass transfer of oligonucleotides in the stationary phase limits the application of these traditional C18 based stationary phases [6,7].", "The development of 2 μm, C18 surface, non-porous polymeric columns, in conjunction with IP RP HPLC offers significant advantages for the analysis of nucleic acids [8–11].", "Rapid, high resolution separation of nucleic acids has been achieved using these non-porous polymeric media.", "The highly mono-disperse nature of the particles results in the minimisation of the diffusion pathways and therefore provides high resolution with rapid analysis times [1–3].", "Furthermore, the column media are robust, being resistant to a broad range of temperatures and pH. This has led to a wide variety of developments in the analysis of nucleic acids using IP RP HPLC, including the sequence-independent sizing of duplex DNA [9], the analysis of oligonucleotides and the separation and purification of single-stranded (ss) DNA [10,12].", "However, a caveat associated with these non-porous particles includes low capacity owing to the limited surface area for interaction with the analyte.", "The application of non-porous polymeric stationary phases has also been extended to analyse larger RNA molecules in conjunction with IP RP HPLC with high resolution, similar to that achieved for larger dsDNA molecules and was demonstrated in the development of an assay for group I intron ribozyme activity [13].", "Further studies have utilised the rapid high resolution separation of RNA to analyse a wide range of RNA transcripts and biological RNAs including ribosomal RNA, mRNA and siRNAs [14–17].", "More recently, macro-porous PS-DVB resins with relatively large pore sizes have been utilised for RNA separations [18].", "Ultra-performance liquid chromatography (UPLC) approaches using 1.7 μm C18 particles have also been employed to improve speed and resolution for short oligoribonucleotides, oligonucleotide therapeutics and the sequencing of synthetic oligoribonucleotides in conjunction with LC–MS analysis [19–21].", "In addition to traditional particular stationary phases, IP RP HPLC in conjunction with a wide range of monolithic stationary phases have also successfully been applied to the high resolution separation of DNA [22] and RNA [16,23].", "Monolithic, poly(styrene-divinylbenzene)-based capillary columns have been utilised for nucleic acid separations interfaced to mass spectrometry in conjunction with MS-compatible mobile phases in a number of different applications [24–27].", "Columns comprising superficially porous particles (SPPs), also termed core–shell particles, solid-core particles and porous-shell particles have been utilised in wide range of studies to analyse biomolecules, demonstrating significant advantages over traditional totally porous particles [28–30].", "The advantages of core–shell particle columns for rapid HPLC analysis of biomolecules including proteins and nucleic acids has been demonstrated, attributing this improved performance to superior mass transfer kinetics [28,29].", "More recently Biba et al., studied the performance of a number of core–shell particle columns in conjunction with IP RP HPLC for the analysis of oligonucleotides [31,32].", "In this study we have utilised superficially porous silica particles (Accucore columns, Thermo Fisher) in conjunction with IP RP HPLC for the analysis of nucleic acids.", "The Accucore columns contain 2.6 μm solid-core particles, with a porous layer of 0.5 μm with an average particle size distribution of 1.12 (D90/D10).", "We have investigated a range of different pore-sizes and phases (C18, C4) for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA.", "Triethylammonium acetate (TEAA, Sigma–Aldrich, UK).", "Tetrabutylammonium bromide (TBAB, Fluka, UK).", "Acetonitrile and water (HPLC grade, Fisher Scientific, UK).", "1,1,1,3,3,3,-Hexafluoro-2-propanol (HFIP, Sigma–Aldrich, UK).", "Oligonucleotides were synthesised by Eurofins Scientific (UK).", "RNase A (Ambion, UK).", "Mobile phases were prepared with HPLC grade solvents (Fisher Scientific, UK).", "Nucleic acids were analysed by IP RP HPLC on a U3000 RSLC UPLC (Thermo Fisher, UK) with the following columns: Accucore C18 150 mm × 2.1 mm I.D. (2.6 μm superficially porous silica particles 80 Å pore size), Accucore C18 150 mm × 2.1 mm I.D. (2.6 μm superficially porous silica particles 150 Å pore size).", "The Accucore columns contain 2.6 μm solid-core particles, with a porous layer of 0.5 μm with an average particle size distribution of 1.12 (D90/D10).", "Accucore C4 150 mm × 4.6 mm I.D. (a research column based on superficially porous silica 4.0 μm superficially porous silica particles 400 Å pore size).", "Chromatograms were recorded using a VWD detector at 260 nm with a 2.5 μl flow cell.", "Tubing was 100 μm I.D. for flow rates of 0.4 ml/min and 180 μm I.D. for flow rates of 1.0 ml/min.", "Buffer A; 0.1 M triethylammonium acetate (TEAA) pH 7.4.", "Buffer B; 0.1 M TEAA, pH 7.4, 25% (v/v) acetonitrile.", "Buffer C; 2.5 mM tetrabutylammonium bromide (TBAB).", "Buffer D; 2.5 mM tetrabutylammonium bromide, 80% acetonitrile.", "Buffer E; 20 mM triethylammonium acetate (TEAA) pH 7.4, 80 mM 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP).", "Buffer F; 20 mM triethylammonium acetate (TEAA), pH 7.4, 80 mM 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP) 25% (v/v) acetonitrile.", "Buffer G; 0.1 M triethylammonium acetate (TEAA) pH 7, 80 mM 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP).", "Buffer H; 0.1 M triethylammonium acetate (TEAA), pH 7.4, 80 mM 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP) 25% (v/v) acetonitrile.", "Gradient 1: HPLC buffers A and B. Linear gradient: 25–35% B in 1 min; 35–70% B in 14 min; 70–100% B in 0.1 min; 100% B for 3 min; 100–30% B in 0.4 min; 25% B for 5 min.", "Flow rate: 0.4 ml min−1.", "Gradient 2: HPLC buffers C and D. Linear gradient: 55% B for 1 min; 50–85% B in 14 min; 85–100% B in 0.5 min; 100% B for 3 min; 100–50% B in 1 min; 55% B for 4 min.", "Flow rate: 0.4 ml min−1.", "Gradient 3: HPLC buffers E and F. Linear gradient 10–30% F in 20 min, 30–90% F in 1 min; 90% F for 3 min; 90–10% F in 0.4 min; 10% F for 5 min at a flow rate of 0.4 ml min−1.", "Gradient 4: HPLC buffers E and F. Linear gradient 10–20% F in 20 min, 20–30% F over 10 min; 30% F to 90%F in 1 min; 90% F for 3 min; 90–10% F in 0.4 min; 10% F for 5 min at a flow rate of 100 μl min−1.", "Gradient 5: HPLC buffers A and B. Linear gradient: 45–50% B in 1 min; 50–80% B in 20 min; 85% B to 100% B in 1 min; 100% B for 2 min; 100–45% B in 0.4 min; 45% B for 4 min.", "Flow rate: 0.4 ml min−1.", "Gradient 6: HPLC buffers A and B. Linear gradient: 35–40% B in 1 min; 40–60% B in 10 min; 60–68% B in 5 min, 68% B to 100% B in 1 min; 100% B for 2 min; 100–35% B in 0.4 min; 30% B for 4 min.", "Flow rate: 1.6 ml min−1.", "Gradient 7: HPLC buffers A and B. Linear gradient: 15–60% B in 12 min; 60–100% B in 0.1 min; 100% B for 2 min; 100–15% B in 0.4 min; 15% B for 4 min.", "Flow rate: 0.4 ml min−1.", "Gradient 8: HPLC buffers A and B. Linear gradient 25–30%B in 1 min; 30–45% B 15 min; 45–100% B in 0.1 min; 100% B for 3 min; 100–25% B in 0.4 min; 25% B for 5 min.", "Flow rate: 0.4 ml min−1.", "Gradient 9: HPLC buffers A and B. Linear gradient 15–20% B in 1 min; 20–75% B 10 min; 75–100% B in 0.1 min; 100% B for 3 min; 100–15% B in 0.4 min; 25% B for 5 min.", "Flow rate: 0.4 ml min−1.", "Gradient 10: HPLC buffers E and F. Linear gradient 15–20% F in 1 min; 20–75% F 10 min; 75–100% F in 0.1 min; 100% F for 3 min; 100–15% F in 0.4 min; 25% F for 5 min.", "Flow rate: 0.4 ml min−1.", "Gradient 11: HPLC buffers C and D. Linear gradient 55–90% D in 15 min; 90–100% D in 0.1 min; 100% D for 3 min; 100–55% D in 0.4 min; 55% D for 5 min.", "Flow rate: 0.4 ml min−1.", "Gradient 12: HPLC buffers E and F. Linear gradient 35–40% F in 1 min; 40–90% F 10 min; 90–100% F in 0.1 min; 100% F for 3 min; 100–35% F in 0.4 min; 35% F for 5 min.", "Flow rate: 0.4 ml min−1.", "Gradient 13: HPLC buffers C and D. Linear gradient 55–80% D in 15 min; 85–100% D in 0.1 min; 100% D for 3 min; 100–55% D in 0.4 min; 55% D for 5 min.", "Flow rate: 0.4 ml min−1.", "Following purification, 1 μg of ssRNA in RNase-free water was incubated with 1 U RNase A at 37 °C for 30 min.", "Subsequently, 500 ng, was analysed using LC ESI MS with an Accucore C18 column (2.6 μm superficially porous silica particles 80 Å pore size 150 mm × 2.1 mm ID) and gradient 4.", "Mass spectrometry was performed on a maXis Ultra High Resolution Time of Flight Instrument (Bruker Daltonics) in negative ion mode with a selected mass range of 300–2500 m/z.", "An ion source voltage of −2000 V and N2 source gas temperature of 300 °C at 6.0 L/h was used to maintain a capillary current of 30–50 nA, with nebuliser gas pressure at 0.4 bar.", "A list of theoretical monoisotopic masses of oligoribonucleotides was generated using Mongo Oligo Mass Calculator (http://rna.rega.kuleuven.ac.be/masspec/mongo.htm).", "The output of this software produces a theoretical sequence ladder of oligoribonucleotides for the ssRNA with all possible chemical termini including; 5′-OH, -phosphate, -cyclic phosphate and 3′-OH, -phosphate, -cyclic phosphate.", "To evaluate the performance and effects of pore size of the solid-core particles for the analysis of oligonucleotides, a 2′-deoxythymine ladder (dT 19–24) was analysed by IP RP HPLC with triethylammnonium acetate (TEAA) as the ion pair reagent in conjunction with C18 2.6 μm solid-core particles with 80 Å and 150 Å pore sizes (see Fig. 1A and B).", "The results show that the pore size of the solid-core particles affects the resolution of the dT ladder.", "Typical peak widths of 0.12 min were obtained on the 80 Å compared to 0.07 min on the 150 Å pore size solid-core particles, demonstrating that increased resolution of the oligonucleotides were obtained on the 150 Å pore size solid-core particles at a flow rate of 0.4 ml/min.", "The 80 Å shows restricted diffusion for the oligonucleotides under these conditions, resulting in broader peaks in comparison to the 150 Å pore size solid-core particles.", "These data are consistent with those observed for the analysis of peptides, where increased resolution is observed on the wider pore sized particles [29].", "Further application using the 150 Å pore size solid-core particles was also examined using an alternative ion pair reagent tetrabutylammonium bromide (TBAB) which enables size-dependent separations of the oligonucleotides (see Fig. 1C).", "Similar resolution was obtained to the weak ion pair reagent (TEAA) with the same column dimensions.", "The results of the oligonucleotide separations using the 150 Å pore size solid-core particles demonstrate that rapid, high resolution separation of oligonucleotides can be achieved in a similar way to those obtained using non-porous particles and UPLC applications.", "Further analysis of smaller oligonucleotides (7–10 mers) revealed that improved resolution was obtained on the 80 Å pore sizes in comparison to the 150 Å pore size particles, in contrast to that observed for the larger oligonucleotides (19–24 mers).", "Analysis of the 7–10 mers was performed using the MS-compatible ion pair solvents (20 mM TEAA + 80 mM HFIP) on the 80 Å column is shown in Fig. 2.", "The separation of small oligoribonucleotides is required for RNase mapping experiments, where large RNA biomolecules are digested with an RNAse prior to liquid chromatography, often interfaced with mass spectrometry analysis [33].", "The application of C18 2.6 μm solid-core particles with 80 Å pore sizes was therefore applied to RNase mapping experiments.", "A 500 nt ssRNA generated via in vitro transcription was digested using RNase A and subsequently analysed using IP RP HPLC interfaced with MS (see Fig. 2B).", "Oligoribonucleotides were identified by comparing their observed and theoretical monoisotopic masses after an in silico RNase A digest and are highlighted in Fig. 2B.", "To evaluate the separation of dsDNA using IP RP HPLC in conjunction with superficially porous silica particles, a pUC18HaeIII digest, consisting of a range of dsDNA fragments (80–587 bp), was analysed.", "Chromatograms of the dsDNA fragments on the 80 Å and 150 Å, 2.6 μm superficially porous silica particles is shown in Fig. 3A/B.", "The results clearly show a significant improvement in the resolution of the dsDNA fragments on the 150 Å pore size solid-core particles in comparison to the 80 Å particles, demonstrating that restricted diffusion for larger MW dsDNA fragments leads to broader peaks.", "Furthermore, the restricted diffusion is more significant for the larger MW dsDNA fragments compared to the smaller MW oligonucleotides previously analysed using the 80 Å particles.", "However, the separation efficiency achieved on the 150 Å pore size solid-core particles is not as high as that typically achieved on non-porous 2 mm PS-DVB particles [3,7].", "Therefore, in an approach to further optimise the separation of the larger dsDNA fragments and limit the restricted diffusion observed on both the 80 Å and 150 Å particles, a research column based on superficially porous silica (4.0 mm superficially porous silica particles with 400 Å pore sizes) was evaluated (see Fig. 3C).", "The chromatogram shows a significant enhancement in the separation of the dsDNA, achieving near baseline separation of the 257/267 bp and 434/458 bp fragments.", "This observation is consistent with previous findings that increasing the pore size (400 Å) results in improved separation of large intact proteins compared to the 80 Å and 150 Å particles [29].", "However, it should be noted that, although the separation of the larger dsDNA fragments is improved using the 400 Å pore size particles, analysis of the oligonucleotides (dT 19–24) revealed a loss in resolution compared to the 150 Å/80 Å particles (data not shown).", "The requirement for high throughput analytical tools that can readily separate, purify and analyse ribonucleic acids (RNA) are assuming increasing significance with the recent discoveries of the diverse and important roles RNA plays in biological systems.", "In particular, the ability to separate and analyse large MW RNAs is challenging.", "However, Fig. 3D shows that the large 3569 nt RNA from bacteriophage MS2 elutes as a narrow peak, demonstrating the high resolution separation of RNA, similar to larger dsDNA fragments, by rapid analysis on 4.0 mm superficially porous silica particles of 400 Å pore size.", "Using stationary phases with different pore sizes will alter the available surface area and therefore affect the overall capacity of the column, retention times and possibly the effective gradient [34].", "Figs. 1–3 demonstrate the importance of selecting an appropriate sorbent pore size for the analytes of interest.", "With the emergence of therapeutic oligonucleotides, including siRNA therapeutics, RNA aptamers and antisense oligonucleotides which are all chemically synthesized, the requirement for high throughput, robust approaches to isolate the therapeutic oligonucleotide from impurities such as failure sequences (typically n-1/n-2, also termed short-mers) and long-mers (typically containing deprotecting groups) is an important analytical challenge.", "The analysis of a typical synthetic oligonucleotide is shown in Fig. 4, demonstrating the ability to resolve the full length product (FLP) from closely related impurities using 2.6 μm solid-core particles with 150 Å pore sizes.", "Therapeutic oligonucleotides are often synthesised to incorporate diverse chemical modifications to confer stability in vivo.", "These modifications include 2′-O-methyl (2′-O-Me), O-ethyl (2′-O-ethyl), O-methoxyethyl (2′-MOE), and fluorine (2′-F) substitutions as well as locked nucleic acids (LNA) and phosphorothioate backbone modifications.", "This synthesis strategy can result in even more complex impurities, including diastereoisomers.", "The separation of phosphorothioate containing oligonucleotides presents additional challenges due to the presence of a large number of diastereoisomers that often causes peak broadening and poor resolution of closely related impurities.", "The application of IP RP HPLC for the analysis of fully phosphorothioate containing oligonucleotides has been previously studied using TEA/HFIP (typically 16 mM TEA/400 mM HFIP) in conjunction with porous C18 particles.", "Under these optimised IP conditions, predominantly size-based separations are performed where the peak broadening effect of the different hydrophobicities of phosphorothioate diastereoisomers is eliminated [35–37].", "Further studies using cyclohexyldimethylammonium acetate (CycHDMAA) in conjunction with monolithic, poly(styrene-divinylbenzene)-based capillary columns have also been used to analyse fully phosphorothioate containing oligonucleotides [38].", "The analysis of an oligonucleotide containing a single phosphorothioate, using 150 Å pore size solid-core particles in conjunction with the weak ion pairing reagent TEAA is shown in Fig. 5A. Short alkyl chains such as those in TEAA, allow the stationary phase to partially retain its hydrophobic or reverse phase properties as TEAA only partially covers the stationary phase.", "Therefore, separation is dependent on both size and base composition.", "The results demonstrate the ability to rapidly separate the two diastereoisomers (RP and SP) that are present similar to previous studies utilising alternative stationary phases [39–41].", "However, analysis of fully phosphorothioate containing oligonucleotides under weak ion pair reagent conditions leads to the appearance of a broad peak due to the presence multiple diastereoisomers which are likely to co-elute with various shortmer and longmer impurities.", "To promote the co-elution of the phosphorothioate diastereoisomers and therefore reduce the peak width of fully phosphorothioate containing oligonucleotides, the analysis was performed in the presence of HFIP and alternatively using strong ion pair reagent tetrabutylammonium bromide (TBAB).", "Using TBAB the longer alkyl chains provide complete coverage of the stationary phase and a dynamic anion-exchange is the predominant mechanism, resulting in size based separation with limited sequence effects.", "Analysis under these conditions promotes size dependent separations and minimises the effects of the diastereoisomers, as shown in Fig. 5B and C. Under these conditions the two diastereoisomers of the singly phosphorothioated oligonucleotide co-elute and as expected the peak width of the fully phoshorothioated oligonucleotide is significantly reduced (see Fig. 5D).", "Although utilising the ion pair reagent TBAB and the addition of HFIP minimises the effects of the diastereoisomers in fully phosphorothioate containing oligonucleotides, their analysis still remains challenging as the effects are not fully supressed.", "This can be seen by comparing the peak width of the fully phosphorothioate oligonucleotide (0.17 min) and fully phosphodiester oligonucleotide (0.11 min) of the same sequence analysed on the same gradient slope (see Supplementary Fig. 1).", "In addition, the ability to separate typical impurities generated during the synthesis of a fully phosphorothioated oligonucleotide was also studied.", "A number of common impurities including the oxidised phosphodiester were synthesised to represent common contaminants (see Table 1).", "To evaluate the ability of the superficially porous silica particles to separate the single phosphodiester containing oligonucleotide (5′-end, 3′-end and the middle), double phosphodiester oligonucleotide and n-1 truncated oligonucleotide from the full length fully phosphorothioate oligonucleotide, the analysis was performed using the previously optimised conditions with the strong ion pair reagent TBAB (see Fig. 6).", "The results demonstrate the separation of the full length fully phosphorothioate oligonucleotide from the single/double phosphodiester impurities and the n-1 truncated species.", "The ability to resolve these common impurities on superficially porous silica particles with 150 Å pore size in conjunction with optimised sequence dependent separations and cost effective solvents, including low amounts of HFIP to TEAA and strong ion pair reagents such as TBAB, provides a significant development in the analysis of therapeutic oligonucleotides including fully phosphorothioate oligonucleotides.", "Superficially porous silica particles enable the rapid, high resolution separation of nucleic acids using IP RP HPLC.", "The pore sizes of the C18 superficially porous silica particles significantly effects the resolution of the nucleic acids.", "Optimum separations of small oligonucleotides such as those generated in RNase mapping experiments were obtained with 80 Å pore sizes and can readily be interfaced with mass spectrometry analysis.", "Improved resolution of larger oligonucleotides (>19 mers) was observed with pore sizes of 150 Å.", "The optimum resolution for larger dsDNA/RNA molecules was achieved using superficially porous silica particles with pore sizes of 400 Å.", "Therefore, it is important to select the appropriate pore sizes of the C18 superficially porous silica particles prior to analysis of nucleic acids.", "The analysis of an oligonucleotide containing a single phosphorothioate backbone modification, using 150 Å pore size solid-core particles in conjunction with the weak ion pairing reagent TEAA enabled the separation of the two diastereoisomers (RP and SP) that are present.", "The use of superficially porous silica particles in conjunction with strong ion pair reagents (TBAB) promotes size dependent separations and was shown to minimise the effects of the diastereoisomers.", "In addition, the ability to separate typical impurities generated during the synthesis of a fully phosphorothioated oligonucleotide using superficially porous silica particles (150 Å pore size) was shown in conjunction with optimised sequence dependent separations.", "The results demonstrate the ability to separate the full length fully phosphorothioate oligonucleotide from the single/double phosphodiester impurities and the n-1 truncated species which are often generated in the synthesis of this important class of therapeutic oligonucleotide." ]	[ "Superficially porous silica particles enable high resolution separation of nucleic acids.", "The pore size of the C18 superficially porous silica particles significantly effects resolution.", "Optimum separations of small oligonucleotides obtained with 80 Å pore sizes.", "Optimum resolution of oligonucleotides (>19 mers) was observed with pore sizes of 150 Å.", "Improved resolution of larger dsDNA/RNA molecules was achieved with pore sizes of 400 Å" ]
Farm-raised tiger shrimp, Penaeus monodon, fed commercial feeds with added organic acids showed enhanced nutrient utilization, immune response and resistance to Vibrio harveyi challenge	The threat of disease in the aquaculture industry, coupled with greater restrictions or ban on antibiotic use, has increased interest in evaluating antibiotic alternatives. A promising alternative is organic acids, which have been used for decades in the livestock feed industry as an antimicrobial and growth promoter. However, very limited information exists on their applications to the shrimp aquaculture industry. The aim of the current study was to examine the potential beneficial effects of a novel microencapsulated organic acid blend (OAB) to the production of tiger shrimp, Penaeus monodon, in earthen ponds at a commercial farm when fed commercial feeds without (Diet A) or with (Diet B) organic acid supplementation at 2% OAB throughout the grow-out period. Farm-raised shrimp were randomly sampled and transferred to the laboratory to examine any effects on nutrient utilization, resistance of shrimp to Vibrio harveyi and associated hepatopancreatic histopathology and phenoloxidase (PO) activity. Results showed that after 22. weeks of culture in ponds, shrimp growth was similar between treatments. Lower nitrite-N and nitrate-N concentrations in the pond water indicated potential improved protein utilization from shrimp fed Diet B. This was supported by data from the digestibility trial, demonstrating crude protein, but also dry matter, ash and phosphorous utilization was significantly enhanced (P. <. 0.05). Total viable bacteria and presumptive Vibrio spp. counts were lower at the end of the grow-out period in the pond water of shrimp fed Diet B. Shrimp fed Diet B showed significantly higher survival to V. harveyi challenge, likely due to enhanced PO activity and less hepatopancreatic damage. Total viable bacterial and Vibrio counts in the hepatopancreas of shrimp fed Diet B were significantly lower compared to Diet A. This study provides the first reported data on the use of dietary organic acids in a commercial shrimp farm setting. The enhancement to nutrient utilization may reduce feeding costs and improve water quality while the higher resistance of shrimp to pathogenic bacteria such as Vibrio spp. may provide shrimp farmers with an effective method to mitigate disease outbreaks in the global shrimp aquaculture industry.	[ "AGRI" ]	[ "Antimicrobials", "Organic acids", "Penaeus monodon", "Shrimp disease", "Shrimp feeds" ]	[ "1104" ]	[ "Diseases in the aquaculture industry are a significant limitation to productivity, and shrimp are certainly no exception with substantial losses due to various bacterial, fungal, parasitic and viral diseases (Flegel et al., 2008).", "The most recent disease to hit the global shrimp industry is the acute hepatopancreatic necrosis disease (AHPND) or more commonly known as early mortality syndrome (EMS) where shrimp mortalities can be 100% with certain strains of Vibrio parahaemolyticus being identified as the causative agents (Tran et al., 2013).", "In response, this has led to the abuse and overuse of antibiotics in the aquaculture industry.", "The uncontrolled use of antibiotics can negatively affect the host species, the environment as well as the health of human consumers which has led to greater restrictions or ban of such practices in many countries (Marshall and Levy, 2011).", "Moreover, even in areas where antibiotics are used, this can potentially depress crustacean growth (Bray et al., 2006) as well as lead to the emergence of antibiotic resistant bacteria, including Vibrio harveyi, and therefore reducing subsequent antibiotic efficacy (Karunasagar et al., 1994).", "For these reasons, it is becoming increasingly imperative to identify suitable antibiotic alternatives.", "While various feed additives have been investigated for use in aquaculture feeds, organic acids are receiving increasing attention due to their strong antimicrobial and prophylactic properties against various pathogenic bacteria (da Silva et al., 2013; Defoirdt et al., 2006; Ng and Koh, 2011).", "Organic acids are “Generally Regarded as Safe” compounds often containing one or more carboxyl groups (–COOH) with antimicrobial properties (Defoirdt et al., 2009).", "Some of the most common are those with short chains (C1–C6) including, formic, lactic, propionic and citric acids and their salts.", "Many of these are known to inhibit various Vibrio strains in vitro, but are highly dependent on the type and level (da Silva et al., 2013; Defoirdt et al., 2006; Ng and Koh, 2011) when tested in vivo with various aquatic animal species.", "It is believed that the primary antimicrobial action of organic acids is by altering the cell cytoplasm pH of bacteria and those that are sensitive to such changes are inhibited, thus reducing harmful bacteria within the gastrointestinal tract of the host animal (reviewed by Booth and Stratford, 2003).", "Therefore a reduction to gastrointestinal bacteria, that often includes pathogenic species, may improve growth and disease resistance in the host animal.", "Meanwhile, organic acids can also lower dietary and gastrointestinal pH, which may chelate or solubilize minerals to make these more available for absorption, as well as break up antinutritional compounds (Hossain et al., 2007).", "The inclusion of organic acids in the diets of red drum, Sciaenops ocellatus, has been reported to increase the activity of several digestive enzymes (Castillo et al., 2014).", "Generally, reports on organic acids improving growth performance and nutrient utilization in aquacultured animals have been positive, but appear to be dependent on the type of organic acids used and host species (reviewed by Ng and Koh, 2011).", "For example, Arctic charr, Salvelinus alpinus, fed sodium-propionate supplemented diets suppressed growth, while dietary sodium-lactate acted as a growth promoter (Ringø, 1991).", "Meanwhile, among different organic acid salts of acetate, propionate and butyrate in the diets of the white Pacific shrimp, Litopenaeus vannamei, sodium-propionate significantly increased apparent gross energy and phosphorus (P) digestibility, while sodium-acetate tended to lower P availability (da Silva et al., 2013).", "In a subsequent study, Da Silva et al. (in press) reported that white shrimp fed dietary sodium butyrate or proprionate showed a modified intestinal microbiota and improved growth performance.", "We recently showed in a series of laboratory-based feeding trials that a novel dietary microencapsulated organic acid blend (OAB) was able to enhance growth, nutrient utilization, immune response, hepatopancreatic integrity and resistance against V. harveyi in L. vannamei (Romano et al., 2015).", "Due to these encouraging laboratory results, field evaluation of the OAB product was deemed as an essential next step.", "To the best of our knowledge, no information currently exists regarding the application of dietary organic acids in commercial shrimp feeds in a commercial farm.", "To investigate the practical applications of organic acids, a unique hybrid experimental design consisting of related experiments in commercial earthen shrimp ponds and in controlled replicated laboratory-based tanks was conducted.", "Tiger shrimp, Penaeus monodon, were cultured in commercial ponds and fed diets with or without supplementations of a novel microencapsulated organic acid blend.", "Over 22 weeks, shrimp growth and various water quality parameters of the ponds were measured.", "At various intervals during the grow-out period, these farm-raised shrimp were transferred to the laboratory and two separate experiments were performed to determine nutrient utilization efficiencies and subsequent resistance to V. harveyi.", "With the incorporation of more controlled laboratory-based experiments using farm-raised shrimp, this helped provide a broader complement of data to the pond trial and a unique opportunity to assess the potential of dietary organic acids to the shrimp aquaculture industry.", "Two earthen ponds in a semi-intensive commercial tiger shrimp (P. monodon) farm located at Penang, Malaysia (5° 23′ 44″ N, 100° 11′ 46″ E) were used in the present study.", "The ponds were about 0.6 ha in size with a depth of approximately 1.2–1.5 m. Each pond was equipped with a properly screened water inlet connected to a reservoir, and water exchanges were performed via a central drain.", "Up to nine paddle-wheel aerators (1 hp) were provided in each pond to maintain strong water currents and continuous aeration.", "Prior to stocking, the ponds were completely drained and pond bottom cleaned through pressure washing to flush out wastes, debris and undesirable aquatic species followed by manually removing any leftover waste in the ponds.", "The ponds were left to dry in the sun for 7–14 days or until the soil cracked.", "After drying, calcium carbonate (CaCO3) was spread uniformly on the pond bottom.", "The ponds were then filled with filtered seawater and tea seed powder (20–30 mg L− 1) was added to eliminate any foreign species in the pond.", "The filled pond was left for seven days before the shrimp post-larvae were stocked.", "Tiger shrimp, P. monodon (specific pathogen free; SPF) post-larvae (PL-12) were purchased from a hatchery at Penang, Malaysia.", "Shrimp post-larvae were transported in plastic bags and upon arrival at the farm, slowly acclimated to the pond conditions for 1 h.", "After acclimation, the PL were released directly into the pond at a density of 160,000 per pond giving a stocking density of 26 PL m− 2.", "This was estimated by individually counting the PL from three randomly selected bags.", "Duplicate groups of 30 PL were individually measured and batch weighed for initial total length and average body weight, respectively.", "The proprietary OAB (Orgacids™-AQUA) was developed and produced in collaboration with Sunzen Feedtech Pte Ltd (Malaysia) and consisted of a blend of four different types of organic acids (formic, lactic, malic and citric acids) microencapsulated within a specialized lipid coating by high speed centrifugal spray chilling.", "The resultant spherical microcapsules were less than 250 μm in size and came in the form of a free-flowing white powder which can be easily incorporated into commercial shrimp feeds.", "A series of five commercial tiger shrimp feed types (from crumble, 0.5–1.1 mm to pellet sizes of Ø1.8 × 2.0–3.5 mm) containing no added OAB (control, Diet A) or 2% OAB (Diet B), respectively, were produced and purchased from Gold Coin Specialities Ltd. (Malaysia) for use at various stages of the grow-out cycle (Table 1).", "The dietary OAB level of 2% was selected based on the optimal results obtained with this dietary inclusion level in laboratory-based feeding trials (Romano et al., 2015).", "All shrimp feed bags were clearly marked as A or B and neither the farmers nor the field researchers were informed of the dietary treatments until all the data were collected and processed to prevent any potential personal biases.", "The feeds were stored in a well-ventilated shed and used within three months from manufacturing date.", "After stocking, shrimp were fed either Diet A or B in one of the two ponds.", "All feeds were manually dispersed to both groups five times daily at 0700, 1100, 1500, 1900 and 2230 h throughout the culture cycle.", "The feeding rate was adjusted daily through visual observations of the feeding trays 2 h after each feeding.", "The amount of feeds used in each pond was recorded throughout the grow-out period.", "After 40 days of stocking, a 10–15% water exchange of the total pond volume was conducted once every three days and CaCO3 was added when required.", "Every fortnight, water quality parameters, including pH, temperature, conductivity, dissolved oxygen, salinity and water transparency of both ponds were measured on site at 1300 h using a portable multi-probe CyberScan PCD 650 water quality meter (EuTech Instruments) at three different locations (~ 20 cm depth from the water surface).", "The salinity was determined using a hand held refractometer, while water transparency was measured using a Secchi disk.", "Meanwhile, the surface water samples from three locations in each pond were collected in 500 mL clean plastic bottles, wrapped with heavy-duty aluminum, kept on ice and transported to Universiti Sains Malaysia for further analysis.", "Total suspended solids (TSS), nitrite-N, ammonium-N and chlorophyll a concentrations of pond water samples were measured following the Standard Methods for the Examination of Water and Wastewater (APHA, 1998), while orthophosphate of the filtered water samples was measured by ascorbic acid method (Boyd and Tucker, 1980).", "Nitrate-N was measured using commercial test kits (JBL GmbH & Co.", "KG, Neuhofen, Germany).", "After six weeks of cultivation, shrimp from both ponds were randomly sampled fortnightly early in the morning at three to four different locations by using a feeding tray (week 6 to week 10), or a cast net (week 12 to week 22) to monitor growth performance.", "The sampled shrimp were then quickly weighed on a digital scale and the shrimp put back into their respective pond.", "Every four weeks, water samples from three locations of each earthen pond were collected in sterile universal bottles at 1100 h and serially diluted (10 folds).", "Aliquots (0.1 mL) were spread plated onto thiosulfate citrate bile sucrose (TCBS) agar (Merck, Darmstadt, Germany) or tryptic soy agar (TSA, Merck, Darmstadt, Germany) to determine presumptive Vibrio spp. and total cultivable bacterial counts, respectively, after being incubated at room temperature for 24 h.", "It was the normal practice for this commercial farm to partially harvest the shrimp for sale to local retail markets and restaurants.", "In order to ensure similar biomass throughout the grow-out period, partial harvest was done alternately from the two ponds.", "This began by week 11, only from shrimp with an average body weight of 20–25 g, and at each partial harvest, their total harvest weights and average body weights were recorded.", "V. harveyi, originally isolated from diseased Talorchestia sp. (ATCC 14126), was used in the disease challenge trial.", "The strain was maintained as a frozen stock in tryptic soy broth with 1.5% (w/v) sodium chloride (NaCl) (TSB +) containing 20% (v/v) glycerol (Qrec, New Zealand) and stored at − 80 °C until needed.", "For the challenge trial, frozen stock cultures were resuscitated in 50 mL of TSB + within a 250 mL conical flask and incubated at 30 °C with orbital shaking at 200 rpm for 18–24 h.", "The bacterial broth was then streak plated onto tryptic soy agar with 1.5% NaCl (TSA +) plates for 18 h at 30 °C and this was followed by inoculating two loops full of pure bacterial colonies in 50 mL of TSB + for 18 h at 30 °C and 200 rpm to achieve an optical density of approximately 0.6 at 545 nm (~ 5 × 108 CFU mL− 1).", "A bacterial pellet from 10 mL of bacterial broth was then harvested by centrifugation at 10,000 rpm for 10 min, followed by two washes of sterile normal saline solution (0.85% NaCl), and re-suspended in 10 mL of sterile normal saline solution.", "This bacterial suspension was used as the inoculum for the challenge trial.", "The bacterial concentration was determined by standard plate count method using TSA plates.", "After 16 weeks of cultivation, adult tiger shrimp (approximately 19–21 g) were collected from both ponds and acclimatized for 15 days in a static system consisting of 10 glass aquaria [68.5 (L) × 29.0 (W) × 45.1 (H) cm] housed within a temperature controlled room.", "Each aquarium was filled with 60 L of sterilized artificial seawater (InstantOcean®) at a salinity of 20 ± 1‰ and continuous gentle aeration was provided via an individual air-stone.", "Each treatment was performed in five replicates with each aquarium containing six shrimp.", "Over the acclimatization period, all shrimp were hand-fed three times daily at 2.5% body weight with their respective diets at 1000, 1400 and 1800 h. Each aquarium received two daily 70% water exchanges (morning and evening) and the pH, dissolved oxygen, temperature, and ammonia-N levels were 7.30 ± 0.05, 5.78 ± 0.06 mg L− 1, 27.9 ± 0.1 °C and 1.03 ± 0.07 mg L− 1, respectively.", "The disease challenge test was carried out by intramuscular injection, with a LD50 of 20 μl (4.94 × 105 CFU g− 1 shrimp) (Liu et al., 1996) of live bacterial suspension (4.95 × 108 CFU mL− 1), into the shrimp between the third and fourth abdominal segments.", "Mortalities were recorded for eight days and any dead shrimp were removed.", "During the challenge trial, the shrimp were fed three times daily, received a 50% water exchange in the morning and evening, respectively and the ammonia-N levels were at or below 1.07 ± 0.03 mg L− 1.", "Phenoloxidase activity (PO) was measured from shrimp after the bacterial challenge test according to Liu and Chen (2004) with slight modifications.", "To obtain the hemolymph, syringes were coated with anticoagulant (30 mM trisodium citrate, 0.34 M NaCl, pH 7.55 and glucose was added until 30‰ was reached), inserted in the ventral sinus and then 40 μl hemolymph was added to 360 μl of the anticoagulant.", "This was then centrifuged at 700 × g for 20 min., the supernatant was removed and the pellet was gently re-suspended with 200 μl cacodylate-citrate buffer, centrifuged a second time and re-suspended with 200 μl cacodylate buffer (0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.01 M calcium chloride, 0.26 M magnesium chloride, pH 7.0).", "Each aliquot (100 μl) was incubated with 50 μl trypsin (1 mg mL− 1) for 30 min at room temperature followed by adding 50 μl of l-DOPA (1 mg mL− 1).", "Five minutes later, 800 μl cacodylate buffer was added and then measured at an optical density of 490 nm.", "A control was used to correct for background interference, which replaced 50 μl trypsin for cacodylate buffer.", "PO activity (unit of optical density at 490 nm) was expressed as dopachrome formation in 40 μl of hemolymph.", "After the hemolymph was collected, the hepatopancreas was dissected for histological examination, immersion fixed in Bouin's solution for 18 h and then transferred to 70% (v/v) ethanol until processing.", "Sections (5–7 μm) were made using a rotary microtome, stained with hematoxylin and eosin and examined under a light microscope (× 20 and × 40 magnification).", "For the digestibility trial, the same commercial diets used in the pond trial were ground into powder and re-pelleted to include 0.5% chromic oxide (Cr2O3) as an indigestible marker.", "The mixing and diet preparation were similar to Ng et al. (2009).", "After 20 weeks of culture, shrimp (30–32 g) were collected from both ponds and transferred into eight 500 L fiberglass tanks (cone shaped bottom) in a closed recirculation aquaculture system (RAS).", "The RAS was fitted with a pump, aerators, sand filters, biofilter, foam fractionators and sedimentation tank housed in a hatchery under natural photoperiod as previously described (Romano et al., 2015).", "Each dietary treatment was performed in four replicates with each tank containing nine shrimp.", "All shrimp were hand-fed four times daily at 2.5% body weight with their respective diets at 0900, 1200, 1500 and 1800 h.", "After a 5 day acclimatization period, the tanks were siphoned for any feces or uneaten food prior to first feeding.", "One hour after each feeding, freshly expelled shrimp feces were carefully siphoned and collected on a fine mesh net at intervals of 30 min until the next feeding.", "Feces were rinsed with freshwater to remove any residual seawater and only intact fecal strands were collected using tweezers and kept at − 20 °C until analysis.", "Daily fecal collections were pooled according to each tank, oven dried and finely ground prior to analyzing their chemical composition and Cr2O3 concentration.", "Throughout the digestibility trial, the pH, salinity, dissolved oxygen, temperature, and ammonia-N levels were measured twice weekly and maintained at 7.25 ± 0.04, 25 ± 1‰, 6.54 ± 0.08 mg L− 1, 29.9 ± 0.21 °C and 0.18 ± 0.05 mg L− 1, respectively.", "The water flow rate was fixed at about 1.92 ± 0.05 L min− 1, and the digestibility trial was conducted for four weeks.", "At the end of the digestibility trial, five shrimp were removed from each tank and sterilized with 70% ethanol to avoid cross contamination.", "The hepatopancreas and intestine were then aseptically dissected, weighed separately and then homogenized in a sterile mortar.", "The homogenates were transferred into sterilized 15 mL polypropylene tubes and diluted to 10 mL with sterile normal saline solution.", "The supernatants were serially diluted with 0.1 mL of the aliquots spread plated in duplicates onto TSA and TCBS agar plates and then incubated at room temperature for 24 to 48 h. Only plates that had between 30–300 colonies were used to determine the number of total cultivable bacteria and presumptive Vibrio spp. counts and the results are expressed as log CFU g− 1.", "The chromic oxide (Cr2O3) content of the feces and feeds was determined according to the wet-acid digestion method described by Furukawa and Tsukahara (1966).", "The phosphorus concentrations of both the feces and feeds were determined by the molybdovanadate method and measured using a spectrophotometer (GENESYS 20, Thermo Electron Corp., Madison, WI, USA) at 450 nm (AOAC, 1997).", "The proximate composition (moisture, crude protein, crude lipid and ash) of the feeds and feces were determined using standard methods in AOAC (1997).", "To measure dietary pH, 5 g of each diet (finely ground) was mixed in 50 mL of distilled water for 5 min and then the water pH was measured using a digital pH meter after prior calibration with precision buffers.", "No statistical analysis of the shrimp growth and water quality data collected from the field trial was performed as only one pond per treatment (no replication) was used.", "However, several measurements were made in each pond for the same parameter to determine the mean and range of values which are presented as mean ± standard deviation.", "For the related laboratory-based trials of the farmed raised shrimp, mortalities in the bacterial challenge test, digestibility data, PO activity and bacterial counts were all subjected to a one-way analysis of variance ANOVA after prior confirmation of variance using the SPSS statistical software (SPSS version 14.0 for Windows).", "Differences between means were determined by independent samples t-test and effects with a probability of P < 0.05 were considered statistically significant.", "The proximate composition of the two commercially-produced shrimp feeds are presented in Table 1 and showed a similar composition.", "The pH was also generally similar, except for the feed type 904 for Diet B which was supplemented with organic acids which showed a pH of 5.88 compared to 6.19 for Diet A which was not added with OAB.", "The range and mean values of various water quality parameters of Pond A (shrimp fed Diet A) and Pond B (shrimp fed Diet B) are shown in Table 2.", "Over the course of 22 weeks, many of the water quality measurements were similar or followed no obvious pattern between Pond A and B. However, there were several notable differences, which included pH, chlorophyll a, and nitrogenous wastes, dissolved oxygen, and TSS.", "In the case of the pond water pH, which was initially similar at 8.08 and 8.15 in Pond A and B, respectively, by the second week the pH of Pond B became higher than that of Pond A and this trend continued until the end of the trial.", "Similarly, dissolved oxygen and TSS were initially similar, but by week 6 generally became consistently higher in the water of Pond B and Pond A, respectively.", "Chlorophyll a was also initially similar between the ponds, however by week 14 and 16 there was a spike, which led to the farmers' adding algaecide to control these levels.", "From week 18 to 22, the chlorophyll a levels in Pond B became over two-fold lower than in Pond A. Meanwhile, the mean ammonia-N levels ranged from over two and 1500 folds higher in Pond A between weeks 8 and 14, but from week 16 onwards the ammonia-N levels in Pond B ranged between 4 and 19 folds higher than in Pond A. Nevertheless, the overall mean ammonia-N level in Pond B was higher than in Pond A since the discrepancy between the ponds in the final weeks were numerically higher (but not proportionally higher), than in the earlier weeks.", "On the other hand, both nitrite-N and nitrate-N were consistently higher in Pond A than in Pond B, leading to an overall mean difference of over six and five-fold, respectively (Table 2).", "The total bacterial count at weeks 8, 12 and 20 was significantly higher in the water of Pond A than of Pond B (Fig. 1), while presumptive Vibrio spp. counts in the water of Pond A was significantly higher at weeks 12 and 20 than in Pond B.", "The only time bacteria or Vibrio spp. counts were higher in Pond B was at week 4.", "The initial size of the shrimp PL when stocked was approximately 0.012 g in both ponds.", "When the shrimp was first weighed at week 6, the mean weight was slightly higher for shrimp sampled from Pond A than Pond B.", "This trend continued from week 8 to 18, however, by the final weeks of the grow-out period, the mean weight of tiger shrimp between Ponds A and B became similar at 33.36 and 31.22 g, respectively (Fig. 2).", "Although the growth was similar, according to the shrimp farmer, the overall production costs and net profitability was slightly better in Pond B than Pond A (per.", "comm.).", "This was due mainly to the fact that less feeds were used in Pond B. Due to the partial harvesting of shrimp practiced by the farmer, it was unfortunate that we were not able to obtain accurate total shrimp harvest data.", "The apparent digestibility coefficient (%) of dry mater, crude protein, crude lipid, ash and phosphorus of diets without (Diet A) or with (Diet B) organic acid blend (OAB) supplementations is shown in Fig. 5.", "With the exception of crude lipid, the apparent digestibility of all nutrients was significantly higher for Diet B compared with Diet A when fed to tiger shrimp.", "The CFU counts of total cultivable bacteria in the hepatopancreas and gut were significantly lower in shrimp fed Diet B (Fig. 6).", "Similarly, presumptive Vibrio spp. counts in the hepatopancreas were significantly lower in shrimp fed Diet B. However, while the CFU counts of presumptive Vibrio spp. in the intestine of shrimp fed Diet A or Diet B were not significantly different, this was numerically higher for shrimp fed Diet A (Fig. 6).", "In the commercial farm feeding trial, after the shrimp were fed diets with (Diet B) or without (Diet A) organic acid supplementation from post-larvae to marketable size, there was no marked effect on the final shrimp size.", "Due to budget and logistical constraints, such as the supply of P. monodon PL from the same source, only one replicate pond per diet was used in the present study.", "Therefore, the results should be interpreted in consideration of the various other factors that can influence shrimp growth in a pond under commercial farming conditions.", "Under controlled laboratory conditions, we previously showed that a 2% dietary OAB inclusion in a soybean meal-based diet was able to significantly enhance the growth performance of white shrimp (Romano et al., 2015).", "When proprionate or butyrate salts were added at 0.5 to 2% in the diets of white shrimp, significant improvement in growth and feeding efficiencies was reported under laboratory conditions by da Silva et al. (in press).", "Over the grow-out period of 22 weeks, Pond B often had the same or better water quality than Pond A. For example, total viable bacterial and Vibrio spp. counts in the water tended to be lower in Pond B, than Pond A, which may have implications for controlling bacterial diseases, including vibriosis.", "The water in Pond B often had higher pH than that in Pond A despite Diet B's having a slightly lower dietary pH due to the presence of organic acids, especially in the last grow-out feed type which was the main feed used, in terms of quantity.", "This appears to indicate that the microencapsulation of the organic acid blend was effective in reducing the leaching of highly soluble dietary organic acids thereby ensuring that most of it got ingested by the shrimp.", "The use of coated organic acids is likely to be particularly important for shrimp that masticate feed, which increases nutrient leaching, as opposed to fish that ingest feed whole.", "One of the most limiting factors in a shrimp farm, besides dissolved oxygen, is nitrogenous waste since this may reduce productivity as well as harm the surrounding environment (Romano and Zeng, 2013).", "While it was found that ammonia-N was higher in Pond B than Pond A which was due to the addition of an algaecide (zeolite) by the farmers in response to a microalgae level spike at week 16, both nitrite-N and nitrate-N levels were consistently lower in Pond B than Pond A. Taking into consideration this finding, along with ammonia-N possibly being influenced by microalgae levels, may indicate that dietary protein was better utilized by shrimp fed Diet B which would lead to less nitrogenous wastes being excreted.", "Indeed, this was later confirmed in the digestibility trial, showing that dietary protein was significantly better utilized in Diet B than Diet A by the farmed shrimp.", "Further studies using more replicate ponds are needed to better understand the potential benefits of dietary organic acid supplementation in commercial shrimp feeds as it relates to growth performance, feed utilization and water quality management.", "In the present commercial pond study, where replication and standardization were difficult, the pond data obtained should be considered in the light of the variability often present in ponds.", "Similar to the results obtained from the replicated laboratory-based part of the present study, it has been previously reported that organic acids can improve protein, dry matter, and/or P digestibility in various other aquatic animals including, Indian major carp, Labeo rohita (Baruah et al., 2007), red sea bream, Pagrus major (Hossain et al., 2007; Sarker et al., 2007), red hybrid tilapia (Koh et al., in press; Ng et al., 2009), beluga sturgeon, Huso huso (Khajepour and Hosseini, 2012), rainbow trout, Oncorhynchus mykiss (Hernández et al., 2012; Pandey and Satoh, 2008), Atlantic salmon, Salmo salar (Lückstädt, 2008), yellowtail, Seriola quinqueradiata (Sarker et al., 2012) and L. vannamei (da Silva et al., 2013).", "However, this can be highly dependent on the organic acid type as well as the aquatic animal species.", "For example, da Silva et al. (2013) showed that propionate was more effective at improving nutrient utilization than acetate or butyrate when incorporated in feeds of L. vannamei.", "Meanwhile, Gao et al. (2011) found that formate or butyrate did not improve P availability to rainbow trout.", "It is believed that an improvement to nutrient availability by dietary organic acids is partly related to reducing gastrointestinal or dietary pH, which may then chelate minerals (thus making them more easily absorbed) or in the case of soybean meal-based diets, to dephosphorylate phytate (thus making P more easily absorbed) (Hossain et al., 2007).", "More recently, Castillo et al. (2014) reported that the inclusion of organic acids in the diets of red drum, S. ocellatus, was able to increase the activity of several digestive enzymes.", "While the applications of organic acids in aquaculture have mostly focused on potential improvements to growth and/or nutrient utilization in fish, their prophylactic efficacy against pathogenic bacteria has been receiving increasing attention recently (Defoirdt et al., 2006; Koh et al., in press; Ng et al., 2009; Park et al., 2011; Romano et al., 2015).", "For example, when formic, acetic, propionic, butyric or valeric acid were added in equimolar concentrations to the culture water, the resistance of Artemia franciscana to the pathogenic bacterium Vibrio campbelli was significantly improved (Defoirdt et al., 2006).", "However, as noted by Defoirdt et al. (2006), on large scale commercial farm operations, the additions of organic acids to the culture water will likely become cost-prohibitive.", "More recently, the prophylactic effectiveness of dietary organic acids in fish has been successfully shown (Koh et al., in press; Ng et al., 2009; Park et al., 2011).", "Ng et al. (2009) demonstrated that dietary OAB or potassium diformate led to improved resistance of red hybrid tilapia to Streptococcus agalactiae and, similarly, Park et al. (2011) showed that the resistance of olive flounder, Paralichthys olivaceus, to Edwardsiella tarda was enhanced when fed OAB supplemented diets.", "Ng et al. (2009) suggested that this was likely due to the antimicrobial activity of the OAB substantially reducing the CFU counts within the intestine post-challenge, particularly since S. agalactiae was not detected in surviving tilapia.", "Later, Koh et al. (in press) reported that dietary OAB supplementation was as effective as conventional antibiotics such as oxytetracycline in the prevention of streptococcis in tilapia.", "In the present study with tiger shrimp, there was a clear correlation between the prophylactic effectiveness of Diet B with protecting the hepatopancreas from bacterial-induced damage as well as preventing PO activity reductions.", "There were significantly lower total cultivable bacteria and presumptive Vibrio spp. counts within both the hepatopancreas and intestine of shrimp fed Diet B in the present study.", "In crustaceans, the hepatopancreas is known to be targeted by V. harveyi (Lavilla Pitogo et al., 1998) as well as inhibiting/altering PO activity (Amparyup et al., 2009; Huang et al., 2013), which are believed to be the main contributors to Vibrio spp. induced mortalities.", "In the present study, it was demonstrated that shrimp fed Diet B had significantly higher survival to V. harveyi challenge than those fed Diet A.", "It is likely that the significantly higher PO activity and less hepatopancreatic histopathological changes, including hemocyte infiltrations, tubule damage/rupture, necrosis and higher R-cell prevalence, which is responsible for lipid storage (Vogt et al., 1985), for shrimp fed Diet B than those fed Diet A led to this result.", "A similar observation was reported for Pacific white shrimp in our earlier disease challenge trials (Romano et al., 2015).", "Considering that Vibrio spp. can lead to significant mortalities, particularly in the hatchery stage or for more advanced post-larvae during times of stress (Karunasagar et al., 1994; Lavilla Pitogo et al., 1998; Sung et al., 2001), these findings may have important implications to controlling the spread of bacterial disease in the shrimp aquaculture industry.", "Moreover, AHPND/EMS which can lead to mass mortalities during the early stages of shrimp grow-out, has been attributed to specific strains of a Vibrio bacterium, V. parahaemolyticus (Tran et al., 2013).", "While the resistance to this pathogen was not tested, it is worth noting that the antimicrobial effectiveness of a blend of organic acids may provide a broader spectrum of antimicrobial protection than the sole use of one organic acid type (Chaveerach et al., 2002; Thompson and Hinton, 1997).", "It is therefore conceivable that the antimicrobial actions of the tested OAB extends to other pathogenic bacteria, such as V. parahaemolyticus, and certainly points to interesting research directions.", "In conclusion, several important benefits were demonstrated in both the pond and laboratory trials.", "The lower bacterial count as well as the generally lower nitrogenous waste in the water of Pond B indicate that organic acids can play a role in expanding the aquaculture industry more sustainably.", "This finding may be due, in part, to improved protein utilization, as demonstrated in the digestibility trial.", "Meanwhile, the improved protein, dry matter, ash and P utilization may have implications for reducing feeding costs as well as mitigating excessive P discharges to the surrounding environment that cause eutrophication.", "Whether the improved feed utilization efficiencies will translate into better FCR and growth in a commercial farm setting remains to be further investigated.", "Finally, the significantly higher resistance of shrimp fed Diet B to V. harveyi challenge demonstrates the effective prophylactic properties of the tested OAB.", "It is anticipated that should shrimp farmers encounter a disease outbreak such as vibriosis, the role of the tested OAB may mitigate productivity losses.", "This is based on significantly better results obtained from the laboratory-based disease challenge tests and further confirmed by the healthier hepatopancreas observed in histological sections.", "Considering that organic acids pose no health risks to the environment, host animal or human consumers, the use of this functional feed additive may help reduce the reliance on antibiotics in the aquaculture industry.", "The shrimp fed Diet A had significantly higher mortalities (50%) than those fed Diet B (30%) after 8 days of V. harveyi challenge (Fig. 3).", "Meanwhile, after 8 days of V. harveyi challenge, shrimps fed Diet B had significantly higher PO activity of 0.125 ± 0.008 units compared to 0.088 ± 0.007 units from shrimp fed Diet A.", "Furthermore, shrimp fed Diet A appeared to have more histopathological damage to the hepatopancreas than shrimp fed Diet B. Damage included tubule degeneration and necrosis, sometimes leading to rupture, hemocyte infiltrations and reduced R-cell and B-cell prevalence (Fig. 4)." ]	[ "Tiger shrimp were fed commercial feeds without or with 2% organic acid blend (OAB).", "Shrimp fed with 2% OAB showed higher protein, dry matter and phosphorus digestibility.", "Shrimp fed organic acid diets showed higher survival after Vibrio challenge.", "Higher shrimp survival may be due to enhanced immune response and lower bacterial infections.", "Organic acids imparted hepatopancreatic protective properties against vibriosis." ]
Tensions between policy and practice: A qualitative analysis of decisions regarding compulsory admission to psychiatric hospital	The use of detention for psychiatric treatment is widespread and sometimes necessary. International human rights law requires a legal framework to safeguard the rights to liberty and personal integrity by preventing arbitrary detention. However, research suggests that extra-legal factors may influence decisions to detain. This article presents observational and interview data to describe how decisions to detain are made in practice in one jurisdiction (England and Wales) where a tension between policy and practice has been described. The analysis shows that practitioners mould the law into 'practical criteria' that appear to form a set of operational criteria for identifying cases to which the principle of soft paternalism may be applied. Most practitioners also appear willing, albeit often reluctantly, to depart from their usual reliance on the principle of soft paternalism and authorise detention of people with the capacity to refuse treatment, in order to prevent serious harm. We propose a potential resolution for the tension between policy and practice: two separate legal frameworks to authorise detention, one with a suitable test of capacity, used to enact soft paternalism, and the other to provide legal justification for detention for psychiatric treatment of the small number of people who retain decision-making capacity but nonetheless choose to place others at risk by refusing treatment. This separation of detention powers into two systems, according to the principle that justifies the use of detention would be intellectually coherent, consistent with human rights instruments and, being consistent with the apparent moral sentiments of practitioners, less prone to idiosyncratic interpretations in practice.	[ "MEDI", "SOCI" ]	[ "Autonomy", "Human rights", "Involuntary treatment", "Mental health legislation", "Paternalism" ]	[ "2734", "2738", "3308" ]	[ "The use of compulsory hospital admission for psychiatric assessment and/or treatment is a relatively common practice in many countries (Riecher-Rossler & Rossler, 2007).", "However, there remains little consensus regarding the circumstances under which it is morally justifiable to use such compulsion, since it deprives the person of their liberty and the legal criteria authorising compulsory admission vary considerably between different jurisdictions (Appelbaum, 1997; Fistein, Holland, Clare, & Gunn, 2009).", "International human rights law requires a legal framework to safeguard the rights to liberty and personal integrity of people affected by mental ill-health by preventing arbitrary detention (United Nations, 1991; World Health Organization, 2003).", "Nonetheless, legal scholars have repeatedly questioned the effectiveness of mental health legislation as a means of protecting the human rights of people receiving psychiatric treatment (Appelbaum, 1997; Gostin, 2008).", "Empirical research also raises questions regarding the effectiveness of much of this legislation as a safeguard for human rights; for example, rates of detention are not necessarily lower in jurisdictions with stringent legal criteria constraining the use of compulsory admission, nor do they necessarily decrease when a jurisdiction enacts new law with stricter criteria (Zinkler & Priebe, 2002; Salize & Dressing, 2004).", "The reasons for this gap between ‘policy’ and ‘practice’ are not fully understood.", "A body of research based upon clinicians' accounts of their decision-making processes suggests that a complex constellation of factors may influence the decision to detain (Bagby, Thompson, Dickens, & Nohara, 1991; Engleman, Jobes, Berman, & Langbein, 1992; Kullgren, Jacobsson, Lynoe, Kohn, & Levav, 1996; Hoge et al., 1997; Sattar, Pinals, Din, & Appelbaum, 2006).", "The role of individual differences in the way risk is assessed by clinicians (Bartlett, 2010) and the role of ‘gut instinct’ based upon professional experience (Glover-Thomas, 2011) have also been highlighted as factors affecting day-to-day mental health decision making.", "Psychiatrists' accounts of the way in which they learn to make these decisions, through observation of the practice of colleagues, and normally without the benefit of formal training in legal principles, has been cited as an explanation of the discrepancy between policy and practice (Wand & Wand, 2013).", "However, there is limited recent observational research describing the processes by which actual decisions to admit are made (Holstein, 1988; Quirk, Lelliot, Audini, & Buston, 2000).", "The aim of this study was to describe the ways in which decisions to detain are made in one jurisdiction (England and Wales) where a tension between policy and practice has been described.", "We sought to understand the reasons behind day-to-day mental health decision making, to describe the principles on which actual decisions were based, and to analyse how and why they might differ from the legal framework that defines the circumstances under which lawful detention may take place.", "In England and Wales, the circumstances under which someone may lawfully be detained in hospital for psychiatric assessment or treatment are defined in the Mental Health Act 1983 as amended 2007 (MHA).", "Most compulsory psychiatric admissions are authorised on the grounds given in Section 2 or 3 of the MHA.", "Two medical practitioners (one of whom has particular expertise in the diagnosis or management of mental disorders) and a specially trained Approved Mental Health Professional (AMHP), who must have a non-medical professional qualification (often, but not necessarily, social work), must agree that the legal criteria for compulsory admission apply.", "Section 2 authorises detention in hospital for a period of up to 28 days, for the purpose of assessment.", "The criteria are that the patient", "is suffering from mental disorder of a nature or degree which warrants the detention of the patient in a hospital for assessment (or for assessment followed by medical treatment) for at least a limited period; and", "he ought to be so detained in the interests of his own health or safety or with a view to the protection of other persons.", "Section 3 authorises detention in hospital for a period of up to six months and can be renewed.", "The criteria are that the patient", "is suffering from mental disorder of a nature or degree which makes it appropriate for him to receive medical treatment in a hospital; and", "it is necessary for the health or safety of the patient or for the protection of other persons that he should receive such treatment and it cannot be provided unless he is detained under this section; and", "appropriate medical treatment is available for him.", "The 2007 MHA amendments came into practice in November 2008 and effectively relaxed the criteria for compulsory admission (Glover-Thomas, 2011).", "These changes were the result of a decade-long debate and were opposed by key stakeholder groups who expressed concerns that the amended Act weakened safeguards for the rights to liberty and self-determination of people at risk of detention (Mental Health Alliance, 2007).", "In contrast to the mental health legislation of many economically developed countries, there is no requirement to establish that the patient poses a risk to the safety of themselves or others, or that they lack the capacity to make a decision to consent to treatment.", "Two years before the amendment of the MHA, the parliament had enacted another new piece of legislation, the Mental Capacity Act 2005 (MCA), which sets out criteria for the provision of care and treatment (for physical or mental ill-health) deemed necessary in the best interests of people who are unable to give consent, as a result of impairment or dysfunction of mind or brain.", "In April 2009, additional safeguards concerning in-patient treatment and residential care for people who lack the capacity to give or withhold consent, the MCA Deprivation of Liberty Safeguards (MCA-DoLS) came into force to ensure compliance with Article 5 of the European Convention on Human Rights, as interpreted through a body of case law (Winterwerp v the Netherlands [1979] ECHR, Litwa v Poland [2000] ECHR, HL v UK [2005] ECHR).", "The deprivation of liberty is said to occur in circumstances where a person is under continuous control and supervision, is not free to leave, and lacks capacity to consent to these arrangements (P v Cheshire West and Chester Council and another and P and Q v Surrey County Council [2014] WLR 2).", "A deprivation of liberty is lawful only if it represents", "a proportionate response to the likelihood of [the patient] suffering harm and the seriousness of that harm", "and if the person authorizing that restriction", "reasonably believes that it is necessary … in order to prevent harm to [the patient]", "Detention under a MCA DoLS authorisation may be considered less stigmatising, as unlike the MHA there is no connotation with detention for public protection.", "However, access to independent review and appeal against MCA DoLS authorisation is less straightforward.", "If a patient objects to the hospitalization or to any of the treatment they will receive there, a MCA DoLS authorisation cannot be granted and detention under the MHA is the only available option.", "Consequently, it appeared that the people who decide whether or not to use compulsory admission for psychiatric treatment would be making those decisions within a relatively complex regulatory framework with two key pieces of legislation, one of which potentially conflicted with their professions' values or their personal moral intuitions (Roberts, Peay, & Eastman, 2002).", "Furthermore, the interface between the two frameworks is complex and poorly understood (Clare et al., 2013; House of Lords Select Committee on the Mental Capacity Act 2005, 2014).", "It remains unclear what the implications of this state of affairs might be for clinical practice.", "Understanding the ways in which the new legislation was implemented in practice could potentially highlight the need for specific training or for further law reform.", "Furthermore, a detailed description of the principles upon which decisions to detain are based in practice, and the way decision makers justify any departure from the legal framework, has broader implications for understanding and addressing the gap between policy and practice that has been observed in multiple studies involving a large number of jurisdictions (Appelbaum, 1997; Zinkler & Priebe, 2002).", "Over a 12-month period, we collected data on the ways in which decisions to detain people under Section 2 or Section 3 of the MHA were made by medical practitioners and AMHPs working in the catchment area of a mental healthcare provider in the East of England.", "The study comprised two components:", "Direct observation of medical practitioners and AMHPs discussing whether adults they had assessed met criteria for compulsory admission and should be detained.", "These discussions were audio-recorded.", "In order to assist interpretation, the lead author (EF) also conducted and recorded brief (15–20 min) semi-structured interviews with the medical practitioners and AMHPs immediately after they had made their decisions, asking about the decision-making process.", "In order to gain a broader understanding of practice than could be obtained through observation of a sample of MHA assessments alone, detailed interviews with medical practitioners and AMHPs, each lasting up to two hours, were also conducted.", "Following the data collection methods used in Biographic-Narrative Interpretive Methodology (Wengraf, 2001), participants were first asked to tell the story of their involvement with compulsory treatment over the course of their working lives.", "They were then asked to describe in more detail up to seven particular incidents of decisions to detain that they had mentioned in their stories.", "This approach was adopted in order to discover the factors that participants consider important when making decisions about detention, rather than imposing an inappropriately narrow, reductionist focus.", "All interviews were audio-recorded.", "Initial sampling was directed towards collecting data from assessments and practitioners that reflected the diversity of situations under which MHA assessments take place, aiming for maximum variability.", "As the project progressed, an iterative approach was adopted.", "Data were analysed as they were collected, building understanding and seeking out confirming and disconfirming cases.", "A thematic analysis of the data was carried out by EF.", "All the audio-recordings were transcribed verbatim, followed by initial line-by-line coding.", "Patterns and relationships between the codes were studied in order to identify themes that arose when participants were making or discussing their decisions about admission to hospital for compulsory assessment or treatment (Braun & Clarke, 2006).", "This initial analysis was used as the basis for an institutional ethnography (Redley & Weinberg, 2007).", "Key themes judged particularly relevant to the research question were re-analysed by EF and described in greater detail, paying attention to the ways in which the participants constructed and characterised the importance of concepts such as ‘mental disorder’ or ‘risk’.", "In order to understand the principles on which participants based their decisions to detain, attention was paid to evidence of responsiveness to proximal interactive cues, evident in the discussions between medical practitioners and AMHPs and to distal institutional mandates, inferred from the researchers' knowledge of the legal frameworks and local policy.", "To improve the credibility of the conclusions, the analysis was reviewed by IC and AH, who attempted to identify and explore alternative ways of understanding the data.", "During the observational component, seven discussions about the use of compulsory admission or treatment were recorded.", "These involved fourteen different practitioners: five AMHPs, five psychiatrists, and four primary care physicians (general practitioners or GPs).", "Three of the seven discussions concerned assessments that had taken place in the patient's home, two had taken place on a mental health in-patient unit, and the remaining two had taken place in the emergency department of a general hospital and in a police custody suite.", "This is representative of the range of settings where MHA assessments occur.", "In five of the seven cases, all three practitioners involved in the discussion reached consensus (in two of those cases, the result was detention under the MHA and in the other three cases voluntary treatment was organized, either in hospital or in the community).", "In the remaining two cases, the medical practitioners recommended detention under the MHA, but the AMHP did not agree that this was necessary and the patient was not detained.", "During the interview component, fifteen psychiatrists and one AMHP provided accounts of a further 112 cases (six or seven from each participant) in which a decision about the use of detention was made.", "Six participants worked in General Psychiatry, five in Older People's Mental Health, two worked with people with Intellectual Disability, one worked in Rehabilitation Psychiatry, one in Liaison Psychiatry, and one in a Forensic Mental Health Team.", "Five participants were women.", "Seven participants had experience of mental health practice outside England and Wales.", "Five participants had more than ten years of professional experience in making decisions about detaining people for compulsory assessment or treatment.", "Seven of the sixteen interviewees expressed concerns about the effectiveness of the MHA as a safeguard for the human rights of their patients: three expressed concerns based primarily on the content of the Act, and the other four on the way they perceived the Act being interpreted in practice.", "One interviewee expressed the view that the Act provided a relatively good safeguard for human rights.", "The remaining eight interviewees were neutral.", "Five key themes emerged from the analysis of the instances and accounts of decision making: (i) diagnosis, (ii) availability of alternatives to detention, (iii) likelihood of response to treatment, (iv) risk assessment, and (v) the patient's capacity to make decisions about treatment.", "An additional theme that emerged was (vi) the degree of difficulty inherent in the decision making.", "These themes are discussed in more detail below, accompanied by illustrative excerpts from the data.", "Diagnosis was raised during all of the discussions recorded in the course of the observational component of the study.", "Before recommending compulsory admission, practitioners explicitly reached a consensus position on the presence of acute mental illness, such as depression, bipolar affective disorder, or schizophrenia.", "In practice, this limitation of detention to cases of acute mental illness appeared to be achieved in two stages: screening and assessment.", "All seven assessments observed in this study were arranged following reports of behaviour that could be associated with a severe depressive episode (self-harm, expressions of suicidal ideation) or behaviour that appeared irrational and could be associated with a psychotic episode.", "At the assessment itself, the purpose of discussing diagnosis appeared to be to establish the presence of acute mental illness, either positively, by gathering evidence of pathognomic symptoms or of pre-existing diagnosis by a trusted authority, or by exclusion of other possibilities:", "Psy4: we even wondered if this was personality disorder …", "GP3: Mm, I was going to say could it be PD [personality disorder], not depression.", "PD with alcohol.", "Psy4: Well this is what we got, you know … But the history didn't support that and {psychotherapist} agreed.", "You know, the history is admission, ECT, sections, hypomanic spell, you know.", "It just doesn't fit with a personality disorder.", "But, you know, in between, reasonable function, but not so much recently.", "This observation was supported by the interview data.", "Practitioners appeared reluctant to use detention in cases involving long-term conditions judged unlikely to respond to compulsory treatment:", "Sectioning people with dementia, on the whole, is a bad thing, because it's not fair.", "It's a different deal, getting sectioned if you've got dementia than if you've got functional illness because if you've got functional illness it's likely that with some treatment you will recover and go back to where you were [Psy9].", "And so a lot of people with learning disabilities [intellectual disability] who'd got in trouble with the law were detained … and one really sometimes struggled to see whether there were genuine benefits that came out of that [Psy6].", "Having agreed upon a diagnosis of mental illness, practitioners discussed whether detention under the MHA was the only appropriate response.", "The possibility of intensive home treatment (crisis care) was raised and considered as an alternative to hospital admission in all cases where the person being assessed had not already been admitted:", "AMHP1: Can she be treated at home?", "This is what I′d like to know.", "Psy2: That is, I think, the big question.", "There is undoubtedly an element of risk.", "Can that risk be sufficiently ameliorated in home treatment or not?", "What do you think?", "Psy1: I′d say no. I think, from the little we know, the picture changes a bit too much.", "An’ I′m not quite sure that home treatment will contain that.", "The AMHP's question about the possibility of home treatment is treated by her colleagues as a call to justify the absence of its use.", "This suggests that home treatment is considered to be the default option.", "A lack of availability of round-the-clock crisis care in the community was not raised as a problem by the participants, suggesting that they perceived this to be a readily available alternative to admission despite the fact that lack of its availability has been raised as a factor contributing to the use of detention in some areas (Mind, 2011).", "Having established the necessity of in-patient treatment, practitioners discussed the need for compulsory rather than informal admission.", "A preference for informal admission was a feature of much of the talk; detention was characterised as undesirable whilst informal admission was characterised as the ‘least restrictive option’, to be pursued whenever possible:", "Psy3 [addressing GP2]: We'll recommend a Section 2 and the AMHP will complete if she doesn't agree to come in when the ambulance arrives.", "AMHP2: We are guided by the principle of the least restrictive alternative.", "We want to give her some choice but we also need to keep her safe…", "Psy3 [addressing AMHP2]: So you said you may decide not to make a recommendation?", "Depending on whether she comes downstairs or not?", "AMHP2: Yes, I think that just depends on whether or not…", "The notion of the ‘least restrictive option’ used by practitioners relates to the legal ‘Principle of Less Restriction’, characterised by the requirement within the MHA Code of Practice in effect at the time the research was conducted (2008: p5, paragraph 1.3) that ‘People taking action without a patient's consent must attempt to keep to a minimum the restrictions they impose on the patient's liberty, having regard to the purpose for which the restrictions are imposed.’", "Subsequently, the Code of Practice has been revised and states (Department of Health, 2015) that:", "‘Where it is possible to treat a patient safely and lawfully without detaining them under the Act, the patient should not be detained’ and ‘If the Act is used, detention should be used for the shortest time necessary in the least restrictive hospital setting available.’", "Participants acknowledged that the cases where the patient had been told that she had no choice but to accept hospitalization, could be viewed as wrongful de facto detention that sidesteps the procedural safeguards of the MHA: it ‘bent the rules somewhat in that it was borderline coercion’ (Psy3) and was ‘a thinly veiled threat’ (GP3).", "In the narrative interviews, five participants described cases where they had difficulty distinguishing the boundary between providing patients with clear information about their options, and introducing an element of coercion that prevented voluntary consent to informal admission.", "In such cases, the notion of a Principle of Least Restriction was frequently raised as a justification for the decision to attempt to proceed with treatment on an informal basis:", "Psy 1: Fortunately, he was persuaded to come into hospital voluntarily, but that was one very key situation where it felt like we would've been pushed to act.", "Psy 7: We often say we don't want to section people because we're concerned about the therapeutic relationship.", "Some of that may also be a natural reticence to use a very restrictive practice when we're not by nature policemen.", "There was very little discussion of the therapeutic purpose of hospitalization in the observed assessments, possibly reflecting an implicit assumption that detention would not be considered for any other purpose (a hypothesis supported by the role of diagnosis in limiting consideration of hospitalization to cases of acute mental illness, discussed above).", "Rather, the practitioners discussed the likely outcomes of the proposed treatment for the patient in question, weighing up the benefits and costs.", "Detention was treated as unjustifiable if the costs to the patient appeared to outweigh the likely benefits:", "Psy 1: Would we bring her into hospital to treat her illness [schizo-affective disorder] or would we be just bringing her in as a short term measure to deal with an ongoing problem, which is her vulnerability and the difficulties she has with coping given her limitations … I quite acknowledge those are concerns, but I don't think those are concerns that we're going to sufficiently mediate by bringing her in right now.", "Psy 2: It's not gonna necessarily help her engage with mental health services in the future if we admit her to a psychiatric ward that's likely to be a fairly rough and ready experience.", "So there are costs to consider to admitting her as well.", "The interview data provide further insights into the intuitions of practitioners regarding the role of the notion of treatability in the justification of detention.", "Detention was frequently characterised as justifiable in cases where the patient responded well to treatment:", "AMHP 5: He [man detained for treatment for schizophrenia] went down to a place [hospital] in London and actually did really well.", "On the other hand, detention was characterised as difficult to justify when no improvement was anticipated:", "Psy 14: It was more and more clear for me that if we detain her [a teenager with conduct disorder], it's a very big label on her and it's not at all needed.", "She does have long-standing issues, but those are all issues that are not something that can be changed, actually, by bringing her into the hospital.", "During the observational component of the study, participants were heard discussing three kinds of risk arising from the patient's condition.", "First, the risks that patients posed to their own safety:", "GP 3: So the choices are really do you take the risk of her running off, absconding one more time, possibly killing herself, taking an overdose, doing something risky, or do you say well look, enough's enough.", "Second, the risks others posed to patients, especially young women patients, all three of whom were deemed to be at risk of ‘exploitation’:", "Psy 3: The fact that she was intending to go to university in the state that she is now clearly rendered her not only a danger potentially to other people, in terms of her erratic behaviour, but certainly to herself and being vulnerable to exploitation and that wasn't acceptable risk for her.", "Third, the risks that the patient posed to others, especially any children living in the household:", "Psy 2: She's sleeping very poorly, she's up in the night, she was up at three o'clock in the morning and in with her children.", "Ah, we just don't know what is … what form her behaviour's going to take.", "Risk assessment also addressed wider notions of ‘best interests’.", "In two cases, for example, the benefit for the patient of having social and family relationships protected from the deleterious effects of mental illness was discussed:", "Psy 3: It was in her best interests according to the legal criteria for her to come in, but was also in her best long-term interests for any deterioration or flare-up not to reach the stage where the option to return [to the family home] would have been precluded.", "This type of discussion could function as a means of acknowledging the views of relatives, which other studies (see, e.g., Kullgren et al., 1996) have identified as a possible influence upon decisions to detain, whilst preserving the principle that the decision to detain should be based upon the interests of the patient.", "The absence of decision-making capacity is not one of the legal criteria for detention under Sections 2 or 3 of the MHA.", "However, during the observational component of the study, the patient's capacity to make decisions about treatment was discussed during all of the cases that resulted in hospitalisation, whether or not the patient was detained under the Mental Health Act.", "In all of the cases where a recommendation for detention was made, the patient was characterised as unable to understand the nature of their illness and/or the need for treatment.", "In the only observed case where all the practitioners agreed that informal admission would be appropriate, the patient was characterised as capable of making the decision to accept treatment:", "Psy5 [addressing his colleagues as they agree to organise a voluntary admission]: I think basically he seemed to be willing to come into hospital informally and it seemed that he understood the reasons for the admission.", "He seemed to be having capacity to make that decision.", "It is striking that the finding of capacity occurred in the case of a patient who readily agreed hospital to admission.", "In all the other cases, the patient had refused hospital admission or asked to be discharged.", "Medical recommendations for detention were made after patients were characterised as failing to understand the reasons why hospitalisation was being recommended.", "However, there was no evidence of formal assessment of decision-making capacity.", "Instead, the absence of capacity appeared to be inferred from failure to agree wholeheartedly to admission or treatment:", "Psy4 [making the case for detention under the MHA]: And you know, there's something almost cognitively lacking in her, in that she'll have, we'll have a long discussion and at the end of it, the ward round, she'll say ‘Can I go home then?’", "This observation raises the possibility that, in practice, incapacity is treated as a necessary condition for justifiable detention, although capacity may be determined on the basis of outcome (i.e., treatment refusals are treated as evidence of incapacity and acceptance of treatment is treated as evidence of capacity) rather than functional ability.", "This hypothesis is supported by the way that incapacity was defined in the interview data.", "Most participants characterised treatment refusal as arising from a ‘lack of insight’ and it was this, rather than incapacity as defined in the Mental Capacity Act that, in their opinion, justified detention for compulsory treatment in their eyes:", "Psy6: I guess one of the striking things when you move from having spent nearly five years in general medicine into psychiatry, is this idea that not everyone wants to see you, as a patient, and that there is the need for the Mental Health Act … I mean she, in a sense, couldn't see that she was unwell, and therefore why should she accept treatment?", "Psy17: She, of course, had absolutely no insight into the fact that she might have a mental illness.", "She wouldn't see anyone from mental health services.", "During the interview component of the study, ten participants recounted cases which they described as straightforward decisions.", "One feature common to the majority of ‘straightforward’ decisions to discharge patients, or not admit them to hospital in the first place, was behaviour that was characterised as an understandable response to a particular social context or environmental trigger rather than a consequence of mental ill-health:", "Psy17: So, for example, people have fallen out with their spouses or partners and they've taken an impulsive overdose or made a threatening statement, they have been picked up by the police and when they've spent a couple of hours in the 136 suite or talking to professionals, they calm down and they realise that perhaps yes, things are not going well in the relationship and they're ready to take advice and ask for help.", "So that's again fairly straightforward, there's not much controversy as to what you do with those things.", "The other common feature in this group of straightforward cases was the emergence of evidence, suggesting that the risk of harm to the patient or others was not as high as it first appeared:", "Psy1: I did a general psychiatric assessment, which was just take her history, find out what actually happened, um, explore motives and suicidality, and it transpired that there wasn't very much there at all.", "She had taken an impulsive overdose.", "She was not suicidal in mood, she was well supported, she wasn't really depressed, it had all been in response to social stress and she was waiting for her parents to pick her up.", "Features common to the majority of ‘straightforward’ decisions to detain were as follows: (1) an uncontested diagnosis of psychosis or severe mood disorder, (2) a high probability of improvement if treated, (3) impaired decision-making capacity resulting from difficulty understanding the need for treatment (as perceived by the clinicians), (4) a high risk of harm to the patient or others, and (5) the presence of significant distress or disability:", "Psy10: He just became very, very psychotic … he thought that he'd cracked some very powerful sort of code and only he knew it.", "And then he felt that there were these women who were interfering … he actually attacked a girl because of that belief.", "Not because of anything else, it was because she was interfering with that process that only he was engaged in.", "And, all of his, like, processes, if you like … to me, he did not have the capacity to make, I think, even small decisions, let alone for his treatment or anything... This was a very unwell man, requires treatment, doesn't have the capacity, and you step in on those grounds.", "Psy4: But her depression was very convincing, very severe and she was quite disabled by it … And she was probably the most ambivalent lady I′ve ever seen.", "She would really request an admission, then no sooner had she got to the ward but she would request discharge.", "And this happened three or four times, and she often would go home and have a relapse and overdose … And I realised that she wasn't in a position, a frame of mind, to make a choice … longitudinally, it became very clear to me that this lady needed to be on section, that she wasn't able to really make these big decisions, and when she made them her suicidality and her depression really sort of confounded the picture and made her change her mind.", "It was straightforward in that it was very clear to me and her GP, because we had been struggling.", "Cases that appeared to cause particular difficulty included those in which one or more of the features of straightforward cases were not unequivocally present.", "Six participants described ‘difficult cases’ where the current degree of mental illness was difficult to assess or the net benefits of admission were difficult to predict.", "This difficulty was also evident in one of the observed cases.", "After the assessment of a young woman with a relapsing and remitting psychotic illness, who was also significantly intellectually disadvantaged, the AMHP commented that she had found the decision difficult because:", "AMHP1: You have to be convinced that she is so ill she needs to be in hospital in terms of her mental illness.", "People have seen her before me; they knew what she's like when she's really ill; this was my first acquaintance with her.", "Five participants described ‘difficult cases’ where the two statutory frameworks (the Mental Health Act and the Mental Capacity Act) both appeared applicable, and a decision about which framework to apply was required.", "This difficulty was also evident in one of the observed discussions:", "Psy2 [addressing Psy1]: I, er, yep.", "I, I′d confirm you were thinking that she should come into hospital but you're thinking that [AMHP1] might not want to proceed with the application on the grounds that she's consenting to an informal admission?", "Psy1: Or that she's, yeah, that she's consenting to an informal admission or that she is not actively opposing if there is a doubt about capacity.", "That's the only thing, I′m just wondering about that point you raised earlier.", "AMHP1: Yes.", "But this is the point about Mental Capacity Act, if somebody's going along with it.", "But then I′m, I myself think that she's, was okay with us, but did say she was not a mental patient earlier on so I′m concerned about…", "Psy2: And we asked her point blank whether she would consider coming into hospital and to be honest she was, I felt at that point her answer was almost unintelligible.", "AMHP1: She's not understanding at all.", "No.", "Psy2: Due to her thought disorder, due to her illness.", "And I, in this circumstance, I think this is clearly a circumstance where the Mental Health Act…", "Finally, six participants described ‘difficult cases’ involving high risks of physical harm.", "It appears that one complex element of decision-making concerns ideas of responsibility and accountability and their relationship to the level of risk posed by the patient.", "Practitioners appeared to view themselves as potentially being held accountable for the behaviour of people they did not detain.", "In most cases, this awareness caused anxiety, and some participants described using detention in cases where they were not completely convinced that it was justified, in order to manage their anxiety:", "Psy17: I think I′m being slightly controversial here, but I think the GP's concern here was more about covering our arses for any potential risk, rather than what was in the best interests of the patient.", "And I was more concerned about the long-term strategy of managing this person, the therapeutic relationship with the team and so on.", "So I think we all had slightly different takes on what would be the best thing to do in this case.", "I think eventually, again I′m being a little bit controversial, I think the GP's fears about a potential nasty incident communicated itself sufficiently to both the social worker and me, and we decided the safest option would be for him to be in hospital.", "The results of this study suggest that, when making decisions about the use of compulsory admission, practitioners employed their own ‘practical criteria’.", "Although the practical criteria are similar to the legal criteria of the Mental Health Act, there are some important differences between policy, as operationalised in mental health legislation, and practice, as enacted in real-life decisions (see Table 1).", "In order to analyse the ways in which practitioners justify their decisions, it is useful to consider the underlying principles upon which the legal and practical criteria may be based.", "Within moral and political philosophy, there are two potential justifications for restricting liberty: the harm principle, which states that it may be justifiable to limit someone's liberty in order to prevent them from harming others, and paternalism or limitation of someone's liberty for their own good.", "Whilst the harm principle remains widely accepted as a justification for the deprivation of liberty, paternalism per se is more difficult to justify in a liberal system (Mill, 1998[1859]; Feinberg, 1984).", "However, a limited form, soft paternalism, is widely (although not universally) accepted.", "Soft paternalism justifies limitations on liberty, for the benefit of the person being limited, provided that they are unable to make a choice that would be consistent with their own interests.", "This lack of decision-making ability may stem from a lack of knowledge about the likely consequences of a particular choice or from an inability to exercise appropriate judgment in making a choice.", "The degree of capacity required to make a choice, is, according to several influential theorists, on a continuum that varies with the potential consequences of the choice to be made (Feinberg, 1986; Eastman & Hope, 1988; Buchanan & Brock, 1990; McMillan & Gillett, 2005).", "Thus, a greater degree of interference with liberty may be justifiable in situations where people are at risk of causing themselves significant harm.", "The legal criteria for authorising deprivation of liberty under the Mental Capacity Act (through the Deprivation of Liberty Safeguards) appear to provide authorisation on the basis of soft paternalism alone.", "Patients can only be hospitalized under this framework if they are unable to make their own decision about the need for care and treatment, and if that care and treatment is necessary in their best interests.", "The legal criteria of the Mental Health Act authorise deprivation of liberty through compulsory admission on the basis of either the harm principle, as patients can be hospitalized if this is necessary for the protection of others, or, alternatively, paternalism as patients can be hospitalized if this is necessary in the interests of their own health or safety.", "The absence of a test of capacity means that the MHA can be used as legal authorisation for exercising hard paternalism, as a person with a mental disorder who is nonetheless able to make his or her own decision about treatment can still be deprived of liberty in the interests of his or her own health or safety.", "Many of the misgivings expressed about the amended MHA's effectiveness as a safeguard against unjustifiable deprivation of liberty can be characterised as concerns about its authorisation of hard paternalism, especially when compared with the approach taken by the MCA.", "The findings of the analysis of the data appear to form a set of operational criteria for identifying cases where the principle of soft paternalism may be applied.", "Furthermore, the descriptions of ‘straightforward’ decisions to detain appear to be clear examples of soft paternalism.", "These observations support a hypothesis that the majority of participants in this study viewed soft paternalism as being both morally permissible and their primary justification for restricting the liberty of their patients.", "Some practitioners seem to have ‘imported’ the concept of ‘best interests’ from the MCA into their decision making about compulsory admission under the MHA.", "This is a logical step to take when following the principle of soft paternalism, provided that care is taken to assess the degree to which the paternalism proposed is actually ‘soft’.", "Without a formal assessment of capacity, and in the context of apparent assumptions that refusal of treatment amounted to absence of insight, which in turn amounted to lack of relevant decision-making capacity, there is a risk that the practitioners would unwittingly make hard paternalistic decisions.", "Some of the participants in this study were also prepared to restrict their patients' liberty according to hard paternalism or the harm principle, particularly when the risk of harm to the patient or others appeared significant.", "However, they did so reluctantly, characterising these decisions as difficult, and suggesting the possibility that, compared with soft paternalism, the harm principle and hard paternalism provide less convincing justifications for detention for psychiatric treatment, at least in the eyes of a group of participants in this study.", "Some even suggested that they did not view detention in these circumstances as fully justified, but rather action that they felt compelled to take in order to defend themselves against liability for any harm that their patients may cause if not admitted to hospital.", "This study is one of very few direct observational studies into the actual practice of assessing adults for compulsory admission for psychiatric treatment (see also Holstein, 1988 and Quirk et al., 2000).", "The observational element of the study provided evidence of the way decisions are actually made, not simply the ways in which practitioners describe their decision-making processes after the event.", "However, MHA assessments are ‘hard to reach’ phenomena and it was not possible to record large numbers of instances of observed decision making.", "Moreover, it is possible that the presence of an observer recording the decision-making process influenced the outcome of the MHA assessments.", "However, rates of completion of recommendations for compulsory admissions at observed assessments (72%) were comparable to the rates recorded by the healthcare provider in the 12 months preceding the study (75%), which suggests that the presence of an observer did not have a significant effect on outcome.", "Interviews provided rich data about a wide range of instances of decision making, especially in circumstances where observational research would not be practical or appropriate.", "Triangulation between the two data sources allowed a more comprehensive description of practice than either observation or interviews alone could provide.", "Whilst a range of medical practitioners were able to participate in interviews, AMHPs, although willing to participate in the observational element of the study, were reluctant to participate in interviews.", "They cited the impact of their workloads, which is plausible given that, at the time of the study, there were unfilled posts in the AMHP rota.", "However, the lack of interview data from AMHPs is an unfortunate limitation.", "This study provides a detailed description of the principles used for decision making by a particular group of medical practitioners and AMHPs.", "Further, wider-reaching research is needed to establish whether these principles are widely shared amongst other practitioners, in England and Wales and in other jurisdictions.", "Some aspects of the observed divergence between legal and practical criteria for admission under the MHA may act as an additional safeguard against arbitrary deprivation of liberty.", "However, other aspects of the divergence may have the opposite effect.", "The tendency to recommend compulsory admission when this is judged to be in the patient's ‘best interests’, rather than necessary in the interests of his or her health or safety, and the tendency to be swayed towards the use of compulsion in cases where there is judged to be a risk of harm to the patient or others, even when other factors are may be equivocal, may raise the likelihood of compulsory admission.", "This risk should be addressed in training programmes for practitioners.", "One key finding of this study was that some practitioners viewed soft paternalism as the best justification for detention and managed their concern that the MHA authorises detention on other grounds by incorporating an informal test of capacity into their decision making.", "However, their conceptualisation of capacity as closely related to acceptance of treatment, may, in practice, limit the effectiveness of their test as a safeguard against unjustifiable deprivation of liberty.", "Once detention is used to safeguard patients' wider ‘best interests’, and not simply in the interests of their health or safety, the possibility of widespread deprivation of liberty opens up unless a coherent and reliable test of capacity is incorporated into the MHA.", "Another key finding was that practitioners departed reluctantly from their usual reliance on the principle of soft paternalism to authorise detention when the risk of harm to the patient or others appeared significant.", "It is possible that the unease expressed by practitioners arises from an awareness that they are stepping outside the usual role of health and social care practitioners to exercise powers usually reserved for the criminal justice system (in the case of enacting the Harm Principle) or powers that are not extended to practitioners treating physical health conditions under the Mental Capacity Act (which does not authorise hard paternalism).", "These observations suggest a potential resolution for the tension between policy and practice: replace the problematic MHA and the MCA Deprivation of Liberty Safeguards, which are already acknowledged as requiring reform (House of Lords Select Committee on the Mental Capacity Act 2005, 2014), with two new frameworks, each with a clear function based upon a single theoretical justification for detention.", "One framework would provide legal justification for detention on the grounds of soft paternalism.", "Currently, soft paternalism can be enacted either through the MHA or the MCA DoLS.", "Patients detained under MCA DoLS have access to fewer procedural safeguards than those detained under the MHA.", "However, patients detained for the purpose of soft paternalism under the MHA do not have the protection of a functional test of capacity and may be subject to greater stigmatisation due to the connotation of the MHA with public protection.", "Procedural safeguards could be provided through a relatively informal system of assessments and hearings, similar to those currently in place to ensure that the MHA is used appropriately.", "With more robust safeguards, a framework justifying detention on the basis of soft paternalism, even in cases where the patient objects, could remain compliant with Article 5 of the European Convention on Human Rights.", "The second framework would provide legal justification for detention for treatment needed to reduce the risk of serious harm to others, arising as a result of mental ill-health, for the small number of people who retain decision-making capacity but nonetheless choose to place others at risk by refusing treatment.", "It could also apply to people who have committed a serious crime as a result of mental ill-health but are more appropriately managed in hospital than in prison.", "A formal system of judicial oversight could provide procedural safeguards, with the role of medical practitioners reduced to providing information on diagnosis, prognosis and risk management, to facilitate judicial decision making.", "This separation of detention powers into two systems, according to the principle which justifies the use of detention would be intellectually coherent, consistent with human rights instruments and, being consistent with the apparent moral sentiments of practitioners, at less risk of idiosyncratic interpretations in practice." ]	[]
Cell biology of Zymoseptoria tritici: Pathogen cell organization and wheat infection	Cell biological research in the wheat pathogen Zymoseptoria tritici (formerly Mycosphaerella graminicola) has led to a good understanding of the histology of the infection process. Expression profiling and bioinformatic approaches, combined with molecular studies on signaling pathways, effectors and potential necrosis factors provides first insight into the complex interplay between the host and the pathogen. Cell biological studies will help to further our understanding of the infection strategy of the fungus. The cellular organization and intracellular dynamics of the fungus itself is largely unexplored. Insight into essential cellular processes within the pathogen will expand our knowledge of the basic biology of Z. tritici, thereby providing putative new anti-fungal targets.	[ "BIOC", "IMMU" ]	[ "Infection biology", "Mycosphaerella graminicola", "Phenotypes", "Septoria tritici blotch" ]	[ "1311", "2404" ]	[ "Septoria tritici blotch (STB) is one of the most devastating diseases of wheat (Dean et al., 2012; Gurr and Fones, 2015).", "Cell biological research on the causative agent, the ascomycete Zymoseptoria tritici (formerly Mycosphaerella graminicola or Septoria tritici), is focused mainly on the host–pathogen interface.", "This commentary aims to provide an overview of the current state of cell biological knowledge across the life history of the fungus Z. tritici.", "Hitherto, our knowledge of this notable crop pathogen has been largely restricted to its invasion strategy.", "In addition, this article summarizes the available knowledge on the organization and function of its cells.", "However, due to space constraints, several important aspects of research on Z. tritici, such as fungicide resistance, genome organization, evolution and population biology are only mentioned briefly here.", "The interested reader is referred to detailed overview articles elsewhere (Cools and Fraaije, 2013; Croll and McDonald, 2012; Orton et al., 2011; Stukenbrock and McDonald, 2008; Suffert et al., 2011; and articles in this special issue).", "Fungi show much morphological diversity.", "Different growth forms include single-celled yeasts, multi-cellular and tip growing hyphae and asexual and sexual spores (Charlile and Watkinson, 1995).", "The vegetative growth forms of Z. tritici fall into all three categories.", "The most common cell type, grown under laboratory conditions, is the macropycnidiospore (Fig. 1A).", "This form is often referred at the “yeast-like” stage.", "However, yeasts are uni-cellular, whereas macropycnidiospore are multi-cellular structures, consisting of 4–8 elongate cells (Fig. 1A, lower panel provides calcofluor staining of septa).", "The individual cells within this multi-cellular structure are ∼1.5–3.5 μm wide and can be up to ∼40–100 μm long (Sanderson et al., 1985; Wiese, 1987; note that data on dimensions varies between reports and that considerable morphological variation is seen in field isolates).", "However, Z. tritici also produces micropycnidiospores, which are small (∼1 μm wide, 5–10 μm long) and uni-cellular structures (Wiese, 1987; Eyal et al., 1987).", "They, therefore, fit the definition of a “yeast-like” growth form.", "These cells are formed by lateral budding from hyphae or macropycnidiospores (Fig. 1A, arrowheads; 1C, arrowheads).", "Neither macro- nor micropycnidiospores are dormant, are formed in asexual fruiting bodies (pycnidia) and are dispersed by rain splash (Sanderson et al., 1985).", "Finally, macropycnidiospores germinate to form thin hyphae, consisting of very elongated cells that extend by polar tip growth (Wiese, 1987; Fig. 1B).", "This morphogenic transition can be triggered in liquid culture upon nutrient deprivation and following an increase in temperature (Mehrabi et al., 2006b; Motteram et al., 2011).", "The ability to grow vegetatively in several growth forms is a characteristic of many pathogenic fungi, with hyphal growth being a prerequisite for invasion of host tissue (Gow, 1995).", "Pycnidia appear as dark spots on the necrotic leaves (Fig. 2A), but can also be found in wheat stubble and debris (reviewed in Suffert et al. (2011)) where they were reported to oversummer on the surface of the soil (Hilu and Bever, 1957).", "Pycnidia develop underneath the stomata of infected wheat leaves and remain embedded in the plant epidermis (Weber, 1922, reviewed in Hilu and Bever (1957), Shipton et al. (1971), Kema et al. (1996a); Fig. 2B).", "They vary in size, usually ranging from 60 to 200 μm (Sanderson et al., 1985), depending on the fungal strain and on the density of the infection (Eyal and Brown, 1975; Kema and Annone, 1991), but also with stomata size variations of wheat cultivars (Rudd, pers.", "Comm.).", "Z. tritici also forms sexual ascospores, which have a role in over-seasoning (Eyal et al., 1987), are distributed by air and can spread the STB over hundreds of kilometers, whereas rain splashed macropycnidiospores dispersed more locally (Sanderson et al., 1985).", "Depending on the environmental conditions and agricultural practice, either asco- or pycnidiospores can be the primary source of inoculum in STB (Eriksen and Munk, 2003; Sanderson et al., 1985; reviewed in Suffert et al. (2011)).", "Ascospores are typically found in stubble and consist of 2 cells (Wiese, 1987).", "They are shorter (10–15 μm) and wider (2–3 μm) than the asexual macropycnidiospores.", "Ascospores are formed in perithecia, which are also sub-epidermal, 48–114 μm in diameter (Wiese, 1987) and appear after the pycnidia on infected leaves (Eriksen and Munk, 2003; Kema et al., 1996a; reviewed in Suffert et al. (2011)).", "Their formation depends on the meeting of strains of opposite mating types (Kema et al., 1996b) and, therefore, their appearance depends on the intensity of the epidemics (Cowger et al., 2002).", "Insight into the regulation of morphology was provided recently.", "Z. tritici contains the mitogen-activated kinase (MAPK) ZtHog1 (named previously MgHog1).", "A zthog1 deletion mutant showed abnormal spore morphology and multiple nuclei, suggesting a role of the MAPK signaling in pycnidiospore morphology (Mehrabi et al., 2006a).", "The morphological transition from “yeast-like” growth to hyphal growth was reported to be under the control of the MAPK and the cyclic AMP pathway (Mehrabi et al., 2006a,b, 2009) and the putative down-stream transcription factor ZtWor1 (Mirzadi Gohari et al., 2014).", "This transcription factor also regulates negatively hyphal fusion (anastomosis), which is found occasionally in early stages in infected leaf tissue, but which becomes very prominent in pycnidia formation (Hilu and Bever, 1957; see below).", "Z. tritici carries 21 chromosomes (Goodwin et al., 2011).", "Eight of the smallest chromosomes appear to be non-essential (dispensable chromosomes) and are absent from some strains collected from the wild (Wittenberg et al., 2009).", "It was found that the dispensable chromosomes are lost frequently in meiosis (Wittenberg et al., 2009).", "The role of these dispensable chromosomes is not clear.", "In other pathogenic fungi, dispensable chromosomes carry genes involved in pathogenicity (Han et al., 2001; Hatta et al., 2002).", "The dispensable chromosomes in Z. tritici contain a high number of repetitive elements (Dhillon et al., 2014) and show a faster rate of evolutionary change (Stukenbrock et al., 2010).", "Thus, the dispensable chromosomes may facilitate rapid adaptation to changing environments (Wittenberg et al., 2009) or participate in development of fungicide resistance (Torriani et al., 2009).", "However, at present, there is no experimental evidence to support such proposed functions.", "Therefore, the biological role of dispensable chromosomes in Z. tritici remains elusive.", "From a cell biological point of view, the infection cycle of Z. tritici can be divided into the following phases: (1) entry of the fungus, (2) colonization of the plant tissue and (3) formation of fruiting bodies.", "Infection begins with the germination of either pycnidiospores or ascospores, both of which are pathogenic (Hilu and Bever, 1957; Eyal et al., 1987; Shipton et al., 1971; Suffert et al., 2011).", "They switch to hyphal growth upon contact with the leaf (Duncan and Howard, 2000; Kema et al., 1996a).", "This transition is essential for fungal infection and was first shown in a deletion mutant, lacking the mitogen-activated protein (MAP) kinase Zthog1 (Mehrabi et al., 2006b) and a mutant defective in protein N-glycosylation (Δztalg2, Motteram et al., 2011; the protein was previously published as MgAlg2).", "Both mutant strains grow as pycnidiospores, but are unable to infect wheat plant tissue.", "As hyphal growth is a pre-requisite for tissue invasion in many fungal pathogens (Gow, 1995), these results suggest that a defect in morphogenic switching precludes plant infection in the mutant strains.", "While the importance of hyphal growth for Z. tritici infection is recognized, reports describing the initial steps of the infection process are not entirely consistent.", "Undoubtedly, Z. tritici hyphae enter the host tissue via substomatal openings (Fig. 3; Hilu and Bever, 1957; Cohen and Eyal, 1993; Kema et al., 1996a; Shetty et al., 2003).", "However, several reports describe the potential of the fungus to invade the epidermis directly, usually at anticlinal walls (Weber, 1922, reviewed in Hilu and Bever (1957), Duncan and Howard (2000), Shetty et al. (2003)).", "These attempts were reported to be mostly unsuccessful, or were not even found in other studies (Kema et al., 1996a), suggesting that they may reflect specialized experimental conditions, strain variations or occur in wounded tissues.", "Another debatable point is the existence of a stimulus that attracts hyphae to the stomata.", "Several studies suggested that invasion of stomata is a random process (Hilu and Bever, 1957; Kema et al., 1996a; Shetty et al., 2003).", "However, Howard and Duncan reported directed growth of hyphae toward the stomata, and suggested that hyphae respond to an unknown “thigmotropic signal” (Duncan and Howard, 2000).", "Surface sensing and “guidance mechanisms” has previously been reported in the rust fungus Uromyces appendiculatus (Hoch et al., 1987).", "Functional characterization of the protein ZtFus3 (named previously MgFus3; Cousin et al., 2006) supports the view that stomata recognition is a non-stochastic process.", "Deletion strains lacking this MAP kinase cannot recognize the substomatal opening, and, consequently, fail to infect the plant (Cousin et al., 2006).", "Interestingly, early infection in the rice blast fungus Magnaporthe oryzae also depends on a Fus3-like MAP kinase, PMK1 (Xu and Hamer, 1996).", "In this fungus, PMK1 is required for the formation of appressoria, which are specialized cells that exert mechanical pressure to force their way into the host (Deising et al., 2000).", "Several morphological studies report appressorium-like swellings at the tip of invading Z. tritici hyphae (Cohen and Eyal, 1993; Duncan and Howard, 2000; Kema et al., 1996a; Fig. 3).", "However, the Z. tritici genome lacks many of the repertoire of genes, known to be involved in appressorium formation (Goodwin et al., 2011).", "This suggests that Z. tritici does not elaborate appressoria (Cohen and Eyal, 1993).", "Thus, the process by which ZtFus3 enables the fungus to locate stomata and thus support early invasion remains elusive.", "Fungal hyphae appear after 12–24 h in the substomatal cavity (Hilu and Bever, 1957; Shipton et al., 1971; Kema et al., 1996a; Duncan and Howard, 2000), from where they colonize the mesophyll tissue of the plant.", "The majority of the hyphae grow lengthwise of the leaf (Hilu and Bever, 1957), and hyphae remain exclusively in the intercellular space of the plant tissue (Hilu and Bever, 1957; Kema et al., 1996a).", "Within the first ∼9–15 days, they form a branched network that colonizes the plant mesophyll (Shetty et al., 2003).", "About 3–11 days after infection, hyphae begin to fill the substomatal space and pre-pycnidia appear in these cavities (Cohen and Eyal, 1993; Duncan and Howard, 2000; Kema et al., 1996a; Shetty et al., 2003; Fig. 3).", "During this course of leaf colonization, the infection remains asymptomatic.", "The leaves appear healthy and dead plant cells are rarely found (Hilu and Bever, 1957).", "This symptom-less “latent phase” (also named biotrophic phase) is unusually extended, and varies between 6 and 36 days (Shipton et al., 1971), depending on wheat genotype-fungal isolate combination (Lee et al., 2014) and, in the field, upon weather conditions.", "However, under laboratory conditions, it lasts usually 9–14 days (Hilu and Bever, 1957; Kema et al., 1996b; Keon et al., 2007; Shetty et al., 2007).", "Z. tritici does not form haustoria or cruder feeding structures (Shipton et al., 1971; Kema et al., 1996a).", "In fact, it is not entirely clear if the fungus lives on plant resources at this stage (Sanchez-Vallet, 2015).", "The fungal genome carries an expanded number of putative peptidases and alpha amylases, and it was suggested that the fungus metabolizes apoplastic proteins or starch, released from the chloroplasts (Goodwin et al., 2011).", "Alternatively, Z. tritici hyphae could access apoplastic sugars, which can reach high concentrations in grass leaves (Tetlow and Farrar, 1993).", "However, no changes in apoplastic nutrient composition were found in apoplastic metabolite analysis (Keon et al., 2007).", "Moreover, the fungal biomass barely increases during the initial days of infection (Keon et al., 2007; Shetty et al., 2007), which suggests that the pathogen utilizes internal stores, such as neutral lipids.", "It was shown that the establishment of this latent phase requires a LysM-motif containing effector protein Zt3LysM (Lee et al., 2014; Marshall et al., 2011; previously published as Mg3LysM).", "Such effectors compete with defence-inducing chitin receptors of the plant for binding to chitin, released from the fungal cell wall (Mentlak et al., 2012; Sanchez-Vallet et al., 2013).", "Indeed, such activity was described for Zt3LysM, but the protein also protects the fungal cell wall from plant-derived hydrolytic enzymes (Lee et al., 2014; Marshall et al., 2011).", "It was shown that plant colonization by the fungus requires the MAP kinase ZtSlt2 (previously published as MgSlt2; Mehrabi et al., 2006a).", "Deletion mutants were able to penetrate normally and appeared in the substomatal cavities.", "However, they remained un-branched and did not progress to invade the mesophyll tissue (Mehrabi et al., 2006a).", "A similar phenotype was observed after deletion of ZtSte12 (previously published as MgSte12; Kramer et al., 2009), suggesting that this transcription factor could function downstream of ZtStl2.", "One possibility is that, in the absence of this pathway, effector secretion is impaired and the fungus is unable to establish the latent phase.", "Future studies are needed to address this question.", "Pycnidia formation is initiated in substomatal cavities (Kema et al., 1996a; Shetty et al., 2003).", "They are formed by extensive growth, branching and extensive fusion of hyphae, which derive originally from 1 or 2 hyphae that invaded the substomatal cavity (Hilu and Bever, 1957: Fig. 3).", "This colonization and the formation of pre-pycnidia occurs at 5–9 days post infection (Hilu and Bever, 1957; Kema et al., 1996a; Duncan and Howard, 2000).", "As these asexual fruiting bodies develop, chlorotic lesions appear, usually followed by necrotic areas that appear at ∼10–12 days post infection (Duncan and Howard, 2000; Kema et al., 1996a).", "This onset of the necrotrophic phase is characterized by disintegration of host tissues (Kema et al., 1996a), and this may be a consequence of extensive programmed plant cell death (Keon et al., 2007; Rudd et al., 2008).", "By this time, fungal hyphae have enlarged significantly (from initially 1 μm to 2.5 μm in diameter), show irregular septation and extended vacuolation (Hilu and Bever, 1957).", "How the necrotrophic phase is initiated is not entirely clear, but plant cell death begins in the vicinity of the substomatal cavity (Shipton et al., 1971; Fig. 3), where most fungal material in concentrated.", "This is consistent with the idea that the fungus secretes toxin proteins that are recognized by the plant and this hyper-activates host defenses (Kema et al., 1996a; Keon et al., 2007).", "Not much is known about such secreted necrosis factors, but candidates could be “necrosis and ethylene-inducing peptide 1” (Nep1)-like proteins (NLPs) – these secreted virulence factors induce necrotic cell death in several host–pathogen interactions (Gijzen and Nürnberger, 2006).", "The genome of Z. tritici contains a single NLP encoding gene, which induces necrosis in Arabidopsis thaliana (Motteram et al., 2009).", "Whilst this protein is highly expressed at the transition from the latent phase to the necrotrophic phase, deletion of the gene did not affect fungal virulence.", "This suggests that other, as yet unidentified, effectors trigger hyper-active plant defenses.", "Interestingly, genes encoding plant cell-wall degrading enzymes are highly expressed at the onset of the necrotrophic phase (Kema et al., 2008).", "This suggests that the fungus directly attacks the host cells, although the genome encodes surprisingly few plant cell wall-degrading enzymes (Goodwin et al., 2011).", "Thus, the role of these enzymes in establishing the necrotrophic phase remains elusive.", "In any case, the release of plant nutrients, such as amino acids and sugars, is thought to provide the basis of massive fungal proliferation and increased biomass, found during the necrotrophic phase (Keon et al., 2007; Shetty et al., 2007).", "Finally, mature pycnidia are formed, which produce the multi-cellular macropycnidiospores that develop from conidiophores (Hilu and Bever, 1957; Kema et al., 1996a).", "They are disseminated through the leaf canopy and to other plants by rain-splash (Suffert et al., 2011, Fig. 3).", "Asexual fruiting body development seems to be controlled by the MAP kinase ZtFus3, as deletion mutants fail to form pycnidia in vitro (Cousin et al., 2006).", "In addition, mutants in the catalytic and regulatory subunit of protein kinase A (ZtTpk2 and ZtBcy1, respectively; previously published as MgTpk2 and MgBcy1, Mehrabi and Kema, 2006) are required for pycnidia formation in planta.", "This suggests that several signaling pathways contribute to this developmental step.", "In summary, the course of infection can be divided in several steps.", "Firstly, spores germinate on the epidermis and the fungus enters via stomata.", "In a second phase, the fungus shields itself from the plant recognition and colonizes the plant mesophyll.", "During this latent phase, changes in fungal biomass are barely detectable, most likely due to a lack of nutrients.", "However, fungal hyphae ramify through the leaf tissue and begin colonize the substomatal cavities around the infection site to initiate the formation of numerous pycnidia.", "Indeed, most recent experimental results, using highly diluted cell suspensions for infection, suggest that a single entry of a hypha is sufficient to induce the formation of large leaf lesions (Gurr and Fones, 2015).", "This supports the notion that extensive invasive growth occurs during the latent phase.", "During the third phase, the fungus strikes and initiates the necrotrophic phase, characterized by collapse of the host cells.", "This provides the nutrients to increase fungal biomass, leading to massive proliferation of pycnidiospores.", "The fourth phase is the release of spores from these fruiting bodies, which enables infection of adjacent plants.", "It emerges that secreted proteins may have a key role in establishing the latent phases and, probably, in initiating the necrotrophic phase (Motteram et al., 2009; Lee et al., 2014; Marshall et al., 2011).", "Currently, we know relatively little about these effectors.", "Bioinformatic approaches have revealed numerous potential candidates for secreted proteins involved in host–pathogen interaction (Morais do Amaral et al., 2012; Gohari et al., 2015).", "Investigating their role during infection will help understanding the cell biology of the wheat-Z.", "tritici relationship.", "In all microbes, the ability to infect the host and cause disease symptoms resides, ultimately, at the cellular level.", "Consequently, cell biology becomes increasingly important in modern plant pathology.", "For decades, cell biological research has emphasized the identification of molecules and their interactions.", "However, profound insight into the dynamic behavior of cells is required to “understand the causes of disease well enough to predict risks, make early diagnoses, and treat effectively” (Pollard, 2013).", "It was suggested that a successful strategy toward a mechanistic understanding follows three consecutive steps: (1) framing a good question, (2) identification of the molecular inventory for the process, and (3) characterization of the function of each molecule to explain its participation in a dynamic system (Pollard, 2013).", "In Z. tritici research, we are at the transition from step 1 to step 2.", "The pathogenic nature of Z. tritici dictates important questions, such as “how is the transition between biotrophic and necrotrophic phase triggered”; “how is morphological transition from yeast-like growth to hyphal growth controlled during early plant infection” or “how does the fungus establish the biotrophic phase in a compatible interaction with wheat”?", "In addressing these, our research has reached phase 2.", "Transcriptional profiling at various stages of pathogenic development (Keon et al., 2007; Kellner et al., 2014; Kema et al., 2008) and bioinformatic prediction of secreted proteins and effectors (Morais do Amaral et al., 2012; Stergiopoulos et al., 2012; Stukenbrock et al., 2011) reveals candidate genes, involved in a particular pathogenicity-relevant process.", "However, phase 3 requires an in-depth functional understanding of individual proteins.", "Targeted gene deletions have provided insight into the regulation of several steps of the infection process (overview in Orton et al., 2011).", "Furthermore, first putative effectors have been identified and their importance in pathogenicity of Z. tritici was assessed (Gohari et al., 2015; Lee et al., 2014; Marshall et al., 2011).", "However, we need cell biological methods, including detailed location studies, to further characterize such identified candidates.", "A good example, to illustrate this point, is the precise timing of effector protein secretion.", "Studies in the corn smut fungus U. maydis (Doehlemann et al., 2009; Djamei and Kahmann, 2012; Bielska et al., 2014a) and in the rice blast fungus M. oryzae (Khang et al., 2010; Dagdas et al., 2012; Mentlak et al., 2012; Ryder et al., 2013; Giraldo et al., 2013) provided detailed cell biological insight into infection structures and invasion strategies of these plant pathogens, using fluorescent dual labelling techniques, and revealed valuable insight into the infection process.", "Similar studies need to be undertaken in the wheat-Z.", "tritici system.", "Surprisingly little is known when it comes to cellular organization and dynamic processes in Z. tritici cells.", "Early studies provide details on dimension and number of septa in spores (Sanderson et al., 1985; Wiese, 1987), and more recent work defines the molecular role of signaling pathways in the switch from “yeast-like” growth to hyphal growth (Mehrabi et al., 2006a,b, 2009).", "However, dynamic cellular pathways, such as membrane trafficking or cytoskeletal organization, are virtually not addressed.", "How can knowledge about fundamental cellular processes in the pathogens cell biology inform strategies toward pathogen control?", "As fungi adapt to existing fungicides, cellular processes need to be identified that could provide new anti-fungal targets.", "A good example is the identification of motile early endosome in the corn smut fungus U. maydis.", "The process of fungal early endosome motility was discovered in 2000 (Wedlich-Söldner et al., 2000).", "Subsequent research led to an understanding of the molecular transport machinery (Bielska et al., 2014b; Lenz et al., 2006; Schuster et al., 2011; Wedlich-Söldner et al., 2002; reviewed in Steinberg (2014)).", "It was shown, most recently, that early endosome motility is essential for long-range signaling during infection, which is required for effector production and successful plant infection (Bielska et al., 2014a).", "Thus, 14 years after its identification, fundamental research on motile early endosomes has revealed unexpected and important insights into the strategy of host invasion in U. maydis.", "While it remains to be seen if this process supports infection in Z. tritici, this example illustrates the principle point.", "We need to understand the invasion strategy and essential cell biology of the fungus to develop new ways of defence against the pathogen.", "Together with existing strength in population biology, biochemistry and molecular plant pathology, further discoveries in the fundamental cell biology of Z. tritici promise to help defeating STB." ]	[ "Cell biology of the infection begins to shed light on the host–pathogen interaction.", "The cell biology of the fungal pathogen is highly understudied.", "Intensified cell biology research promises new fungicide targets and will help mode-of-action studies." ]
Enteroviral infections in the pathogenesis of type 1 diabetes: new insights for therapeutic intervention	The development of islet autoimmunity and type 1 diabetes has long been linked with enteroviral infection but a causal relationship has proven hard to establish. This is partly because much of the epidemiological evidence derives from studies of neutralising antibody generation in blood samples while less attention has been paid to the pancreatic beta cell as a site of infection. Nevertheless, recent studies have revealed that beta cells express specific enteroviral receptors and that they can sustain a productive enteroviral infection. Importantly, they can also mount antiviral responses which attenuate viral replication and may favour the establishment of a persistent enteroviral infection. Together, these responses combine to create the Trojan horse by which enteroviruses might precipitate islet autoimmunity.	[ "PHAR" ]	[]	[ "3002", "3004" ]	[ "Human beta cells are known to be susceptible to infection with HEVs, particularly members of the Coxsackievirus B family.", "Thus, isolated human islets can be productively infected with a range of different EV-B family members (CVBs and Echoviruses, many of which have been associated with T1D; Table 1) in vitro.", "Furthermore, among the various islet cells, it is the beta cells that are preferentially susceptible to infection [32–34], and this leads to a dramatic decrement in glucose-induced insulin secretion [21•,35••,36].", "Tropism of HEVs for the islets has also been demonstrated in vivo in the pancreata of neonates who died following a lethal CVB infection [6,34,37] and in the pancreas of individuals with T1D [38,39].", "This then raises the question: `so why the beta cells?’", "The tropism of enteroviruses for the beta cell is likely to be driven by at least two factors; first, these cells express receptors necessary for the binding and subsequent internalisation of the virus and secondly, they contain specific host factors which the virus can hijack to facilitate successful infection, replication and, perhaps, persistence.", "This latter point is interesting since the traditional view states that enteroviruses are not likely to establish persistent infections and this concept will be explored further below.", "The various potential receptors utilised by enteroviruses and their expression in human islets are summarised in Table 2 but one that is receiving particular attention is the Coxsackie and Adenovirus Receptor (CAR).", "This molecule is utilised as an entry vehicle by many of the viruses that are associated with T1D in epidemiological studies and, very recently, we have shown that a specific isoform of CAR, having a unique C-terminal PDZ binding domain (CAR-SIV) is selectively and highly expressed within the beta cell [40••].", "Studies by Ylipaasto et al. have also demonstrated that infection of human islets with CVB4 and CVB5 was effectively prevented in the presence of an antibody that blocks CAR [41].", "Intriguingly, in our work, the subcellular localisation of CAR-SIV was unusual in that it was not present primarily at the plasma membrane of beta cells, as might be expected, but rather it was located mainly in insulin secretory granules.", "This unexpected localisation implies that the virus could selectively enter the beta cell by a Trojan horse mechanism in which secretory granule proteins are hijacked as they emerge onto the cell surface during exocytosis, such that virus particles are then internalized by the endocytic machinery during membrane recovery (Figure 1).", "In support of this, electron microscopy studies by Frisk et al. of human islets infected with CVBs clearly show the presence of viral replication complexes and newly synthesised virions at, or near, insulin granule membranes [35••].", "In recent years, a series of critical host factors required for successful HEV infections have been identified.", "These include PLA2G16 [42••] which is essential for virion-mediated genome delivery into the cytoplasm; phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P), which are critical for the generation of specialised organelles required for efficient viral replication [43,44]); polypyrimidine tract-binding protein 1 (PTBP1), which is utilized by the virus to promote cap-independent translation of viral RNA [36] and heat shock protein 90 (HSP90), which is required for the correct processing of the capsid precursor P1 [45].", "Importantly, many of these proteins are expressed in human beta cells and play a key role in pathways unique to beta cells (reviewed in [46]).", "For example, PTBP1, has an important role in glucose-stimulated cap-independent translation of insulin granule proteins [36]; PI4KIIIβ acts as a metabolic sensor in beta cells and regulates the priming of secretory granules [47] and HSP90 is a chaperone that regulates surface expression of ATP-sensitive potassium (KATP) channels [48].", "Together these results suggest that human beta cells express specific virus entry receptors as well as key host factors that aid the virus at various points in its lifecycle.", "This may therefore help to explain their exquisite sensitivity to infection.", "In parallel with these considerations, it is also important to note that beta cells are terminally differentiated and studies of their neogenesis and proliferation suggest that these processes are vanishingly rare in humans after the age of 10y [49,50].", "This means that the human host must develop strategies to effectively manage, or preferably clear, any beta cell viral infection, whilst doing everything possible to minimise the destruction of these largely irreplaceable cells.", "In this context, it is well known that beta cells are extremely sensitive to interferons (IFNs), the principal anti-viral cytokines produced in response to an infection [51,52].", "As viremia (viruses in the bloodstream) must occur before infection of the beta cells it is likely that any cells targeted during the initial acute phase of infection will elaborate IFNs.", "Thus, the pancreas will be exposed to IFNs prior to any encounter with the virus and this may serve to prime the beta cells to resist infection.", "Indeed, pre-treatment of islets with Type I and III IFNs promotes an anti-viral state and significantly reduces viral replication following infection in vitro [51,52].", "However, IFNs do not necessarily block viral entry, which could yield a scenario in which virus has entered the cell, yet the host cell has succeeded in upregulating a range of anti-viral proteins that will counter any attempt by that virus to establish a productive, lytic, infection [53].", "Conceivably, a battle then ensues between the host (beta) cell and the virus which culminates in a mutual compromise where viral persistence is established and the host cells remain viable (Figure 2).", "In support of this hypothesis, risk-associated single nucleotide polymorphisms (SNPs) for T1D are found in key anti-viral response genes such as IFIH1 and TYK2.", "Individuals carrying these SNPs exhibit altered IFN responses [54–60,61••] and the risk variants have been associated with an increased frequency of HEV infection [62].", "Furthermore, of the 51 identified candidates genes associated with T1D, 42 are expressed in human beta cells and when Ingenuity Pathway Analysis was performed on these genes, the three highest scoring canonical pathways were — Interferon signalling, Role of JAK1, JAK2 and TYK in interferon signalling and Role of pattern recognition receptors in recognition of virus and bacteria [54].", "These pathways are all activated in response to viral infections and this provides a possible link between genetic predisposition to T1D and host anti-viral responses.", "Traditionally, HEVs are thought to induce an acute infection in which large numbers of new viral particles are rapidly synthesized and release from infected cells to spread to other nearby host cells.", "One of the most effective host mechanisms to control HEVs infection is the generation of neutralising antibodies which help to clear the virus from the circulation and affected tissues.", "However, there is mounting evidence that HEVs can evade these primary defense mechanisms to establish a lower level, persistent, infection under certain circumstances.", "Indeed, this type of infection has now been associated with several diseases such as Chronic Fatigue Syndrome (CFS) [63]; chronic myocarditis and dilated cardiomyopathy [64] and Amyotrophic Lateral Sclerosis (ALS; reviewed in [65]) as well as Type 1 diabetes.", "In order to understand this previously unrecognized aspect of EV biology, a number of mechanisms have been proposed to explain how the virus might persist.", "These include the activation of processes to restrict viral RNA replication, including by deletion of nucleotides from within the 5′UTR of the viral RNA genome [64,66–68] and equalization of the proportions of positive and negative strands to form double stranded RNA (dsRNA) molecules [69,70].", "A key additional requirement is the need for the virus to minimise host cell lysis, which would not only promote inflammation and the activation of antiviral immune cells, but also lead to the release of free virus particles that are susceptible to neutralization by anti-EV antibodies.", "One mechanism by which this may be avoided is suggested by recent evidence that HEVs, including CVB3, can be shed from cells within extracellular vesicles [71,72].", "In principle, this could shield the virions from neutralizing antibodies and provide a means by which they can evade immune surveillance.", "Two main strategies are being explored to tackle HEV infection in T1D; vaccination and treatment with anti-viral agents.", "Both have the potential to slow disease progression, yet each also has significant obstacles that must be overcome before it could be utilized in clinical practice.", "Islet autoimmunity in at-risk children peaks at two different ages and the specificity of the first autoantibody also differs at each age [73].", "The first peak of autoimmunity occurs during the second year of life and is associated with the development of insulin autoantibodies (IAA), while the second is seen between 3 and 5y and is associated preferentially with the emergence of GADA autoantibodies [73].", "Given that these initial signs of islet autoimmunity occur early in life, children would probably need to be vaccinated within the first few months of life in order to offer effective protection against HEV infection.", "This will require the development of safe and effective vaccines that can target multiple HEVs associated with the disease and there is precedent for this approach given the proven success of neonatal vaccination against poliomyelitis (another enterovirus).", "Moreover, encouraging progress has already been made on this front, with a new formalin-inactivated CVB1 vaccine successfully developed and tested in animal models [74,75••,76].", "Multivalent CVB1-6 vaccines are also now being generated (Hytönen and Flodström-Tullberg, personal communication).", "Epidemiological data support the idea that a vaccination approach might be effective as a means to reduce the incidence of T1D since Finnish children infected early in life with CVB3 or CVB6 appear to be immuno-protected against a subsequent infection with different HEVs which might, otherwise, precipitate T1D [7].", "On the basis of such evidence, one company, `Provention’ has recently announced exciting plans to launch a first phase clinical trial to assess the safety and efficacy of a CVB vaccine in humans, with the intention of developing an effective approach to reduce the incidence of T1D (www.proventionbio.com).", "Alternative approaches also being explored include the development of virus like particles (VLPs) as antigens.", "These resemble the viral capsid of HEVs but do not contain infectious genome [77].", "Vaccines and/or VLP rely predominantly on the host developing neutralizing antibodies against virus.", "These will therefore be most effective when given to individuals prior to any exposure to diabetogenic viruses and will hopefully ensure that the immune response is sufficiently robust to prevent the spread of infectious virus to the pancreas.", "What, though, might be done to tackle infection in people who already have evidence of islet autoimmunity and/or clinical diabetes and who may be harbouring a persistent infection?", "This could be a fertile realm for anti-viral agents; although at the present time very little is known about whether these are effective against persistent enteroviral infections.", "Anti-viral agents are available (many of which have been re-purposed from use in other conditions), or are in development, that are effective against HEVs associated with T1D (comprehensively reviewed recently in [78]), but the majority of these have been tested only under acute infection settings.", "These agents can be subdivided into two broad categories; those that target viral proteins and others which affect host proteins required for efficient viral replication, translation and release.", "Examples of the former include pleconaril which targets the viral capsid (reviewed in [79]); fluoxetine, commonly known as Prozac, which inhibits the viral protease 2C; and Gemcitabine, which binds to the viral RNA-dependent RNA polymerase, 3Dpol [80].", "The second group includes Enviroxime, which targets the PI4K pathway [79].", "Encouragingly, recent evidence has suggested that fluoxetine is effective against persistent enteroviral infection in cell models [81], but more research is required to test the activity of other drugs in this setting.", "Extensive efforts are underway to identify new anti-viral agents and these are aided by an increasing knowledge of the structure and function of viral proteins, as well as the identification of essential host factors.", "A further avenue of exploration is the use of combinations of different anti-viral agents that have additive or synergistic responses, which together can increase anti-viral potency, minimise the emergence of resistance and reduce drug toxicity/side effects.", "This could be achieved by, for example, combining one drug that targets a viral protein, with another that targets an essential host factor.", "Alternatively, since it is well established that some anti-viral drugs have a low barrier to resistance (meaning that a single mutation within the virus can rapidly lead to drug resistance) whereas, for others, this is much higher, a combination approach employing each type of reagent might also yield clinical benefit.", "In summary, evidence for a viral aetiology in T1D is a long-established concept that has remained unproven.", "Nevertheless, supportive evidence continues to emerge at increasing pace and effective strategies which would minimize the risks deriving from HEV infection in susceptible individuals are being developed with increasing momentum.", "Arguably, it is only when the outcomes of these studies are known that it will be possible to confirm once-and-for-all whether T1D has an enteroviral component.", "We are pleased to acknowledge financial support via a JDRF Career Development Award (5-CDA-2014-221-A-N) to SJR, an MRC Project Grant (MR/P010695/1) to SJR & NGM and project grants from Diabetes UK (15/0005156 & 16/0005480) to NGM & SJR.", "S.J.R and N.G.M. wrote the manuscript and are the guarantors of this work.", "Papers of particular interest, published within the period of review, have been highlighted as:", "• of special interest", "•• of outstanding interest" ]	[ "Enteroviral infection has been long-associated with type 1 diabetes in epidemiological studies.", "β-Cells express a specific enteroviral receptor isoform, CAR-SIV, mainly on secretory granules.", "β-Cells respond to enteroviruses by allowing the establishment of a persistent infection.", "Enteroviral vaccines are under development that might be effective in type 1 diabetes." ]
Narratives of scarcity: Framing the global land rush	Global resource scarcity has become a central policy concern, with predictions of rising populations, natural resource depletion and hunger. The narratives of scarcity that arise as a result justify actions to harness resources considered ‘underutilised’, leading to contestations over rights and entitlements and producing new scarcities. Yet scarcity is contingent, contextual, relational and above all political. We present an analysis of three framings – absolute, relative and political scarcity – associated with the intellectual traditions of Malthus, Ricardo and Marx, respectively. A review of 134 global and Africa-specific policy and related sources demonstrates how diverse framings of scarcity – what it is, its causes and what is to be done – are evident in competing narratives that animate debates about the future of food and farming in Africa and globally. We argue that current mainstream narratives emphasise absolute and relative scarcity, while ignoring political scarcity. Opening up this debate, with a more explicit focus on political scarcities is, we argue, important; emphasising how resources are distributed between different needs and uses, and so different people and social classes. For African settings, seen as both a source of abundant resources and a site where global scarcities may be resolved, as well as where local scarcities are being experienced most acutely, a political scarcity framing on the global land rush, and resource questions more broadly, is, we suggest, essential.	[ "SOCI" ]	[ "Africa", "Land rush", "Narratives", "Politics", "Resources", "Scarcity" ]	[ "3312" ]	[ "Understandings of what has been called the ‘global land rush’ – the large-scale acquisition of land and other resources by governments, agribusiness companies and financiers often in overseas territories – have often been cast in terms of ‘scarcity’.", "A number of overlapping narratives are at play.", "Commodities, be they food, feed or fuel, are deemed scarce, and therefore sought in areas where land and water in particular are seen to be relatively abundant.", "Advocates argue that this involves taking advantage of global comparative advantages of demand and supply to realise a ‘win-win’ situation, in which commodities are supplied to those who need them, while those who have the resources to produce them profit as well (e.g. Bell et al., 2012).", "However, others argue that, as the world runs out of resources, increasing competition potentially leads to processes of exclusion and conflict (e.g. Le Billon, 2012).", "Some narratives also suggest that, if environmental limits are exceeded, dangers may arise, as we transgress some earth system boundaries (e.g. Rockström et al., 2009).", "What does the deployment of the term ‘scarcity’ in these narratives imply?1", "What are the theoretical underpinnings of different versions of scarcity?", "And are there any alternatives to mainstream versions?", "In this paper, we explore how scarcity is represented in policy debates, by whom and to what ends.", "Through a qualitative review of key documents and other sources, we interrogate the knowledge politics of the global land rush when investment activity and international concern were at their peak, during 2007–2013.", "We argue that notions of scarcity are presented as a deliberate political strategy, justifying resource control, appropriation, dispossession, population restrictions and the securing of exclusionary property rights (cf. McCarthy and Wolford, 2011; Hildyard, 2010; Mehta, 2010a, 2001; Hartmann, 2010; Xenos, 1989).", "Pointing out the political nature of scarcity, however, is not a call to relativism – real, material scarcities clearly exist – but an acceptance that meanings and interpretations are co-constructed in particular policy settings, in arenas of power and contestation (Bakker and Bridge, 2006; Bridge, 2009).", "There are winners and losers from different policy narratives, as they have concrete effects, and shape outcomes of struggles over resources.", "Scarcity narratives do not merely describe, but justify changes in access to and control over resources, in ways that reallocate scarcities across regions and populations.", "The policy and business narratives explored in this paper have fundamentally affected the direction and outcomes of land investments, and the wider governance responses, since the height of the global land rush.", "Narratives and their underlying framings therefore really matter, making a deeper look at the narratives being deployed in policy arenas around the global land rush vitally important.", "The paper starts with an introduction to the global land rush in order to set the scene.", "We then discuss three theoretical framings of scarcity derived from classical political economy, contrasting absolute, relative and political scarcity.", "Next, we discuss our methodology and how we explored framings and narratives of scarcity across 135 reports and other sources focusing on land investments and agricultural development, particularly in Africa.", "The following section presents the results of our analysis, identifying four core themes of a dominant narrative of the global land rush.", "The narrative is based on an underlying framing of scarcity that combines absolute and relative interpretations to the almost total exclusion of the political.", "We discuss why and how politics are excluded.", "In the final section, we turn to an exploration of what an alternative political scarcity framing might look like, and its implications for the global land rush debate.", "The recent land rush, sparked by the global financial, food and fuel crises of 2007–2008, is but the latest in a series of cycles of land acquisition in Africa – first in the colonial period when Africa was partitioned by global powers, and then in the 1980s with the far-reaching economic liberalisation (Moyo et al., 2012).", "Various models of large-scale commercial farming have been established (Hall et al., 2017; Smalley, 2013), intensifying land transactions and, generating tenure insecurity and land conflicts in many countries (White et al., 2012; Edelman et al., 2013).", "Economic liberalisation policy reforms resulted in the increased financialization of capital and an expansion of markets and speculative activities around land globally (Fairbairn, 2015).", "In addition, this period has seen a transformation of the world economic and political order with the rise of the BRICS (Brazil, Russia, India, China and South Africa), allowing them to become key players in an increasingly multi-polar world with wide engagements in land and agriculture in Africa (Scoones et al., 2016; Hall, 2011).", "The food and fuel price spikes in 2007–2008 provoked global alarm.", "A long period of cheap resources seemed to be over.", "This hit consumers hard, with food riots occurring in a number of countries.", "The UK’s former chief scientific adviser, John Beddington, captured the imagination by describing a coming together of forces – rising demand for food, water and fuel caused by population growth, urbanisation and consumption changes, increasing shortages of those resources and the challenges of climate change – that threaten to create a ‘perfect storm’ on a global scale (Beddington, 2009).", "References to resource scarcity became commonplace at this time.", "Announcing a new report in 2011, the UN’s Food and Agriculture Organization (FAO) claimed:", "“Widespread degradation and deepening scarcity of land and water resources have placed a number of key food production systems around the globe at risk, posing a profound challenge to the task of feeding a world population expected to reach 9 billion people by 2050” (FAO, 2011a, para. 1).", "Over the past decade, investments in large-scale commercial farms – often linked to ‘water grabs’, to guarantee irrigated production – have accelerated (Borras et al., 2011; Mehta et al., 2012; Hall et al., 2015).", "As we show below, such investments have often been justified in terms of ‘scarcity’, with overseas investments focused on stable, secure and sustainable supplies of food and fuel.", "These investments have involved both food crop and biofuel production, and have occurred on a large scale, although their extent and performance is widely disputed (Oya, 2013; Scoones et al., 2013).", "A large proportion of these are in Africa, where the debate over the pros and cons has been intense (Deininger and Byerlee, 2012; Cotula et al., 2014; Pedersen and Buur, 2016), linked to wider discourses around investment, growth and Africa’s rise (Obeng-Odoom, 2015a).", "Globally, the policy debate intensified, with much discussion about the drivers and consequences of the rush for scarce resources.", "The European Commissions’ Report on Development for 2011/12, Confronting Scarcity: Managing Water, Energy and Land for Inclusive and Sustainable Growth, commented:", "“Projected scarcities of food, water and energy, and the search for investment opportunities to satisfy food security in an increasingly global market, have led to growing pressure on land worldwide … Large-scale land acquisitions are just one manifestation of the increased pressure on land” (EU, 2012: 87).", "Equally, the Washington-based International Food Policy Research Institute (IFPRI) observed:", "“Increased pressures on natural resources, water scarcity, export restrictions imposed by major producers when food prices were high, and growing distrust in the functioning of regional and global markets have pushed countries short in land and water to find alternative means of producing food” (IFPRI, 2009: 1).", "The food-fuel-financial crisis of the late 2000s thus galvanised a series of scarcity narratives justifying interventions around land and resources.", "What were the underlying framings that guided these narratives, and from where were they derived?", "The next section explores three frames for understanding scarcity, each suggesting very different implications.", "How scarcities are understood – where, at what scale, in what timeframe, for whom, in what context – is crucial for the conceptualisation of and response to the global land rush.", "Ideas about scarcity are of course fundamental to understandings of economics and politics.", "From Thomas Malthus’ treatise on population first published in 1798 to Lionel Robbins’ famous 1932 definition of economics – as the ‘science which studies human behaviour as a relationship between ends and scarce means which have alternative uses’ (Robbins, 1932: 15) – to the environmentalist arguments from the Club of Rome on the ‘limits to growth’ (Meadows et al., 1972) and the newer incarnations defining planetary boundaries (Rockström et al., 2009), commentators have repeatedly related our understanding of human progress to notions of abundance and plenty, contrasted with dearth and want.", "In this section, we offer three contrasting framings of scarcity, derived from classic political economy: Thomas Malthus and conceptions of absolute scarcity; David Ricardo and ideas of relative scarcity; and Karl Marx and what we call political scarcity.", "It is these three frames, and their intersections, adaptations and reinterpretations, that in turn helped guide our analysis of sources on the global land rush.", "In the last major ‘resource crisis’ of the 1970s, arising from dramatic oil price shocks, thinking was heavily influenced by the birth of the environmental movement and the idea of there being ‘limits to growth’.", "Elements of this thinking have regained popularity, with the idea of ‘planetary boundaries’ (Rockström et al., 2009; Steffen et al., 2015).", "A clear influence on this work is the intellectual legacy of Thomas Malthus, who is widely credited for introducing the conception of scarcity into economics (Gammon, 2010).", "Malthus is associated with the concept of absolute scarcity, meaning an immutable physical limitedness of natural resources that are subject to increasing demands from human society.", "Despite his acknowledgement of variable qualities of land, he claimed there were limited ‘inventions’ available to agriculture, so significant increases in production to offset the population crisis were unlikely.", "Rather, ‘the power of population is indefinitely greater than the power in the earth to produce subsistence for man’ (1970 [1798]: 71).", "From the 1950s onwards, Malthusian ideas informed debates about population growth, especially in developing countries (Mamdani, 1981; Peacock, 1953), and perhaps particularly in populous India and Bangladesh (Paarlberg, 2010).", "Concerns were expressed that overpopulation could create social instability, with countries described as ‘population powder kegs’ (Michaelson, 1981).", "There were increasingly persuasive ideas about planetary biophysical limits, the interconnectedness of whole ecosystems (Odum, 1969) and the environmental impacts of human activity, such as pollution (Carson, 1962).", "Much of this work originated in the conservation movement and the maturing discipline of ecology (Ross, 1975).", "Academics and policymakers began to discuss the earth’s ‘carrying capacity’ (Seidl and Tisdell, 1999) and debate the optimal or maximum human population of the earth (Fraser, 1971).", "A Malthusian collapse of society seemed more likely than before: ‘The human race has expanded to a point of near saturation of its habitat (the earth)’ (Fraser, 1971: 4).", "Concerns over a declining availability of farmland were intensified by the incidence of famines and poverty in the midst of economic development (Sen, 1981).", "In the Club of Rome’s The Limits to Growth, Donella Meadows and colleagues worried about widespread malnutrition and predicted ‘a rather sudden and uncontrollable decline in both population and industrial capacity’, based on the simulations of their systems model (Meadows et al., 1972: 23).", "They argued that, with exponential growth, ‘one can move within a very few years from a situation of great abundance to one of great scarcity’ (1972: 51) and that arable land was too scarce in absolute terms for productivity increases through intensification to be anything more than a temporary measure.", "The ‘neo-Malthusian’ ecologists and economists came to be synonymous with catastrophic predictions and an emphasis on overpopulation and the finite nature of resources (Lipton, 1989).", "Such ideas are visible in Paul Ehrlich’s The Population Bomb (1968), in Garrett Hardin’s (1968) ‘tragedy of the commons’ thesis linking resources and property rights and in work on land degradation and links between resource scarcity and conflict (Homer-Dixon, 2010).", "Such neo-Malthusian ideas influenced welfare, population, conservation and development policies (McCarthy and Wolford, 2011; Hartmann, 2010); sometimes with extreme Malthusian positions arguing for population control and strong, centrist, state-led intervention to offset the crisis (e.g. Brown, 1995).", "While somewhat more nuanced, contemporary discourses on population, resources and development continue to be framed by (neo-) Malthusian ideas.", "The idea of ‘planetary boundaries’, while eschewing a fixity in boundaries, still has at its core the idea of limits and absolute scarcity.", "For example, Johan Rockström and colleagues (2009: 48) warn that ‘humanity may be reaching a point where further agricultural land expansion at a global scale may seriously threaten biodiversity and undermine regulatory capacities of the Earth System’.", "Equally, as we show below, the scarcity narratives, so common in the debate about the global land rush, also have clear resonances with earlier debates, being premised on the notion that there is limited land and water for humanity to use.", "Many who emphasise the limits to the earth’s capacity to support human activity do not adopt a simple Malthusian perspective, but argue for the potential for transformation, including through technological innovation.", "Scarcity is therefore relative to use, and so scarcity is economic, and relative rather than absolute.", "‘Scarcity does not mean mere infrequency of occurrence,’ explains Robbins (1932: 45), ‘… it means limitation in relation to demand.’", "The neoclassical theory of scarcity, especially as it relates to natural resources, was influenced by earlier work by classical economists, and David Ricardo in particular.", "Writing in the early nineteenth century, Ricardo emphasised that farmland varies in quality.", "Furthermore, agricultural productivity is influenced not only by the quality of the land, but also by the amount of financial capital, and the ‘skill, ingenuity and instruments in agriculture’ applied (Ricardo, 1821).", "Thus, whereas for Malthus the great threat to society was population growth, Ricardo was more concerned about the progressive decline in the quantity and quality of farmland (Hussen, 2013).", "A relative scarcity framing suggests that society responds to shortage, through price signals, with institutional and technological change.", "This may involve substitution of the scarce resource, whether staple foods, energy sources or labour; increased recycling of the resource and extraction of lower quality sources; or technological change to increase the efficiency of extraction (Neumayer, 2000).", "Stefan Baumgärtner and colleagues (2006: 489) explain how ‘In [neoclassical] economics, it is generally assumed that continuous substitution is always possible, at least on the margin’.", "For much of the late nineteenth and twentieth centuries, this optimism appeared to be justified.", "This was a time of cheap natural commodities, technological leaps and discoveries of new oil and mineral reserves (Barbier, 2011).", "In agriculture, the Asian Green Revolution could be seen as the ultimate example of a technological response to scarcity (Hayami and Godo, 2005).", "Perhaps the high water-mark of this technological optimism was the 1963 publication of Scarcity and Growth, in which Harold Barnett and Chandler Morse (1963) argued that the absolute scarcity hypothesis should be rejected because the costs of extracted natural resources in the United States had declined since 1870.", "However, few today would claim that resources are infinitely substitutable or that technological improvements will always offset resource limits.", "Instead, many commentators would argue for some acknowledgement of limits, while arguing for technological solutions.", "In the field of agriculture, the work of Esther Boserup on technological innovation (Boserup, 1993, 1981) and Yujiro Hayami, Vernon Ruttan and Hans Binswanger (Hayami and Ruttan, 1985; Binswanger and Ruttan, 1978) on labour intensification as a response to resource pressure have been especially influential, entrenching the iconic role the 1960s–1970s Asian Green Revolution has played in development thinking.", "Michael Lipton (1989) suggests that Malthus’s scarcity narrative and neoclassical models of technological response to scarcity are part of the same approach.", "In his view, society can respond to a Malthusian situation of population growth putting pressure on land and food supplies through technological innovation to produce more food (Boserup, 1981) or increase labour productivity (Hayami and Ruttan, 1985; Binswanger and Ruttan, 1978).", "New institutional economics further suggests that changes in institutional arrangement through shifting transactions costs can result in changes in relative scarcity (Obeng-Odoom, 2016).", "None of these conceptualisations of scarcity – whether absolute or relative or some compromise between them – takes account of the political nature of scarcity: how scarcity is perceived and ‘manufactured’ to suit particular interests (Mehta, 2001); how narratives of scarcity are deployed in political contests over resources (Scoones, 2010); how historical inequalities due to colonialism, exploitation and elite control have affected patterns of resource access and control (Mehta, 2005); and how such patterns are distributed between different groups of people, with real winners and losers in resource struggles (Mehta, 2010a,b).", "In this view, scarcity is not independent, but is constructed in relation to historically-specific patterns and forces of production, distribution and consumption (Perelman, 1979).", "Resources are produced and they are relational; just as humans are part of nature, interlinked through complex metabolic relations (Moore, 2011).", "A relational perspective on scarcity, centred on its social and political dimensions, also challenges a ‘systems’ understanding of scarcity, where dialectical power relations are hidden from view (Harvey, 1974).", "When scarcity is seen as political, embedded in the functioning of capitalism, it follows that states of scarcity that define people’s material needs can be transcended following social transformation of relations of production and ownership (Xenos, 1989).", "Enclosures, and appropriations of resources as individual, private property, for example, are the direct result of such capitalist processes, creating scarcities for some, but resource access for others, differentiated by class, gender, ethnicity and other social axes of difference.", "Under contemporary financialised, globalised capitalism, speculative surplus capital in search of secure investments can, it is argued, lead to ‘accumulation by dispossession’ (Harvey, 2003; Hall, 2013), as new enclosures are created through processes of private accumulation (White et al., 2012; Obeng-Odoom, 2015b).", "Contributions from political ecologists, sociologists and anthropologists extend our understanding of political scarcity, focusing on knowledge politics and how scarcities are constructed discursively (Perreault et al., 2015; Mehta, 2010b; Peet and Watts, 1996).", "The political ‘manufacture’ of scarcity is seen to frame policy discourse and action, with questions raised over how claims about resource scarcity are made (Mehta, 2001).", "A focus on the micro-practices of resource use and management equally show how resource scarcity looks very different from the local level than when viewed with a global gaze (cf. Scoones, 2010; Mehta, 2005).", "Thus a focus on ‘global’ food security may do little to alleviate scarcities of food at a local level (Nally, 2015).", "Such contemporary critical social science analysis highlights the relational, social and political dimensions of scarcity in ways that go beyond the more structural, class analytics of earlier work, and look at how knowledge and practice, constructed across scales, intersect with structural dynamics.", "Table 1 offers a highly simplified summary of these three framings of scarcity, indicating the type of themes we aimed to identify in our document analysis.", "Through a detailed analysis of a range of sources, we explored a set of narratives relating to the global land rush and interrogated how the narratives are framed, examining their construction, the actors involved, the interests implicated, and what silences and exclusions are created.", "We define a narrative, following Roe (1994, 1991), as a storyline, one with a beginning (the definition of the problem), a middle (the elaboration of its implications) and an ending (the proposed solutions).", "The power of narratives is often in their simplicity, their invocation of metaphors and images, their sense of urgency, and thus their political appeal and their ability to enlist followers.", "Narratives are created in a social and a political milieu by coalitions of actors with interests and positions.", "Narrative analysis thus seeks to define some clear storylines and compare them, tracing the actors who tell the stories and the interests that are associated with them (Keeley and Scoones, 2003).", "Narratives are underpinned by framings, defined here as the conceptual constructs that inform and drive narratives (Druckman, 2011; Entman, 1993).", "In the previous section, we have identified three contrasting framings of scarcity – absolute, relative and political.", "Framings may draw on deeper cultural understandings, as well as debates in public and academic discourse (Benford and Snow, 2000).", "Discursive analysis, alive to the use of narrative and framing, reveals that policy interventions are mutually constructed with social, technological, economic and political processes (Shackley and Wynne, 1995; Irwin and Wynne, 2003), with epistemic and socio-political orders intertwined through a process of co-production (Jasanoff, 2004).", "Such a constructivist perspective points to the knowledge politics inherent in defining policy positions, and the patterns of exclusion that play out.", "These processes of constructing policy inevitably involve practices that provide legitimation and authority for preferred narratives.", "In resource assessments that define scarcity for example, the use of models and other ‘calculative devices’ (Callon and Muniesa, 2005) has enormous power.", "Particular technologies, such as satellite imagery and Geographic Information Systems, may present data in particular ways, at certain scales; again, providing forms of evidence that define certain narratives (Bridge, 2015; Robbins, 2003).", "Based on initial searches of material on the global land rush, we first identified five categories of organization publishing material on the land rush, and the main actors in each category2: (i) International organizations (e.g. World Bank, FAO, EU, IFPRI, UK Foresight); (ii) African regional organizations (e.g. African Development Bank, NEPAD, the Southern African Development Community); (iii) Investors and financiers (e.g. Chayton Capital, Rabobank, International Finance Corporation); (iv) Agribusinesses (e.g. Syngenta, Cargill, Illovo Sugar); and (v) NGOs and civil society organizations (e.g. Oxfam, ActionAid, GRAIN, African Biodiversity Network).", "We identified the most frequently cited documents from organizations across our five actor categories covering the period from 2007 to 2013.", "This period covers the start of the food-fuel-financial crisis and the policy responses to it, as well as an intense time of actual and speculated land acquisition in Africa.", "Those core documents were then used as the starting point for snowball sampling of references to other sources.", "In this way the sense of overlapping ‘discourse coalitions’ (cf. Hajer and Versteeg, 2005) emerged around each of the actor groups.", "We complemented this with Google searches of keywords identified from our initial document analysis.", "Our sampling was not comprehensive, and was restricted to English language documents that were available online.", "The total of 135 was reached when the identification of new material had tailed off significantly for each actor group, suggesting we had reasonably good coverage.", "The sample for each group was not the same, but we believe is broadly representative of sources from each actor group for the study period.", "The total was distributed as follows: international organizations (N = 37); African policy (N = 18); investor (N = 31); agribusiness (N = 19) and NGO/civil society (N = 30).", "The full list of documents analysed is presented in the online Supplementary Material linked to this article.", "The result was 135 documents and other sources that deal with the land rush and with agriculture and natural resources more generally, particularly in Africa.", "Most set forward either public policy or investment recommendations for the global or regional community.", "For each of the sources, we identified the narratives on land and its use, the actors involved and their associated interests.", "Looking at the sources of evidence presented, we examined how the narratives were generated, and what sources were used.", "And, digging deeper, we explored which of the three underlying scarcity framings (or combinations) were evident.", "To help understand the authors’ underlying perspectives on scarcity, we looked for key words and phrases that signify particular positions, such as ‘yield gap’ or ‘limits’.", "Through the document analysis, we examined how the three framings discussed above contributed to the construction of narrative storylines, and how these frames are in turn constructed through various narrative devices and sources of evidence.", "Our analysis of each document was qualitative, and we did not undertake any quantitative assessment of frequency of use of terms and phrases, nor did we derive a quantitative assessment of the relative importance of different framings and narratives for different actor groups.", "We use exemplar quotes in our assessment below, derived from many possible alternatives, but believe these provide good illustrations of the narrative being discussed.", "Our assessment emerges from deep immersion in the many thousands of pages of material, and knowledge of the wider land rush debate in this period (e.g. Hall et al., 2015; White et al., 2012).", "Despite the broad range of actor groups represented in the material, our analysis revealed remarkable convergence by most of them (except civil society and NGOs) on a single overarching narrative, expressed through four dominant narrative themes.", "These themes broadly relate to the narrative structure, with a beginning that highlights the problem of limits and urgency; a middle, which presents a context of relative abundant and idle land; and an end, centred on solutions around investment and capturing comparative advantage.", "While the overall narrative storyline was common to all actor groups except civil society organizations and NGOs, different actors had different emphases, as discussed below.", "In our analysis, in relation to each theme, we ask what versions and combinations of the scarcity frames introduced above are evident; which are the dominant ones; how are they expressed; and what frames are excluded?", "All five actor groups use the same narrative theme of crisis to open their position on the global land rush.", "The sense of limits, tipping points, irreversibility and boundaries being reached is repeatedly emphasised across the sources.", "These broadly draw on a (neo-)Malthusian framing, highlighting absolute scarcity.", "The narrative often starts with ‘the challenge’, usually focused around the forecast need to feed 9 billion people by 2050.", "A confluence of factors is pointed to, which the actors argue will result in potential catastrophe unless urgent responses follow.", "For example: ‘The global food system will experience an unprecedented confluence of pressures over the next 40 years….", "This is a unique time in history – decisions made now and over the next few decades will disproportionately influence the future’ (UK Foresight, 2011: 9, 13).", "The FAO’s 2011 review of The State of the World’s Land and Water Resources for Food and Agriculture argued: ’In some … areas, the accumulation of environmental impacts in key land and water systems has now reached the point where production and livelihoods are compromised’ (FAO, 2011b: 4).", "The CEO of Unilever, Paul Polman, emphasised ‘natural limits’:", "“Food security has to be seen as part of the wider question of how we can live sustainably within the natural limits of the planet … We are already consuming natural resources at a rate faster than the planet’s capacity to replenish them” (Polman, 2011).", "Some offer distinctly Malthusian overtones.", "In a 2012 newsletter to investors, Jeremy Grantham, the co-founder of the investment management firm GMO, said:", "“We are five years into a severe global food crisis that is very unlikely to go away.", "It will threaten poor countries with increased malnutrition and starvation and even collapse.", "Resource squabbles and waves of food-induced migration will threaten global stability and global growth.", "This threat is badly underestimated by almost everybody and all institutions with the possible exception of some military establishments … We simply cannot have exponential growth on a finite planet” (Grantham, 2012: 2, 14).", "The imperative for action, and the justification for their business contributions, is emphasised by large agribusiness.", "Two of the largest, Syngenta and Cargill, offered this on their websites:", "“Over the next 20 years, we will need to feed another 1.8 billion people.", "Calorie demand will grow even faster, as diets in some countries increasingly shift to meat.", "In much of the world, agricultural land is limited and water scarce.", "So tomorrow’s growers will have to produce much more food and animal feed with today’s limited natural resources” (Syngenta, 2013, paras. 5–6).", "“By 2050, an anticipated 70 percent boost in global food production will be necessary to meet the world’s growing demand for food.", "To protect the environment, most of the increase in food production will need to come from increased yields and productivity rather than from the use of additional land” (Cargill, 2011, para. 4).", "Of the iconic figures that are repeated continuously in narratives on the global land rush, two stand out: the estimate of a human population of nine billion by 2050 and the need to double food production in the same period.", "These figures are repeatedly cited, although their origins remain obscure.", "For example:", "“Every day sees 220,000 new mouths to feed, meaning 80 million additional people a year.", "Global population today is about 7 billion.", "By 2050, it will probably balloon up to 9.3 billion.", "According to FAO, ‘the world must double food production by 2050’.", "But already in 2011, some 950 million people experienced hunger.", "During the same period, 5 million babies died from hunger.", "Can we produce enough food for all?", "Will we run out of land?”", "(SIFCA, 2012: 10)", "Thus, with different emphases and intentions in mind, the sources across all actor groups highlighted how crises were imminent, storms were in the offing, boundaries were being transgressed and urgent action was required, supported by iconic facts and figures.", "For international organizations, investors and agri-businesses, this justified intervention and investment; for NGOs and civil society the urgency of approaching limits was deployed to spur social action and shifts in development paradigm.", "Sometimes, as in most of the commentaries from African regional policymakers, this urgent scenario of crisis was presented in more local or regional terms; for all other actors, it was presented as a more global challenge.", "At this point in the story the actors diverge, and so too does the underlying framing.", "Most of the actor groups now go on to contrast the global scenario of absolute scarcity and impending crisis, with relative local abundance of a fundamental resource – land – in sub-Saharan Africa.", "In justifying the need to invest in land, particularly in large-scale land deals, particular representations of land and its use are offered.", "Relative to the growing shortage of land globally, land in target investment areas was described in most sources as abundant, empty, idle and underutilised.", "Scarcity (global) and abundance (local, African) are thus juxtaposed as part of the narrative.", "This narrative theme is exemplified in the highly influential World Bank studies that argued for investments in areas where land is ‘available’ and has high ‘potential’.", "For example: ‘Areas [in sub-Saharan Africa] of low population density with good agricultural potential represent untapped reserves for continued expansion of area’ (World Bank, 2007: 231).", "What is meant by ‘available’ and ‘potential’ is highly disputed (see below), yet the argument took hold.", "As regions and countries compete for investment, the idea of ‘untapped potential’ becomes significant.", "This is an argument put forward by African regional actors keen on attracting investment.", "The President of the Economic Community of West African States (ECOWAS) told audiences in China of well-watered arable land in West Africa, noting that ‘ECOWAS governments are willing to promote collaborations to unlock the huge untapped potentials and are hereby extending an invitation to Chinese private sector investors to invest in commercial farming and agro-processing’ (ECOWAS, 2011: 9).", "Other African regional organizations agreed.", "The African Union’s Comprehensive African Agricultural Development Programme (CAADP) argued that ‘… there is substantial untapped potential for the development of the continent’s water and land resources for increasing agricultural production’ (NEPAD, 2003: 24) and the United Nations Economic Commission for Africa (UNECA) concurred: ‘The continent is endowed with many natural resources, including plentiful land and fertile soils, oil and minerals’ (UNECA, 2013: 8).", "While abundance, emptiness and under-use was emphasised, again based on figures of often unknown origin, there are qualifications offered by some.", "Some African policymakers and analysts acknowledged water scarcity, soil degradation and declining per-capita land availability across the region.", "Meanwhile, aware of the wider debate about ‘land grabbing’, some international organizations, such as IFPRI (2009), FAO (2011b) and the World Bank (2011, 2009), were careful to note that some ‘available’ land might already be used by poor people or pastoralists, be degraded, or require massive investment to make productive.", "Such sensitivities were shown by private investors too.", "The CEO of Emergent Asset Management was, for example, quoted as saying: ‘We are not taking land away.", "We buy or lease operational farms that are undercapitalised, or we start projects on land that is fallow to produce food, which in itself creates many local jobs’ (Whitby, 2010: 44).", "The narrative theme on abundance, emptiness and under-use was therefore selectively applied.", "It appears more prominently early on in our period of analysis, before media exposure of ‘land grabs’ heightened awareness of problems.", "For many African regional organizations and policymakers, making the case for investment remained high on the agenda, but for international organizations and investors/agribusinesses greater caution is shown, even if the basic argument for large-scale investment – taking advantage of under-used resources – was still deployed.", "After telling a story about global crisis and local abundance, most narratives end with a suggested technical and investment solution, arguing that there is a way out from the Malthusian bind.", "In this way, the underlying framing combines an element of absolute scarcity with the technological optimism of relative scarcity.", "Yet there is little room for politics in these commentaries.", "Some actors, such as the World Bank and the former head of the UN World Food Programme, acknowledged inequalities in food entitlements and purchasing power; however, while access, equity and distribution are (almost) universally acknowledged as important, the mainstream discourse does not separate these social concerns from an emphasis on increased food production as a solution.", "Thus production is presented as the answer to poverty and food insecurity both locally and globally, to be achieved largely through improvements in technology and greater investment.", "This narrative theme dominates the sources from international organizations, investors and agribusiness actors.", "They propose a reliance on technology to intensify agricultural production of food and animal feed, and thereby mitigate or escape the limits imposed by natural resource scarcity and keep ahead of population growth.", "For example: “With the closing of the land frontier across much of the developing world and continuing strong demographic pressures, gains in land productivity – and sustainable land management – will become fundamental” (World Bank, 2007: 227) or “If used in proper association with suitable technologies [land and water] have the capacity to enable global agricultural production to continue outpacing the growing demand despite the declining per capita availability” (NEPAD, 2003: 23).", "A recurrent metaphor is the idea of a ‘gap’ between a potential level of agricultural production and the actual yields achieved by farmers.", "This yield gap is seen to be particularly large in Africa.", "The work of Günther Fischer, Mahendra Shah and colleagues at the International Institute of Applied Systems Analysis (IIASA) in Austria has been especially influential (Fischer and Shah, 2010).", "Quotes from the reviewed sources that illustrate the concern with solving Africa’s yield gap include: ‘If all current land and water were managed optimally, output could double in the regions where the yield gap is less than 50 percent’ (FAO, 2011b: 35), and ‘Many developing regions … have large gaps relative to their potential.", "In sub-Saharan Africa, for example, crop yields reached only about 27 percent of their economic potential in 2005’ (FAO, 2012: 105).", "Two economists from the International Finance Corporation called for ‘massive’ amounts of investment in agriculture to meet the scarcity challenge.", "They argued:", "“You’ve probably already heard the Malthusian projections targeting our planet’s finite capacity to feed a growing population – projected to reach 9 billion by 2050 – in the face of dwindling resources of suitable land and water in productive climates … by working together, the public and private sectors can help deliver abundant, affordable, and nutritious food for all” (Vegarra and Moses, 2012: 6).", "The African Development Bank was equally assertive on the need for technical solutions:", "“Clearly, raising agricultural productivity including that of smallholder farmers is a key component in reducing poverty and hunger in Africa … In the long term, enhancing agricultural productivity together with mitigating and adapting to climate changes should be the primary focus of food security initiatives” (Salami et al., 2011: 3).", "While broad narratives of scarcity framed the problem, the directions of future pathways of development were more open to debate, and reflected particular interests.", "Not surprisingly, agribusiness companies argued for their own technologies, while financial institutions emphasised investment.", "Many of the actors, such as the World Bank, went beyond mere technological optimism to call for an accompanying improvement in incentives, institutions and infrastructure, echoing the success of the Green Revolution in Asia.", "And many in the international agencies, under labels such as ‘sustainable intensification’, hedged their bets, and avoided the more political discussions about the direction of technological development and its distributional consequences.", "Yet nearly all actors saw the solution as a technical and institutional fix.", "The same group of actors frequently presented the argument for intervention in terms of a ‘shared’, ‘global’ crisis, with solutions being forged through ‘partnerships’ and ‘inclusive’ approaches to development.", "Africa was singled out as a particularly promising investment destination, and a source for ‘feeding the world’.", "The former head of the UN World Food Programme, Josette Sheeran, noted: ‘You can look at hunger as a Malthusian nightmare, or you can look it as a tremendous opportunity because everyone has to eat’ (Hotter, 2012).", "Similarly, the landmark EU report commented:", "“[C]ompeting claims [for land] place a high value on natural resources.", "This presents real opportunities for economic growth in countries endowed with vast natural resources, and in particular those with a large productivity gap – which is the case of many of the poorer countries, particularly in sub-Saharan Africa” (EU, 2012: 88).", "One result of the ‘technical apparatus’ (Fine, 2010) of scarcity-centred economics is the idea of comparative advantage, and so the justification for intervention in a place (Africa), in favour of the globe, as part of ‘shared development’.", "This narrative theme is particularly appealing to global investors and agribusinesses who were keen to justify their interventions in Africa in particular.", "As in the narrative theme addressing land abundance, Africa’s potential was much hyped (Obeng-Odoom, 2015a), particularly in relation to land and water-constrained countries wishing to meet their food, feed or fuel requirements.", "Debates about the opportunities and dangers of land investments were enhanced by the mapping efforts of the Land Matrix group (Anseeuw et al., 2013; Rulli et al., 2013), with data from its public database frequently being quoted, despite many questions being raised about its veracity, at least initially (Scoones et al., 2013).", "The argument goes that there is mutual advantage – and profit – to be gained.", "For example, a global investor commented: ‘We are positive about the role that Africa, with its vast agricultural potential and resources, could play in addressing the growing global demand for food’ (Mouton, in Zeder Investments, 2012: 6).", "Similarly, the EU argued: ‘While land deals give rise to concerns they also provide opportunities.", "Investors may introduce new technologies and skills, expedite the development of contextualised production systems with higher productivity, and spark innovation’ (EU, 2012: 5).", "In finance and investment circles, land was projected as the ‘new gold’ (Brown, quoted in Buckholtz and Delay, 2012), and a safe and secure investment with good returns.", "For example:", "“Agcapita believes farmland is a safe investment, that supply is shrinking and that unprecedented demand for ‘food, feed and fuel’ will continue to move crop prices higher over the long-term” (Agcapita, 2013) and “We expect strong investment performance to continue across the world as fundamentals of food production, security and [demand for] renewable energy all impact on the finite area of global farmland” (InvestAg Savills, 2011: 4).", "Africa was singled out as a particularly promising investment destination, with land as an ‘asset class’ (Schaffnit-Chatterjee, 2012: 1).", "For example: ‘Africa has a tremendous future in terms of agriculture.", "Africa could feed much of the world’ (CEO of Aslan Global Management, quoted in Charles, 2012), and: ‘Looking at global agriculture from a long-term perspective it seems that Africa has a huge opportunity to feed both itself and the world in the coming decades’ (GreenWorld, 2013).", "Some of the thinking behind these statements is revealed in a 2011 research report from Rabobank, a significant financier of international agricultural projects.", "This argued that corporations must rethink their commodity sourcing strategies in this new and unprecedented era of scarcity.", "Because the world’s farmers had become squeezed by low producer prices on one side and high input prices on the other, went the argument, they were unlikely to be able to respond to price rises by increasing production.", "Therefore, in order to secure supply of food and other agri-commodities, corporations turned to investing in land, working directly with farmers, setting up greenfield production and other operations along the supply chain.", "Actors involved in large-scale farmland acquisition ‘all recognise the over-arching rationale that scarcity will become an increasingly regular feature of agricultural commodity markets in the future’ (Rabobank International, 2011: 18).", "The previous section offered an overview of the dominant narrative themes seen consistently across four of our actor groups – international organizations, African regional organizations, investors/financiers and agribusinesses.", "These were underpinned with framings that combine absolute scarcity with economic ideas of relative scarcity and the importance of technological change and investment in addressing limits.", "This section asks: how were these narratives constructed, and what political effects did this have?", "What exclusions of alternative framings resulted?", "The section proceeds then to identify two marginal narratives identified during our analysis and associated with the NGO/civil society actor group.", "We explore how these offer hints of an alternative political scarcity framing, but also point to its limits.", "A variety of approaches to constructing the dominant narrative themes is observed in the sources investigated.", "One example is the deployment of iconic figures, based on population projections, food balance estimates, assessments of land degradation extent, or assumed productivity and yield gaps.", "These are frequently repeated in the sources, yet their origins and basis of calculation are rarely addressed.", "They become iconic markers for a position, but their validity goes unquestioned, despite often large bodies of research that dispute their utility (cf. Leach and Mearns, 1996; Stott and Sullivan, 2000; Scoones, 2001; Keeley and Scoones, 2003).", "The power of global, aggregated assessments, and the scenario modelling often associated with these, is also enormously powerful, and becomes central to global discourses (Hulme et al., 2011; Scoones, 2009), including around the global land rush.", "Figures and maps on land use and availability are particularly important in the evidence base for the dominant narratives identified.", "These are often generated at large scales, extrapolating from limited data inputs.", "Assessments of utilisation are based on factors visible from satellite imagery, and so may exclude uses such as mobile pastoralism or shifting agriculture.", "The models from IIASA and the Land Matrix became especially important in the construction of land policy narratives – from those favouring large-scale investments and from those opposing alike.", "Yet the limitations of such assessments, while widely discussed (cf. Scoones et al., 2013; Oya, 2013), was rarely acknowledged in the sources.", "Again, a simple figure or a startling map is sufficient to make the point, reinforcing a narrative position.", "A similar problem arises from arguments around yield and productivity ‘gaps’.", "The assumption that a new type of investment, portrayed as efficient, modern and high-tech, can overcome such assumed gaps is often made.", "Yet the notion of a yield gap has been widely critiqued (Sumberg, 2012).", "Estimates are usually derived from comparing on-station agronomic conditions with the field settings of farmers.", "While in theory much higher productivity levels can be reached, this requires a whole series of technological, agroecological and economic conditions that may never be realistically achieved in field settings, no matter what the scale or level of capitalisation of the farm.", "Modelling the availability of land only on the basis of what can be seen in satellite images is obviously prone to error.", "Equally, developing yield models based on potentials rather than likely levels, given differences in resource quality, infrastructure and technology attributes and so on, adds another level of uncertainty.", "Yet these models, particularly when they appear in multi-coloured maps or dramatic graphics, have enormous power, and the uncertainties and qualifications are buried in the footnotes.", "Models are of course only constructs that may or may not have some analytical utility in thinking about more complex phenomena.", "But when their assumptions are flawed – whether in terms of the behaviour of individuals in perfect markets or the availability and potential of land – they become potentially dangerous and misleading, obscuring other insights.", "As the Committee on Food Security’s High Level Panel report on land concluded: ‘The satellite and aerial imagery used in bio-physical surveys is blind to the rights and institutions that govern how land is actually used on the ground’ (HLPE, 2011: 9).", "Parables of success in other places – in this instance most frequently the example of the Asian Green Revolution – are often paraded as part of the construction of narratives.", "These provide a counterpoint and illustration of an alternative that justifies the solution presented in the narrative.", "Success stories are usually problematic as comparators, as they emerge from very particular conditions; often ones that have much larger variation than is admitted (Sumberg et al., 2012).", "As a rhetorical technique they are important, but as sources of evidence they are often found wanting.", "The case of the Asian Green Revolution is a case in point.", "As decades of research has shown, there have been huge variation in outcomes, by location, groups of people and so on, with a whole array of ‘green revolution’ pathways evident across Asia (Harriss-White and Harriss, 2007; Hazell, 2009), with no justification for the use of a singular ‘Asian’ success story to contrast with, or advocate for, the African setting (Thompson and Scoones, 2009).", "Thus a series of models, metrics and assessments are generated by certain socio-technical practices, including the use of satellite imagery, geographic-information-system models and yield assessments, which generate eye-catching and persuasive figures.", "These forms of data act as ‘anchoring devices’ (Van der Sluijs et al., 1998) in the boundary work that goes on between science and policy in the construction of knowledge for policy (Gieryn, 1999).", "Figures, maps and models are presented as objective and scientific, part of evidence-based policy.", "This process of ‘mutual construction’ (Shackley and Wynne, 1995) or ‘co-production’ (Jasanoff, 2004) generates a knowledge politics that creates particular types of ‘biopolitical’ knowledge, knowledge subjects and social and political orders.", "These forms of governmentality (cf. Foucault et al., 1991; Jessop, 2006) necessarily define the world – and in this case in relation to resource scarcity – in a way that excludes other versions and realities.", "In the period between 2007 and 2013, the dominant narrative, generated through a variety of mechanisms and promulgated by four major, and powerful, actor groups, justified an argument about the necessity of land investment in Africa.", "Deploying representations of scarcity in different ways, this provided the core discursive support for the global land rush at its peak.", "By identifying the dominant narrative and its underlying framing, a key question raised is: what is excluded?", "Across the sources demonstrating the key narrative themes, a ‘political scarcity’ framing was almost completely absent.", "Only the NGOs and civil society groups (9 sources) embraced a political understanding of scarcity within their framing of the global land rush.", "Two narratives were identified: one focused on ‘food sovereignty’ and local economic solutions; and the other on global inequalities of production and consumption.", "The food sovereignty narrative tells a story of local scarcities, arguing that these can be addressed through local, indigenous economic development.", "It rejects a wider, global discourse of scarcity, presenting it as a political construction that results from the inequities of global capitalism.", "Such a position was articulated by AFSA (the Alliance for Food Sovereignty in Africa) (2011) and La Via Campesina (2012), for example, and Friends of the Earth Africa (2010:5), who argued: “Food is a natural right and agricultural products should not be treated as commodities whose ultimate purpose is the generation of business profits rather than meeting needs of the people.", "Family and small-scale farmers should be encouraged and strengthened”.", "The narrative concerned with global inequalities also highlights the importance of local farming solutions.", "Scarcities, it is argued, are created through over-consumption in some parts of the world, and are imposed on others.", "The focus turns to demand-side changes, including a reduction in resource consumption in rich countries.", "For example, Oxfam (2011:4) argued: “Appalling inequities … plague the food system from farm to fork”, while the IF campaign launched in 2013 claimed that “…rigged rules and deep inequalities… allow a few to make billions while leaving hardworking poor farmers – especially women and their children – and vulnerable and ordinary people everywhere to face the highest prices in a generation” (Enough Food For Everyone IF, 2013: 12).", "As noted earlier, in both cases the political discourse was combined with a premise of ‘absolute scarcity’ in terms of ecological or planetary limits, which must be addressed through local, often technical agroecological, solutions in the case of food sovereignty; or approaches to redistribution in the narratives focused on global inequalities.", "In both cases they focus on local-level solutions – creating new forms of local economy and food sovereignty or addressing over-consumption in particular locales – without necessarily attending to wider structural, political conditions (Bernstein, 2013).", "While they reject globalised versions of scarcity, their frames nevertheless often draw on arguments of limits, and their solutions focus on technological, investment and institutional options, even if these are alternative to the mainstream.", "Thus agroecology is advocated rather than high-tech, industrial agriculture, but the framing of the wider problem often remains technology-centred.", "Despite the NGO and civil society organization authors positioning themselves as in opposition to ‘land grabs’ and large-scale capitalist agriculture, the articulation of a political scarcity framing therefore remains underdeveloped.", "Across our sources a dominant set of narrative themes emerged, constructed through a range of mechanisms and promoted by a powerful group of actors, and underpinned by a framing that draws on theoretical concepts of absolute and relative scarcity.", "An alternative, often poorly articulated, challenge to this is found among sources produced by NGOs and civil society organizations.", "While containing elements of the dominant narrative themes, notably a diagnosis of problems centred on an absolute scarcity theme of ecological crisis and a commitment to technical solutions of different sorts, these narratives did contain a more political dimension, pointing to the consequences of inequality and the advantages of locally-driven development.", "But in relation to the framing of political scarcity introduced earlier, only certain elements were included.", "What then might an alternative narrative, informed by a more comprehensive political scarcity framing, entail, and how might this challenge the dominant powerful narratives of the global land rush?", "To articulate a political framing of scarcity, and associated narratives for policy, means going beyond the empirical material, and returning to our earlier outline of what a political scarcity framing implies.", "At the centre of a political framing of scarcity is the contention that we must restructure the relationships between resources, the state and society.", "If scarcities are constitutive of social, political and economic relationships within society, a new relationship between nature, society and economy has to be negotiated.", "In a political view, as elaborated above, scarcity is constructed in relation to historically-specific patterns and forces of production, distribution and consumption.", "In this way, capitalism generates scarcities through processes of accumulation, but such scarcities have an impact on profits, and so subsequent differential patterns of accumulation across societies and between nations.", "This way of framing scarcity requires paying attention to how resources and the associated flows of benefits are distributed.", "Scarcity for one person may be abundance for another, and scarcities are indeed constructed through unequal access, across all scales (Mehta, 2010a).", "Scarcity as a relational concept cannot be apprehended only by simple metrics or models that focus on absolute scarcities (resource availability, gaps, limits and so on).", "Such assessments need to be embedded in a much deeper understanding of resource control (Peluso and Lund, 2011) and access (Ribot and Peluso, 2003).", "We must ask how resources are distributed across different groups within society, and across the globe, and through what social relations and institutions, in whose interests and with what material effects (Li, 2014).", "Scarcities thus have class, gender and generational dimensions that have major consequences, suggesting a political ecology perspective that takes resource dynamics seriously, but locates these in wider social-political understandings (Peet and Watts, 1996; Rocheleau et al., 1996).", "Scarcities of course emerge historically, and in particular places.", "In Africa, present scarcities are rooted in colonialism and are perpetuated through unequal trade relationships, often reinforced by aid and development interventions that favour one resource use or user group over others (Moyo et al., 2012).", "Scarcities have therefore been created by such imbalances, through excess production and consumption in some parts of the world, and poverty and inequality in others, for example (Jackson, 2011).", "Framings that accept a position of relative scarcity may argue for technological and market interventions to overcome absolute scarcities, but such interventions also may generate new social and political scarcities.", "Technologies and markets are never neutral, yet proponents neglect the inevitable favouring of certain actors and disadvantaging of others.", "The mantras of ‘sustainable intensification’ or ‘responsible innovation’, for example, carry politics and power with them, and can act to exclude as well as include (Loos et al., 2014; Stilgoe et al., 2013).", "Debating the directions of development and their distributional implications is a vital step (STEPS, 2010).", "We must always be clear about the political consequences of scarcity-driven interventions on the structural relations within a society, set within the wider political economy that defines these.", "Scarcity is not universal, given, fixed or determining, but context-specific, socially constructed, politically contested, variable and dynamic; and therefore, always subject to contestation, negotiation and contentious politics, as different groups mobilise around resource issues (Scoones, 2016).", "A political scarcity lens therefore draws attention neither just to finite limits (which of course exist), nor just to the potential opportunities of transformation through markets and technologies (also important in some contexts), but also to the relational qualities of multiple scarcities, being constructed in particular ways by different actors with particular interests.", "Such an approach goes substantially beyond the narratives promoted by NGOs and civil society organizations during 2007–2013 towards a more comprehensive political scarcity framing at the heart of a critique of the global land rush and its dominant framings.", "This has major implications for the debate on Africa’s land rush.", "A switch of focus from land availability or yield gap assessments, with the assumption that improved efficiency and production will arise from a certain style of investment, to a political scarcity framing would emphasise investing in the negotiating capacities of different actors, and most notably disempowered local communities, whose perspectives are currently excluded from the models, assessment and technical appraisals of most policy debates and investment initiatives.", "A technical understanding of absolute scarcity can feed in, to define limits, tipping points and trade-offs, but not dominate.", "Instead, a more open deliberation around how scarcities are understood by different actors would allow more focused debates on the direction of development (for example, is large-scale farm investment the most appropriate option in this setting?)", "and the distribution of benefits (who wins and loses from different investment models?).", "Our analysis of the 135 sources showed the dominance of absolute and relative scarcity representations of Africa’s land rush, and an absence of nuanced political scarcity interpretations, even within alternative positions critical of land investments.", "Recognising that scarcity narratives are constructed does not mean that scarcities are not ‘real’; rather, we show that policy narratives are subject to processes of construction, and that a knowledge politics is at play, with very tangible, material effects.", "Framings and narratives matter in policy and investment behaviour and, as we have seen, some have greater influence than others, with direct effects on how problems are understood and solutions designed and promoted – by governments, development agencies and private-sector actors.", "Opening up this debate, with a more explicit focus on political scarcities is, we argue, urgently required.", "For African settings – seen as both a source of abundant resources and a site where global scarcities may be resolved, as well as where local scarcities are being experienced most acutely – a political scarcity framing on the global land rush, and resource questions more broadly, is essential." ]	[ "Three framings of scarcity are identified: absolute, relative and political.", "A qualitative analysis of 135 global land rush sources from 2007–13 is undertaken.", "Narratives on limits, land-use, investment and comparative advantage are identified.", "These emphasise framings of absolute and relative scarcity not political scarcity.", "The implications of a political scarcity framing are explored." ]
SDNET2018: An annotated image dataset for non-contact concrete crack detection using deep convolutional neural networks	SDNET2018 is an annotated image dataset for training, validation, and benchmarking of artificial intelligence based crack detection algorithms for concrete. SDNET2018 contains over 56,000 images of cracked and non-cracked concrete bridge decks, walls, and pavements. The dataset includes cracks as narrow as 0.06 mm and as wide as 25 mm. The dataset also includes images with a variety of obstructions, including shadows, surface roughness, scaling, edges, holes, and background debris. SDNET2018 will be useful for the continued development of concrete crack detection algorithms based on deep convolutional neural networks (DCNNs), which are a subject of continued research in the field of structural health monitoring. The authors present benchmark results for crack detection using SDNET2018 and a crack detection algorithm based on the AlexNet DCNN architecture. SDNET2018 is freely available at https://doi.org/10.15142/T3TD19.	[ "MULT" ]	[]	[ "1000" ]	[ "The SDNET2018 image dataset contains more than 56,000 annotated images of cracked and non-cracked concrete, bridge decks, walls, and pavements.", "Its purpose is for training, validation, and benchmarking of autonomous crack detection algorithms based on image processing, deep convolutional neural networks (DCNN) [8], or other techniques.", "Such techniques are increasing in popularity in the structural health monitoring field.", "Continued advancement of crack detection algorithms requires an annotated diverse image dataset [9], which has not been available until now.", "Images of bridge decks were taken at the Systems, Materials, and Structural Health (SMASH) Laboratory at Utah State University, where a number of full scale bridge deck sections were stored.", "Images of walls and pavements were taken on Utah State University campus.", "Table 1 lists the number of cracked, non-cracked, and total sub-images of each type included in SDNET2018.", "The sample images in Fig. 1 show the range of crack widths, surface conditions, and other environmental factors represented within SDNET2018.", "Images are 256 × 256-px RGB image files in .", "jpg format.", "Each image is classified as cracked or non-cracked and stored in a corresponding folder within the repository.", "Images are organized into three sub-directories: P for pavements, W for walls, and D for bridge decks.", "Each subfolder is further organized into sub-sub-directories with the prefix C for cracked and U for uncracked (e.g.,:/D/CD for images of bridge decks with cracks).", "With the exception of segmentation into sub-images as discussed above, the images have not been modified from their original state.", "SDNET2018 images were taken with a 16-MP Nikon camera at a working distance of 500 mm without zoom.", "The sensitivity was 125 ISO and the image resolution was 4068 × 3456 px.", "The surface illumination was between 1500 and 3000 lx.", "Each full image was segmented into 256 × 256-px sub-images.", "Each image represents a physical area of approximately 1000 mm × 850 mm and each sub-image represents a physical area of approximately 60 mm × 60 mm.", "The authors analyzed the SDNET2018 dataset using the AlexNet DCNN architecture in fully trained (FT) and transfer learning (TL) modes using the computational setup and procedure described by Dorafshan et al. [8].", "Benchmarking results, including the sizes of the training and testing datasets, number of epochs required for training, and accuracy of classification of the testing dataset, are presented in Table 2." ]	[]
Nitrate removal effectiveness of fluidized sulfur-based autotrophic denitrification biofilters for recirculating aquaculture systems	There is a need to develop practical methods to reduce nitrate-nitrogen loads from recirculating aquaculture systems to facilitate increased food protein production simultaneously with attainment of water quality goals. The most common wastewater denitrification treatment systems utilize methanol-fueled heterotrophs, but sulfur-based autotrophic denitrification may allow a shift away from potentially expensive carbon sources. The objective of this work was to assess the nitrate-reduction potential of fluidized sulfur-based biofilters for treatment of aquaculture wastewater. Three fluidized biofilters (height 3.9m, diameter 0.31m; operational volume 0.206m<sup>3</sup>) were filled with sulfur particles (0.30mm effective particle size; static bed depth approximately 0.9m) and operated in triplicate mode (Phase I: 37-39% expansion; 3.2-3.3min hydraulic retention time; 860-888L/(m<sup>2</sup>min) hydraulic loading rate) and independently to achieve a range of hydraulic retention times (Phase II: 42-13% expansion; 3.2-4.8min hydraulic retention time). During Phase I, despite only removing 1.57±0.15 and 1.82±0.32mg NO<inf>3</inf>-N/L each pass through the biofilter, removal rates were the highest reported for sulfur-based denitrification systems (0.71±0.07 and 0.80±0.15gN removed/(L bioreactor-d)). Lower than expected sulfate production and alkalinity consumption indicated some of the nitrate removal was due to heterotrophic denitrification, and thus denitrification was mixotrophic. Microbial analysis indicated the presence of Thiobacillus denitrificans, a widely known autotrophic denitrifier, in addition to several heterotrophic denitrifiers. Phase II showed that longer retention times tended to result in more nitrate removal and sulfate production, but increasing the retention time through flow rate manipulation may create fluidization challenges for these sulfur particles.	[ "AGRI" ]	[ "Autotrophic", "Denitrification", "Fluidized biofilter", "Mixotrophic", "Recirculating aquaculture", "Sulfur" ]	[ "1104" ]	[ "The global demand for food protein must be balanced with increased concern for the environmental impact caused by these production systems.", "Land-based closed-containment aquaculture using recirculating aquaculture systems (RAS) are uniquely poised to produce highly desirable and valuable food products while also maintaining a small environmental footprint.", "However, while most RAS are designed to remove solids and recycle water back to the fish culture tanks (Summerfelt and Vinci, 2008; Timmons and Ebeling, 2010), the inability of these systems to remove nitrate–nitrogen from the water significantly arrests this industry's ultimate economic and environmental sustainability.", "For these aquaculture systems to more completely address environmental issues, it is now critical that efforts focus upon the reduction of nitrogen species in effluent waters.", "Importantly, the ability to confidently and consistently remove nitrate nitrogen from RAS effluent may allow expansion of this industry into locales currently bound by stringent water quality standards and may potentially allow increased reuse of treated effluents.", "There is a crucial need to develop practical and cost effective methods to reduce RAS nitrate–nitrogen loads to allow their maintained or increased productivity simultaneously with attainment of water quality goals and good environmental stewardship.", "A major disadvantage of this process is that autotrophs generally grow at a slower rate than heterotrophs, thus have lower denitrification rates (Sahinkaya and Kilic, 2014).", "Major by-products of concern from sulfur-based autotrophic denitrification are sulfate and acidity (Sahinkaya and Kilic, 2014) with 4.57 mg CaCO3 alkalinity consumed and 7.54 mg sulfate produced for each mg NO3–N reduced (Sahinkaya et al., 2014).", "Many sulfur-based denitrification studies use a mix of sulfur and limestone or dolomite to buffer pH and alkalinity decreases (Sahinkaya and Kilic, 2014; USEPA, 1978).", "The presence of sulfur in the system combined with low-oxygen conditions could also lead to sulfide production, though not as a result of Eq. (1).", "An additional challenge is that elemental sulfur is relatively water insoluble, meaning it has a limited microbial availability at room temperature.", "Batchelor and Lawrence (1978) outlined that for elemental sulfur-based denitrification to proceed, three steps were necessary: (1) the sulfur must be solubilized, and (2) nitrate must be transported from solution to the biofilm surface, where (3) it can be transported through the film so it can be denitrified.", "Sulfur-based autotrophic denitrification in static beds has proven successful for treating nitrate in groundwater, landfill leachate, and wastewaters (Koenig and Liu, 1996; Lee et al., 2008; Shao et al., 2010), and this approach presents a unique option for treatment of aquaculture effluents (Sher et al., 2008).", "Nitrogen removal rates from previous laboratory studies are generally on the order of 0.1–0.4 g N/(L d) (Lampe and Zhang, 1996; Sahinkaya and Kilic, 2014; Sahinkaya et al., 2014).", "Nitrogen (N) removal performance may be limited by N loading with Kim et al. (2004) observing a decline in N removal beyond loading rates of 2.5 kg NO3–N/(m3-d), and Koenig and Liu (1996) noting that areal based loading rates (g N/m2-d) were their limiting factor in a packed sulfur bed.", "In an aquaculture application, Sher et al. (2008) reported the use of autotrophic denitrification provided a dual benefit for recirculated waters; not only were nitrate levels brought under control, but the oxidation of sulfide in the anaerobically digested sludge helped safeguard against sulfide toxicity within the system.", "Fluidized bed reactors are a proven aquaculture water treatment technology due to their plugging prevention, ease of maintenance, low cost and efficient treatment (Summerfelt, 2006).", "Because fluidized sand biofilters are common in this industry, their application as fluidized sulfur autotrophic denitrification reactors could be a natural extension of the technology.", "Fluidized sulfur biofilters have been researched at the lab scale, with Kim et al. (2004) showing higher N removal rates from fluidized sulfur beds than packed sulfur beds.", "This was due to the absence of clogging and good nitrate transfer to the sulfur surface in the fluidized system.", "In previous work, Christianson and Summerfelt (2014) determined fluidization velocities of commercially-available sulfur flakes, grains, and powder, and concluded the grains provided the most realistic option for full-scale testing of a fluidized sulfur-based denitrification biofilter.", "The objective of this work was to assess the nitrate reduction potential of fluidized sulfur-based biofilters for treatment of aquaculture wastewater.", "Three fluidized sulfur biofilters (285 L, height 3.9 m, diameter 0.31 m; Fig. 1) were operated at The Conservation Fund's Freshwater Institute (Shepherdstown, West Virginia, USA) for 253 days to quantify nitrate removal from aquaculture wastewater (Phase I: 225 days, 13 March 2014 to 23 October 2014; Phase II: 28 days, 24 October 2014 to 20 November 2014).", "During Phase I, the three biofilters were operated in triplicate fashion, each fluidized at 37–39% expansion with a hydraulic retention time (HRT) of 3.2–3.3 min and a hydraulic loading rate of 860–888 L/(m2 min) based on the mean flow rate of 63–65 L/min.", "Two Phase I study periods of relatively consistent influent nitrate concentrations were selected for analysis; Periods 1 and 2 allowed evaluation at influent concentrations of 2.0–5.0 and 7.6–17 mg NO3–N/L, respectively (days 57–92 and 190–225, respectively; six sample events each).", "Phase II utilized a different flow rate in each biofilter to assess the impact of HRT on nitrate removal (i.e., no replication; 42–13% expansion; 3.2–4.8 min HRT; 67–43 L/min flow rate; influent 8.5–15 mg NO3–N/L).", "The waste and wastewater treatment system and biofilter design has been previously described by Tsukuda et al. (2015) (Fig. 2).", "In short, waste sludge from the production of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), was concentrated via microscreen drum filters and radial flow settlers and was pumped to a series of gravity thickening settling cones.", "A holding tank for the supernatant overflow from the settling cones fed the three fluidized denitrification biofilters.", "Overflow from each biofilter was treated using a radial flow settler.", "Biofilter bed height (2.82 m; biofilter volume 0.206 m3) was controlled with a shearing pump at the top of each biofilter.", "The static sulfur bed depth was approximately 0.9 m, although the sulfur grains in all three biofilters were replenished on days 181 and 198 following an undetected wash-out (68 kg or approximately 0.75 m S per biofilter total replenished).", "Biofilter influent nitrate levels were manipulated by dosing a concentrated sodium nitrate (NaNO3; 34.0 g NO3–N/L) solution into the supernatant holding tank.", "The spring water feeding the RAS was naturally alkaline (≈275 mg CaCO3/L), resulting in high alkalinity of flows.", "The sulfur grains had effective and calculating sizes of 0.30 (D10) and 1.31 mm (D90), respectively, and a uniformity coefficient of 3.1 (Georgia Gulf Sulfur, Customer Code 1660, distributed by Prince Agri-Products, Inc., Quincy, Illinois, USA; Christianson and Summerfelt, 2014).", "This is smaller than reported particle size ranges for other sulfur-based denitrification studies as most have used grains ranging from 2 to 16 mm (Koenig and Liu, 1996; Oh et al., 2003; Sahinkaya et al., 2014).", "Sahinkaya and Kilic (2014) reported using the most comparable size (0.5–1.0 mm grains) in a packed column study, and in the only reported fluidized bed study, Kim et al. (2004) used 2.0–3.35 mm sulfur grains.", "The smaller grain size used here provided a desirable high specific surface area (SSA bed: 4110 m2/m3) relative to, for example, a 4.4 mm mean particle size sulfur product that had a 1363 m2/m3 SSA (Koenig and Liu, 1996).", "Elemental sulfur powder was initially used in the fluidized biofilters, but was discontinued due to fluidization and wash-out challenges.", "Lampe and Zhang (1996) similarly reported difficultly with powdered sulfur in a batch reactor (i.e., uniform mixing was problematic).", "Water quality samples were collected from a sampling valve located at the back of each of the three biofilters and directly from the influent supernatant tank (i.e., effluent sample values were pooled as replicates during Phase I, n = 3; influent samples, n = 1).", "Water chemistry was analyzed weekly onsite, and both study phases followed the same sampling routine.", "Samples were analyzed for chemical oxygen demand (COD), carbonaceous biochemical oxygen demand (cBOD5), total ammonia nitrogen (TAN), nitrite–nitrogen (NO2–N), nitrate–nitrogen (NO3–N), total nitrogen (TN), alkalinity, pH, sulfate (SO42−), sulfide (S2−), total suspended solids (TSS), total phosphorus (TP), and dissolved reactive phosphorus (DRP) using methods from APHA (2005) and Hach Company (2003).", "Temperature, dissolved oxygen (DO), and oxidation reduction potential (ORP) were assessed at least twice weekly.", "Measurements were made directly from the supernatant tank (influent) and the open biofilter tops (effluent) utilizing both inline and handheld probes (HACH HQ40d Portable meter with either HACH IntelliCAL LDO101 or MTC101 ORP/redox probe; HACH pHD sc Differential ORP sensor with HACH sc100 controller; HACH Advanced LDO Process Dissolved Oxygen Probe with HACH sc200 controller).", "Flow rate was measured along the influent pipes to each biofilter and adjusted at least twice weekly concurrently with temperature, DO, and ORP readings, as well as prior to the weekly water chemistry sampling event (Dynasonics DXN Portable Ultrasonic Measurement System).", "Samples for screening the potential denitrification community were collected from all three biofilters on the final day of Phase I Period 2 (day 225).", "Biofilm attached to the sulfur media were detached by vigorously vortexing a sample of sulfur media/biofilter water in 50 mL sterile plastic conical tubes for 5 min.", "The resulting suspensions of detached surface layer (SL) biofilms were centrifuged at 10,000 × g at 4 °C for 20 min prior to DNA extraction.", "Following SL biofilm detachment, the media were directly used for DNA extraction of inner layer (IL) biofilm.", "The genomic DNA were extracted from each reactor's SL and IL biofilm using a PowerSoil DNA Extraction Kit (MO BIO Laboratories, Inc., Carlsbad, CA) following the manufacturer's protocol.", "The concentration and quality of extracted DNA were determined by absorbance at 260 nm and 260/280 nm ratio, respectively (NanoDrop 2000c UV-Vis spectrophotometer; Thermo Fisher Scientific, Inc., Wilmington, DE).", "Isolated DNA was stored at −20 °C.", "Microbial community DNA extracted from three biofilters were pooled in equal quantity and used to amplify nosZ fragments which encode the catalytic subunit Z of nitrous oxide reductase.", "Primers nosZ-F (5′-CGYTGTTCMTCGACAGCCAG-3′) and nosZ-R (5′-CATGTGCAGNGCRTGGCAGAA-3′) yielding approx. 700 bp fragments (Rösch et al., 2002) were used.", "PCR reaction mixtures were prepared to contain 2× Taq PCR Master Mix (QIAGEN, Gaithersburg, MD), 6 pmol of each forward and reverse primers, and 100 ng of genomic DNA in a final volume of 20 μL.", "The PCR amplification was carried out as following: initial denaturation step at 94 °C for 4 min; one cycle at 94 °C for 20 s (denaturation), at 65 °C for 30 s (annealing), and at 72 °C for 40 s (elongation); two cycles at 94 °C for 20 s (denaturation); at 62 °C for 30 s (annealing); 72 °C for 40 s (elongation); three cycles at 94 °C for 20 s (denaturation); at 59 °C for 30 s (annealing); at 72 °C for 40 s (elongation); five cycles at 94 °C for 20 s (denaturation); at 57 °C for 30 s (annealing); at 72 °C for 40 s (elongation); twenty four cycles at 94 °C for 20 s (denaturation); at 55 °C for 30 s (annealing); at 72 °C for 40 s (elongation); and then, final extension at 72 °C for 10 min in a PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA).", "A negative control prepared without DNA was included in every PCR reaction performed to test for false positives caused by contamination.", "PCR products were separated and visualized by electrophoresis in 1.2% agarose gel stained with EtBr, and were purified from excised gel slices (about 700 bp size band) using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA).", "Purified SL and IL nosZ amplicons were ligated into pCR4_TOPO vector, and vector with insert were transformed into OneShot TOP10 chemically competent Escherichia coli cells using TOPO TA Cloning Kit following the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA).", "Ninety-six total clones were randomly selected from each SL and IL nosZ library and were cultured for plasmid preparation.", "Plasmid DNAs were purified (Agencourt SprintPrep 384 HC Kit, Agencourt Bioscience, Beverly, MA) and sequencing was performed using an ABI PRISM genetic analyzer (Applied Biosystems, Foster City, CA) with T7 and T3 primers provided in the cloning kit (Invitrogen) at the Biological Analysis Service Laboratory, Institute of Marine and Environmental Technology (Baltimore, MD).", "Sequences were edited and assembled using Sequencher software (Gene Code Corp., Ann Arbor, MI, USA), were analyzed using the Basic Local Alignment Search Tool (BLASTn; http://www.ncbi.nlm.nih.gov/BLAST), and were compared with available sequences in the GenBank database to create neighbor joining phylogenetic trees to aid the selection of the closest reference sequences.", "The 41 partial nosZ sequences that were generated in this study have been deposited in GenBank database under accession numbers KT252910 to KT252950.", "Nitrate reduction was observed during both Phase I periods (Fig. 3a).", "Although differences between influent and effluent nitrate concentrations were relatively small (Table 1; 1.57 ± 0.15 and 1.82 ± 0.32 mg NO3–N/L for the two periods, respectively), the high flow rates and compact biofilter volume resulted in mean removal rates of 0.71 ± 0.07 and 0.80 ± 0.15 g N removed/(L bioreactor-d) for the two periods, respectively.", "This is much higher than the previously reported range of 0.1 to 0.4 g N/(L d) for sulfur-based denitrification (Lampe and Zhang, 1996; Sahinkaya and Kilic, 2014; Sahinkaya et al., 2014), but similar to the low end of the range for fluidized sand biofilter heterotrophic N removal rates of 0.86–1.74 g N/(L d) (or 35.8–72.6 mg NO3–N/(L h); reviewed by van Rijn et al., 2006).", "Relative to previous experiments with these biofilters, Tsukuda et al. (2015) reported removal rates of 0.4 g N/(L d) when they were operated with fluidized sand.", "Christianson and Summerfelt (2014) reported sand was much less expensive than sulfur products for fluidized biofilters on both a volumetric and surface area basis ($70–$200/m3 vs. >$1000/m3, respectively; $0.02/m2 surface area vs. ≈$0.30/m2 surface area, respectively), though a fluidized sand biofilter would also require purchase of a carbon source to fuel denitrification.", "Influent loading averaged 1.46 and 5.82 g N/(L d) for Periods 1 and 2, respectively.", "Nitrate removal efficiencies averaged 50 ± 4.6% and 16 ± 3.2% for the two Phase I study periods, with the relatively high efficiency for Period 1 due to the low influent nitrate concentration.", "Theoretically, the production of sulfate is proportional to the extent of autotrophic denitrification, thus sulfate production may be the best indicator of this process (Oh et al., 2003; Sahinkaya et al., 2014).", "Based on Eq. (1) and average removals of 1.57 and 1.82 mg NO3–N/L, Periods 1 and 2 should have produced an average of 11.8 and 13.7 mg SO42−/L. However, only 2.7 ± 2.0 and 6.1 ± 1.6 mg SO42−/L were produced during these two periods, with no statistically significant difference between influent and effluent sulfate concentrations for either Periods 1 or 2 (Table 1; Fig. 3b).", "This is an indication that some of the N removal was potentially due to heterotrophic denitrification in addition to autotrophic.", "Just as the elemental sulfur was converted to sulfate, some sulfide present in solution was also oxidized (Fig. 3c; Table 1; mean removal: 6.19 ± 1.82 and 8.64 ± 1.04 μg S2−/L).", "Sher et al. (2008) observed that a RAS sludge digestion basin also provided autotrophic denitrification treatment with sulfide as the electron donor.", "Dual functionality of nitrate and sulfide removal would be a more significant benefit for RAS waters being recirculated to fish culture tanks as compared to the treatment of effluent waters here.", "Reduced alkalinity, another indicator of autotrophic denitrification, was observed here with average decreases of 16 and 12 mg CaCO3/L from the two Phase I study periods (Table 2).", "Others have reported significant drops in alkalinity during sulfur-based denitrification studies (Koenig and Liu, 1996), and this may be the largest operational challenge of such a system (Kim and Bae, 2000).", "The naturally alkaline spring water used in the on-site RAS here was considered well-buffered enough to not require alkalinity addition as Furumai et al. (1996) reported the optimum alkalinity for sulfur-based autotrophic denitrification was 150–240 mg/L.", "Based on Eq. (1), removal of 1.57 and 1.82 mg NO3–N/L should have resulted in alkalinity consumption of only 7.2 and 8.3 mg CaCO3/L for Periods 1 and 2, respectively.", "Likewise, based on N removal rate (0.71 and 0.80 g N/(L d)), alkalinity consumption should have been 3.2 and 3.7 g CaCO3/(L d) although it averaged 7.1 ± 2.7 and 5.3 ± 2.3 g CaCO3/(L d) for the two periods.", "The simultaneous occurrence of heterotrophic denitrification would have reduced alkalinity consumption rather than increased consumption, and while nitrification can consume alkalinity, there was no consistent change in TAN concentrations across the biofilters.", "Degradation of possible accumulated sludge within the biofilter may have consumed some alkalinity, although this could not be verified.", "The variability in alkalinity standard error complicated further analysis.", "No major pH changes were observed with the influent and effluent both averaging between 7.33 and 7.39 for both periods (Table 2).", "Others have observed notable pH decreases (Koenig and Liu, 1996; Sahinkaya and Kilic, 2014) with nitrite accumulation possible at pH below 7.4 (Furumai et al., 1996).", "There was no accumulation of nitrite here as levels were generally slightly reduced over the biofilters (Table 1; Fig. 4).", "Water temperature between the influent and effluent did not notably vary, although a seasonal trend was observed.", "Temperatures peaked between days 100–150 (20 June 2014–09 August 2014) during the warmest time for these greenhouse-run experiments (Fig. 5a).", "As expected, effluent DO concentrations were reduced to less than 1.0 mg DO/L when the columns were operating as intended (Fig. 5b), and to less than 0.5 mg DO/L during both analysis periods (Table 2).", "This indicated a strong aerobic and/or facultative anaerobic component existed within the biofilters.", "Facultative heterotrophic denitrifiers use free oxygen as their electron acceptor while it is available, because oxygen is a more energetically favorable electron acceptor than nitrate.", "Thus, heterotrophic denitrification and the use of nitrate as an electron acceptor is reduced when free oxygen is still present.", "Autotrophic denitrification has been documented under both aerobic and anaerobic conditions (Zhang and Lampe, 1999).", "The potential impact of the sulfur wash-out was evident as early as day 150 when effluent DO levels increased; additional sulfur was added on days 181 and 198.", "Oxidation reduction potentials were highly variable though always negative, and were reduced slightly across the biofilter during Period 1 but increased during Period 2 (Table 2; Fig. 5c).", "This increase in ORP across the biofilters was mainly apparent because the influent ORP was more reduced during this period; influent water quality throughout the experiment was variable and somewhat uncontrollable due to the nature of this production aquaculture facility's waste stream.", "The term “mixotrophic denitrification” refers to the simultaneous occurrence of heterotrophic and autotrophic denitrification (Oh et al., 2003; Sahinkaya and Kilic, 2014).", "With this relatively high COD and cBOD5 wastewater, it is likely mixotrophic denitrification was occurring.", "Oh et al. (2003) observed addition of a variety of soluble organic sources (methanol, ethanol, acetate) did not inhibit autotrophic denitrification, although supplementation of organic carbon in excess did decrease sulfate production.", "Balancing the autotrophic/heterotrophic reactions can reduce the alkalinity requirement caused by autotrophic denitrification due to alkalinity produced by heterotrophs (Kim and Bae, 2000; Lee et al., 2001; Oh et al., 2003).", "Because heterotrophs grow faster than autotrophs, some organic carbon forms may be preferentially utilized before sulfur in a mixotrophic denitrification reactor (Sun and Nemati, 2012).", "Availability of the electron donor may play a role in this as limited dissolution of solid sulfur particles can limit denitrification, especially at higher N loading rates (Kim et al., 2004).", "Suitable COD:NO3–N ratios for heterotrophic denitrification are on the order of 3:1 to 6:1(van Rijn et al., 2006), and influent values here averaged 74 ± 7.5 and 7.2 ± 1.3 COD:NO3–N for Periods 1 and 2, respectively, more than sufficient to fuel heterotrophic denitrification (cBOD5:NO3–N of 24 ± 4.9 and 3.3 ± 0.5).", "However, during Periods 1 and 2, COD was only reduced 8.5 ± 19 and 2.4 ± 3.0 mg COD/L, respectively, and cBOD5 concentrations were not reduced across the biofilters (Table 1).", "The COD:NO3–N utilization ratios were 5.4 and 1.3 mg COD consumed per mg N removed for the two periods, respectively.", "The very low utilization ratio for Period 2 potentially indicated relatively more of the N removal was due to autotrophic vs. heterotrophic denitrification compared to Period 1.", "The absence of measureable cBOD5 reductions was likely due to the extremely short HRTs.", "While it is likely that heterotrophic denitrification did account for some of the nitrate removal, internal cycling of solids may have complicated the COD balances.", "Sequence analysis of 96 randomly selected clones from each the biofilm surface layer (SL) and inner layer (IL) nosZ libraries revealed fourteen unique operational taxonomic units (OTUs) for SL and nine for IL (Table 3).", "The % Clone of similar sequences in a library were calculated for SL-nosZ and IL-nosZ, separately (Table 3, upper for SL-nosZ and lower for IL-nosZ).", "The nosZ library clones in the SL belonged to: Alphaproteobacteria (19.6%), Betaproteobacteria (76.5%), and unclassified bacterium (4.3%); and in the IL belonged to: Alphaproteobacteria (2.2%), Betaproteobacteria (17.5%), and unclassified bacterium (80.4%).", "Similarly to previous fluidized sand biofilter denitrification studies (Tsukuda et al., 2015), the denitrifying microbial population containing the nosZ gene in the SL was more diverse than in the IL.", "Here, more than 80% of IL-nosZ clones were closely related to the uncultured bacterium clone 2–80 (Accession JF509076.1).", "This lack of diversity may have been the result of lower DO and higher sulfur availability (electron donor) in the IL.", "Uncultured bacterium clone 2–80 were also found in the SL biofilm, but their much higher % clone in the IL (4.3 vs. 80.4% in SL vs. IL, respectively) may be an indication there are sulfur-utilizing autotrophic denitrifiers that have not yet been isolated or identified.", "The microbial communities indicated that the encoding key enzyme for denitrification (nosZ) in the SL was largely from Azoarcus, Thauera and Paracoccus spp., which are known as heterotrophic denitrifiers, and their presence suggests the geochemical conditions near the SL were suitable for heterotrophic denitrification compared to conditions in the IL.", "In contrast, nosZ sequences belonging to Thiobacillus denitrificans, an obligate chemolithoautotrophic denitrifier, were dominant in the IL denitrifying microbial community suggesting IL provided a suitable cultivating environment for autotrophs, although this was only 4.3% of the IL-nosZ clones (80.4% were uncultured bacterium clone 2–80).", "The optimal growth temperature of T. denitrificans is between 28 and 32 °C (Shao et al., 2010), and lower water temperatures here (13–22 °C; Fig. 5a) may have influenced this relatively low percentage.", "Among known autotrophic denitrifiers, the obligate chemolithoautotroph, T. denitrificans, was the first to be isolated and characterized, is capable of utilizing thiosulfate, tetrathionate, thiocyanate, sulfide and elemental sulfur as the electron donor for denitrification, and is the most commonly reported autotrophic denitrifier (Park et al., 2010, 2011; Chen et al., 2013; Xu et al., 2014).", "The detection of nosZ genes from autotrophic denitrifiers in both SL and IL biofilms strongly indicated the capability of fluidized sulfur biofilters to cultivate and enrich autotrophic denitrifying bacteria for removal of nitrate, even under relatively short HRTs compared to packed sulfur reactor studies.", "In addition, the co-existence of autotrophic and heterotrophic denitrifiers suggests these reactors provided conditions to cultivate both types of bacteria which can offer unique and efficient mixotrophic nitrate removal (Oh et al., 2001).", "When each biofilter was operated independently, N removal and sulfate production showed a weakly increasing trend at increasing HRTs (Fig. 6a and b).", "Based on the regression slope (−0.0405 g N/(L d) per L/min of flow rate), decreasing the flow rate approximately 20 L/min would provide an additional 0.81 g N removed/(L d) which equated to an additional 167 g N removed/d for these biofilters.", "Removal of sulfide also tended to increase at higher HRTs, though this regression was even less strongly correlated (Fig. 6a).", "For fluidized systems, this reduction in flow rate to achieve a longer HRT is a tradeoff resulting in less fluidization of the sulfur particles, and thus the HRT would need to be increased via a larger biofilter.", "A recommended 60% expansion, as was modeled in Christianson and Summerfelt (2014), would have required a flow rate of over 80 L/min and yielded an HRT of only 2.5 min in these biofilters.", "Other reported HRTs for packed or continuously stirred sulfur denitrification reactors have been on the order of 3 to 24 h (Lampe and Zhang, 1996; Lee et al., 2001; Sahinkaya and Kilic, 2014; Sahinkaya et al., 2014).", "Koenig and Liu (1996) reported the required HRT for complete N reduction depended upon the sulfur particle size, and showed greater than 30 min was required for a 40% nitrate removal efficiency using their smallest sulfur size fraction (2.8–5.6 mm) in packed beds.", "At a loading of 2.2 kg N/(m3-d), Kim and Bae (2000) reported an HRT of 2.34 h in a packed bed provided complete denitrification.", "Loading during Phase II was between 2.7 and 6.7 kg N/(m3 biofilter-d), thus a greater HRT, in a packed bed at least, would have been required for complete N removal.", "The only comparable fluidized bed study reported an HRT of 0.19 h (empty bed contract time) and bed expansion of 25–30% (2–3.35 mm sulfur particle size; Kim et al., 2004).", "Under these conditions, greater than 90% removal efficiency was achieved from an influent concentration of 20 mg NO3–N/L.", "However, Kim et al. (2004) also reported a decline in N removal-performance when N loading exceeded 2.53 kg N/(m3-d), as the present study did.", "Ideally, this study would have been improved if the biofilters were 2–3 m taller or if a slightly smaller-sized sulfur particle could have been identified, because both options would have increased the HRT within the denitrification bed.", "Despite only removing 1.57 ± 0.15 and 1.82 ± 0.32 mg NO3–N/L each pass through the biofilter during Phase I, removal rates were the highest reported for sulfur-based denitrification systems (0.71 ± 0.07 and 0.80 ± 0.15 g N removed/(L bioreactor-d)).", "Lower than expected sulfate production indicated some of the nitrate removal was due to heterotrophic denitrification although there was no statistically significant decrease in COD or cBOD5 concentrations between the influent and effluent.", "Mixotrophic denitrification was verified via the presence of both heterotrophic and autotrophic denitrifiers.", "Phase II tended to indicate that longer retention times may result in more nitrate removal and sulfate production, but increasing the retention time through flow rate manipulation may create fluidization challenges for these sulfur particles.", "Operationally, the sulfur particles will degrade over time, and optimizing the balance of fluidization velocity versus HRT may be challenging." ]	[ "Autotrophic denitrification treatment is underexplored in aquaculture applications.", "Wastewater nitrate removal in fluidized sulfur biofilters was evaluated.", "Nitrate removal rates were high due to heterotrophic and autotrophic activity.", "Longer retention times tended toward more N removal and sulfate production." ]
Strontium isotope evidence for human mobility in the Neolithic of northern Greece	Strontium isotope ratios are widely used in archaeology to differentiate between local and non-local populations. Herein, strontium isotope ratios of 36 human tooth enamels from seven archaeological sites spanning the Early to Late Neolithic of northern Greece (7th–5th millennia B.C.E.) were analysed with the aim of providing new information relating to the movement of humans across the region. Local bioavailable 87Sr/86Sr signals were established using tooth enamel from 26 domestic animals from the same Neolithic sites. 87Sr/86Sr values of faunal enamel correlate well with predicted strontium isotope ratios of the local geology. This is consistent with animal management occurring at a local level, although at Late Neolithic sites strontium isotope values became more varied, potentially indicating changing herding practices. The strontium isotope analysis of human tooth enamel likewise suggests limited population movement within the Neolithic of northern Greece. Almost all individuals sampled exhibited 87Sr/86Sr values consistent with having spent their early life (during the period of tooth mineralisation) in the local area, although movement could have occurred between isotopically homogeneous areas. The strontium isotope ratios of only three individuals lay outside of the local bioavailable 87Sr/86Sr range and these individuals are interpreted as having spent their early lives in a region with a more radiogenic biologically available 87Sr/86Sr. Mobility patterns determined using Sr isotope analysis supports the current evidence for movement and exchange observed through studies of pottery circulation. Suggesting limited movement in the Early and Middle Neolithic and greater movement in the Late Neolithic.	[ "ARTS", "SOCI" ]	[ "Animals", "Greece", "Human", "Mobility", "Neolithic", "Strontium isotopes" ]	[ "1204", "3302" ]	[ "The application of strontium isotope analyses to archaeological skeletal remains can provide information regarding the movement of humans and animals by comparing the strontium isotope signature of an individual to the biologically available signature determined by the surrounding biosphere (Bentley, 2006).", "Given suitable variation in the biologically available signatures in a region, strontium isotope ratios are able to differentiate between local and non-local populations.", "This can be used as complementary evidence to that obtained from the traded and exchanged goods, identified through material cultural remains, which together can provide a record of movement and exchange networks across a region.", "Strontium isotope analyses have thus been widely utilised to explore the mobility of early Hominins (Balter et al., 2012), Pliocene mammals (Hoppe et al., 1999), Neanderthal populations (Britton et al., 2011) and Pre-historic human and animal mobility (Grupe et al., 1997; Viner et al., 2010; Bentley, 2013; Boric and Price, 2013; Giblin et al., 2013; Gerling, 2015; Henton et al., 2017).", "Evidence for movement and exchange has been identified in the Greek Neolithic from the movement of material culture, including pottery, stone tools and shell ornaments.", "The study of fine-decorated pottery assemblages has shown that the circulation of pottery in northern Greece was very limited (Pentedeka, 2011; Urem-Kotsou et al., 2014).", "Due to the low amount of pottery found in the Early Neolithic (EN) it is difficult to ascertain if exchanges of pottery took place (Perlès and Vitelli, 1999).", "However, indications of pottery exchange, in the form of imported pottery is found in the Middle Neolithic (MN) where stylistic developments were shared, and small-scale exchanges took place (Perlès and Vitelli, 1999; Pentedeka, 2011; Urem-Kotsou et al., 2014).", "Analogous traits between vessel technology, shapes and cultural styles infers networks of communication and exchange operational on regional and inter-regional levels at this time (Çilingiroglu, 2010; Dimoula et al., 2014; Urem-Kotsou et al., 2016).", "Analysis of lithics from across the period have shown that stone tools were produced in the vicinity, from locally sourced raw materials (Perlès and Vitelli, 1999; Pentedeka, 2011).", "In the Late Neolithic (LN), common pottery wares (black burnished and red slipped) are widespread, thus making it difficult to determine if the pots are locally produced or exchanged (Perlès and Vitelli, 1999).", "Widespread distribution of materials with limited production sites, such as obsidian, flint and jasper have been identified in the LN (Perlès and Vitelli, 1999; Milić, 2014) as have the movement of stone and spondylus shell ornaments, which circulated over large distances (Pentedeka, 2011; Veropoulidou, 2014).", "This demonstrates contact between human groups, however, given the small sample size and the number of sites and locations, any suggestion of diachronic change should arguably be made more cautiously.", "In contrast, 87Sr/86Sr values from human and animal teeth can be used to infer mobility over longer distances.", "Thus far, the use of strontium isotope ratios to identify mobility during the Neolithic of Greece has been restricted to Crete.", "This small scale study demonstrated that mobility in Crete during the Neolithic was limited or that movements occurred between areas of homogenous underlying geology (Triantaphyllou et al., 2015).", "To date, no isotopic studies have been conducted on other Neolithic Greek assemblages and aside from movements identified through material culture (Perlès, 2001; Urem-Kotsou et al., 2014; Triantaphyllou et al., 2015) little is known about human mobility in northern Greece.", "A greater number of mobility studies using strontium isotope ratios have been conducted on Bronze Age tooth enamel and modern reference material from sites situated in the southern Greek mainland and Crete (Nafplioti, 2008; Nafplioti, 2009, 2011).", "Consequently, this study of strontium isotope analysis on human tooth enamel aims to shed light on the extent of mobility throughout the Neolithic of northern Greece.", "The study area in the central and western regions of Greek Macedonia comprises fertile basins separated by mountain ranges.", "Three broad NE-SW trending geographical ranges give rise to three geological zones bounded by faults which share a common history of deposition and formation (Higgins and Higgins, 1996).", "The western Pelagonian zone, comprising Triassic and Jurassic limestones and marbles deposited over gneiss.", "The central Vardar zone, once part of the Tethys Ocean, is made up of Mesozoic deep-water sedimentary and ophiolites, occasionally overlain by limestone and Eocene sediments.", "The eastern Serbo-Macedonian massif is dominated by metamorphic and plutonic rocks uplifted and faulted by Alpine compressions (Higgins and Higgins, 1996).", "Strontium isotope analyses were performed on 36 human tooth enamels from seven archaeological sites spanning the Early to Late Neolithic of northern Greece (Fig. 1).", "The sites in this study were chosen to chronologically span a large period of the Neolithic and, in addition, cover a range of geographical environments and terrains from coastal to inland locations and fertile basins to mountainous localities (Fig. 1).", "This allowed temporal study of mobility and comparisons between settlements which are in geographically close proximity with those that are spatially apart.", "The majority of the human remains studied comprise single burials with minimal grave goods located within the settlements (Triantaphyllou, 2001; Bessios et al., 2005; Triantaphyllou, 2008; Ziota et al., 2013).", "However, at two of the study sites, Stavroupoli and Toumba Kremastis Koiladas, human burials comprise disarticulated remains and articulated burials are rare (Chondrogianni-Metoki, 2001; Triantaphyllou, 2002, 2004, 2008); at Toumba Kremastis Koiladas many of the dead were cremated (Chondrogianni-Metoki, 2009).", "Given the complexity of the regional geology, it is difficult to accurately predict the bioavailable 87Sr/86Sr values in the vegetation local to each Neolithic site.", "For LN Makriyalos, values estimated from modern vegetation (of deep-rooted trees and ground vegetation, n = 20), collected from within 15 km of the archaeological site and determined by Vaiglova et al., 2018, were used for this purpose.", "These values are also extended to and combined with the information from geographically and geologically similar sites of Revenia and Paliambela to establish a local 87Sr/86Sr ratio.", "For six other sites, bioavailable 87Sr/86Sr values were estimated from tooth enamel from archaeological specimens of domestic animals recovered by excavation.", "Cattle were excluded as they have been shown to be herded over long distances through-out Europe during the Neolithic (Knipper, 2009; Towers et al., 2010; Viner et al., 2010; Sjögren and Price, 2013; Gerling et al., 2017).", "For this reason, teeth of sheep, goats and pigs were selected where possible for the estimation of locally bioavailable Sr values.", "There is a possibility that these species can be transhumant; seasonal movement of ovicaprids has been detected throughout prehistory (Bocherens et al., 2001; Henton, 2012; Makarewicz et al., 2017; Makarewicz and Pederzani, 2017) however, this is not always the case (Bogaard et al., 2014; Gerling et al., 2015; Marciniak et al., 2017).", "In the Neolithic of Northern Greece there is no evidence for or against the adoption of transhumance for sheep/goat and we will have to experimentally determine (by way of agreement with either pigs or cows) whether this behaviour is practiced and therefore whether they can be used as a local reference for these sites.", "Sample preparation, Sr purification and isotope ratio measurement closely follow the procedures outlined in Haak et al. (2008), Lewis et al. (2014) and Lewis et al. (2017).", "Sample preparation and analyses were conducted in the Bristol Isotope Group, School of Earth Sciences, University of Bristol, UK.", "The exterior of a tooth was cleaned with a stainless-steel grinding burr and a small piece of enamel (~10 mg) removed from the crown of the tooth using a diamond tipped cutting wheel and any dentine adhering to the enamel was removed.", "Enamel were then rinsed with ultrapure water (18.2 MΩ) and dried.", "Super high purity acids were used throughout the strontium chemistry and diluted to the desired concentrations using ultrapure water.", "Rinsed enamel were then weighed into a clean PFA beaker and dissolved in 7 M HNO3, an aliquot of the dissolved enamel solution equivalent to 3 mg of solid enamel was taken for ion exchange chromatography.", "Strontium was separated from the enamel matrix using Sr Spec.", "resin (Eichrom 50 100 μm particle size; Horwitz et al., 1992).", "Clean Sr Spec.", "was loaded on to 100 μL PFA micro columns for Sr separation, dissolved enamel aliquots were loaded in 0.5 mL 3 M HNO3, the enamel matrix was eluted in 2 mL 3 M HNO3 and strontium was then collected in 1.5 mL ultrapure water.", "Strontium isotope analyses were carried out using a ThermoFinnigan Triton Thermal Ionisation Mass Spectrometer (TIMS).", "Sr fractions were loaded on to rhenium filaments in nitric acid form with 1 μL of tantalum pentachloride (TaCl5) and 1 μL of 10% phosphoric acid following a modified version of Birck (1986).", "Triton amplifier gains were determined at the start of each sequence of analyses and all beams were collected on 1011 Ω amplifiers.", "Filaments were heated automatically, including source tuning, and analysis started once a beam current of 80 pA 88Sr was reached.", "Faraday cups were set to collect 84Sr, 85Rb, 86Sr, 87Sr and 88Sr as a static analysis and data was collected in 30 blocks of 12 cycles with a 4.194 s integration time per cycle.", "Values were corrected for mass fractionation using exponential mass fractionation law and 86Sr/88Sr of 0.1194 (Nier, 1938; Russell et al., 1978).", "87Sr beams were corrected for isobaric 87Rb using the 85Rb/87Rb value of 2.59265 (Steiger and Jäger, 1977) which is adjusted for instrumental mass bias.", "All recorded values were normalised to a known standard NIST SRM 987 87Sr/86Sr value of 0.710248 (Avanzinelli et al., 2005).", "Long term reproducibility of NIST SRM 987 measurements made over the course of this study is 0.71022 ± 0.00005 (2SD; n = 24).", "The reproducibility of a NIST SRM 987 passed through each batch of chemistry is 0.71024 ± 0.00001 (2SD; n = 6) and modern marine shell measured with each batch of enamels is 0.70916 ± 0.00004 (2SD; n = 6).", "‘Bioavailable strontium’ is the range of local strontium available to plants and animals within an ecosystem, which is delivered to the consumer with an accompanying 87Sr/86Sr isotope value (Bain and Bacon, 1994).", "Ecological studies have shown the strontium isotope ratio of terrestrial herbivores broadly reflects that of the surrounding bedrock values (Capo et al., 1998).", "The local environmental strontium isotope ratio value is made up of terrestrial sources primarily from mineral weathering of surrounding bedrock and atmospheric strontium derived from continental dust and precipitation.", "At coastal locations sea-spray can also contribute to the local 87Sr/86Sr ratio values (Miller et al., 1993; Capo et al., 1998; Vitousek et al., 1999; Whipkey et al., 2000).", "Human and animal tooth enamel from several sites appear to have values close to that of seawater (0.70918–0.79020; McArthur et al., 2001).", "A sea-spray affect has been observed at distances of up to 50 m from the coast (Whipkey et al., 2000) and on islands in the Outer Hebrides where biosphere samples reflected seawater strontium isotope ratio rather than the more radiogenic underlying gneiss and granite bedrock (Montgomery and Evans, 2006).", "As rainwater is derived from seawater, high levels of precipitation (>c. 2000 mm a−1) can result in saturation of the soil and contribute to the bioavailable strontium pool (Evans et al., 2010).", "The humans and animals from Revenia, Paliambela and Makriyalos situated a few km from the coast at the time of occupation (Pappa and Besios, 1999; Krahtopoulou and Veropoulidou, 2016) each display 87Sr/86Sr values close to that of seawater.", "At Stavroupoli the 87Sr/86Sr values of humans and animals are more radiogenic than that of seawater despite the close proximity of the site to the coast (ca. 3.5 km; Grammenos, 2006).", "The average rainfall for all settlements would be between 350 and 500 mm per annum (Panagos et al., 2016), hence, high rainfall means that it is unlikely that the 87Sr/86Sr values are influenced by soil saturation.", "The results presented in Table 1 show that, for the majority of sites, the 87Sr/86Sr values of Neolithic domestic animals fall within the predicted strontium isotope ratios of the surrounding geology, inferring that animal management most likely occurred at the local level.", "In fact, the 87Sr/86Sr values of the faunal samples are far less varied than the values estimated from the bedrock.", "The results also show there is no difference between the transhumant and non-transhumant species.", "This demonstrates that the local bioavailable Sr isotope baseline should ideally be determined from small fauna and plants, rather than extrapolating from bedrock as this is likely to underestimate the presence of ‘non-local’ individuals.", "The exception to this is the site of Stavroupoli, a LN tell site spread over 10 ha (Table 1).", "Here, the 87Sr/86Sr values of pig (0.71066), sheep (0.71113) and goat (0.71040), are consistent with the surrounding bedrock, which comprises Holocene coastal and Miocene lacustrine and terrestrial deposits.", "However, a more radiogenic 87Sr/86Sr value (0.71255) was determined for the cattle tooth which is consistent with both Cenozoic and Mesozoic metamorphic rocks located to the east of the site (Fig. 1).", "Mesozoic metamorphic and igneous rocks approximately 3 km east of the settlement, provide a nearby likely candidate for the more radiogenic bioavailable 87Sr/86Sr value.", "Although this distance is still very close to the site the more radiogenic Sr ratio observed in the cattle could be either a result of cattle grazing on pastures in an area of more radiogenic 87Sr/86Sr or animal management strategies such as movement between the coastal flats surrounding Stavroupoli and the slopes of the Mount Chortiatis to the east (which comprise Cenozoic and Mesozoic metamorphic rocks).", "The seasonal movement of animals between lowlands and mountainous regions has been observed elsewhere during the Neolithic in Europe (Balasse et al., 2002; Bentley et al., 2003; Bentley and Knipper, 2005; Gerling et al., 2017; Makarewicz, 2017).", "Although it is difficult to be certain due to the very small sample size, it is also possible that the cow was not raised within the local geologic area and was perhaps brought to Stavroupoli through exchange.", "Cattle management strategies throughout the Neolithic have been shown to vary, possibly as a result of adaptation to the local environment.", "For example, in EN Scandinavia (Gron et al., 2016) and LN Britain (Viner et al., 2010) cattle have been shown to have been driven over long distances.", "There is also evidence of both local management of livestock (Giblin et al., 2013) and transhumance strategies (Bentley et al., 2003; Stephan et al., 2012).", "The strontium isotope analysis of human tooth enamel undertaken herein suggests that population movement within the Neolithic of northern Greece was highly restricted as almost all populations investigated have 87Sr/86Sr values consistent with individuals having spent their early life in the local area or moving between areas with similar geology and thus similar strontium isotope ratios.", "An example of the latter possibility is observed in the 87Sr/86Sr values of the humans from the sites of Revenia and Makriyalos which are in close geographical proximity and have similar bioavailable 87Sr/86Sr, which could potentially mask movement between them (Table 2).", "During the EN there is little variability in human (average 2SD = 187 ppm; n = 10) and faunal (average 2SD = 202 ppm; n = 6) strontium isotope values.", "A similar trend is observed in the MN fauna (average 2SD = 146 ppm; n = 4).", "Towards the LN, however, there is more variation in strontium isotope values for both the fauna (average 2SD = 687 ppm; n = 14) and humans (average 2SD = 2607 ppm; n = 26) possibly reflecting changes in subsistence strategies and mobility behaviour.", "For example, the variance apparent in the human strontium isotope values for LN Makriyalos could be a consequence of a larger variability in the faunal samples due to changes in herding strategies or movement of people from an area with a similar underlying geology with differing Sr isotope values.", "Strontium isotope analyses of human tooth enamel from the northern Greece Neolithic sites demonstrate that most of the individuals sampled exhibited 87Sr/86Sr values consistent with having spent their early life in the ‘local’ area, although movement could have occurred between isotopically homogeneous areas.", "However, there are a few instances where the 87Sr/86Sr values of the sample population deviate from the bedrock geology, implying that these individuals spent their early life outside the local area from which their skeletal remains were ultimately recovered.", "A notable departure from the general trend of local origin is seen in the population from Kleitos.", "The site is located on Pleistocene lacustrine and terrestrial sediments.", "Jurassic and Cretaceous hills sit close to the north of the site (Fig. 1).", "The 87Sr/86Sr values of the local bioavailable Sr estimate at Kleitos are consistent with the upper range of Mesozoic sediments and lowest estimates of Cenozoic sediments and in broad agreement with a sediment source for the Pleistocene age lacustrine and terrestrial deposits (Voerkelius et al., 2010).", "Consistent with the general trend for Neolithic northern Greece, five of the individuals investigated displayed strontium isotope values that lie within the local 87Sr/86Sr range determined by the faunal teeth, suggesting that all were resident in the area at the time of tooth mineralisation, or had travelled from an area with a similar bioavailable strontium isotope ratio.", "Interestingly, however, the strontium isotope ratios of three individuals from Kleitos lie outside of the local bioavailable 87Sr/86Sr range (Fig. 2).", "These three individuals are interpreted as having spent their early lives on a more radiogenic geology.", "The 87Sr/86Sr values of the possible migrants are consistent with values established for Lower Palaeozoic rocks (Voerkelius et al., 2010).", "The closest Lower Palaeozoic rocks lie approx. 60 km the north and north-east.", "The tooth with the most radiogenic 87Sr/86Sr value (0.71467) comes from KL-1, an individual of 6 years of age and of unknown sex.", "Enamel was sampled from the mandibular premolar, which mineralises between 1.5 and 7 years (Logan and Kronfeld, 1933; Gustafson and Koch, 1974; Schroeder, 1991).", "This suggests that the child had lived in a different geologic environment for the majority of their young life.", "Individual KL-2, a male in his mid-30's to mid-40's in age exhibited a slightly less radiogenic 87Sr/86Sr value (0.71285).", "Enamel was sampled from the maxillary right 2nd molar, which mineralises between 1.5 and 7.5 years (Logan and Kronfeld, 1933; Gustafson and Koch, 1974; Schroeder, 1991).", "This indicates that the individual was also a non-local during infancy.", "Finally, individual KL-6 is slightly more radiogenic than the local strontium range (0.71089), the individual is estimated to be 15 years of age and of unknown sex.", "Enamel was sampled from the maxillary left 1st incisor, which mineralises between 3 months and 6 years of age (Logan and Kronfeld, 1933; Gustafson and Koch, 1974; Schroeder, 1991).", "The interpretation for this individual is more nuanced.", "It is possible that this individual has had a variable diet with some strontium being sourced from the local area and some from a non-local source either as a direct function of mobility, imported foodstuffs or the individual may have been raised in a region of discrete geology with a bioavailable 87Sr/86Sr of ~0.7109.", "This value would be consistent with Cenozoic age sedimentary rocks.", "The strontium isotope ratios of five individuals from Makriyalos lie outside of the determined local isotopic range (Fig. 2), but within the estimated 87Sr/86Sr values for Miocene age uplands to the west of the site (0.70901 to 0.71100; Voerkelius et al., 2010).", "Makriyalos, Paliambela and Revenia sit approx. 2–3 km form the same Miocene age upland, so one might expect the biologically available strontium isotope ratios the sites to be similar (~0.7092 to 0.7096).", "This interpretation is supported by the analysis of modern plants used to determine the local bioavailable strontium isotope ratio in this study (0.70900 to 0.70974; Vaiglova et al., 2018) Teeth MAK-5, 6, 9, 10 and 12 all belong to adults, suggesting movement from an area of a similar geology after tooth mineralisation.", "The strontium isotope values for the individuals at Nea Nikomedeia lie outside the estimated 87Sr/86Sr values for the area for Cenozoic sediments (0.70901 to 0.71100; Voerkelius et al., 2010), but lie in the range predicted for Mesozoic geology (0.70701 to 0.70900), which is consistent with the sediments which form the alluvial plain.", "The geology surrounding Nea Nikomedeia indicates that the site sits on Pliocene and Quaternary rocks, with alluvial sediment deposits consisting of lacustrine silts and terrestrial clays (Fig. 2).", "The alluvial plain was formed during the Holocene by the deposition of sediment from four of the surrounding rivers from the Cretaceous and Jurassic uplands to the West (Bintliff, 1976) and thus the source of bioavailable strontium at the site.", "Although direct determination of local bioavailable 87Sr/86Sr values from local vegetation would be required to confirm this.", "Therefore, the individuals at Nea Nikomedeia have 87Sr/86Sr values consistent with individuals having spent their early life in the local area.", "In this investigation strontium isotope ratios have been used to explore the movement of Neolithic people in the north of Greece.", "Baseline bioavailable 87Sr/86Sr values calculated from faunal tooth enamel were shown to fall within with predicted 87Sr/86Sr values of the surrounding bedrock, suggesting that animal management occurred on a local level, although differences in herd management practices became more evident through more varied strontium isotope values at LN sites, which is potentially indicating of changing herding practices leading to a larger variability in the strontium isotope values of the faunal samples.", "Analyses of human tooth enamel suggests that overall population movement within the region was limited as at almost all sites most individuals exhibit 87Sr/86Sr values consistent with them having spent their early life in the local area from where their remains were recovered; although the potential for movement between geologically homogenous areas exhibiting similar strontium isotope ratios must always be considered.", "The strontium isotope ratios of three individuals at Kleitos lie outside of the local bioavailable 87Sr/86Sr range.", "These individuals are interpreted as having spent their early lives on a more radiogenic geology, providing evidence of mobility in Greece determined using strontium isotope ratios.", "The results determined using Sr isotope analysis strengthens the current archaeological evidence for movement and exchange observed through studies of pottery circulation.", "With both suggesting limited movement in the Early and Middle Neolithic and greater movement in the Late Neolithic." ]	[ "Sr isotope ratios were determined for 36 human and 26 faunal tooth enamel samples.", "Seven sites spanning the EN to the LN of northern Greece.", "87Sr/86Sr values of human tooth enamel shows that population movement was limited.", "Three individuals identified as ‘non-locals’.", "Sr ratio of faunal enamel infers animal management occurred on a local level." ]
Machine Learning Methods for Histopathological Image Analysis	Abundant accumulation of digital histopathological images has led to the increased demand for their analysis, such as computer-aided diagnosis using machine learning techniques. However, digital pathological images and related tasks have some issues to be considered. In this mini-review, we introduce the application of digital pathological image analysis using machine learning algorithms, address some problems specific to such analysis, and propose possible solutions.	[ "BIOC", "COMP" ]	[ "Computer assisted diagnosis", "Deep learning", "Digital image analysis", "Histopathology", "Machine learning", "Whole slide images" ]	[ "1303", "1304", "1305", "1311", "1315", "1706" ]	[ "Pathology diagnosis has been performed by a human pathologist observing the stained specimen on the slide glass using a microscope.", "In recent years, attempts have been made to capture the entire slide with a scanner and save it as a digital image (whole slide image, WSI) [1].", "As a large number of WSIs are being accumulated, attempts have been made to analyze WSIs using digital image analysis based on machine learning algorithms to assist tasks including diagnosis.", "Digital pathological image analysis often uses general image recognition technology (e.g. facial recognition) as a basis.", "However, since digital pathological images and tasks have some unique characteristics, special processing techniques are often required.", "In this review, we describe the application of digital pathological image analysis using machine learning algorithms, and its problems specific to digital pathological image analysis and the possible solutions.", "Several reviews that have been published recently discuss histopathological image analysis including its history and details of general machine learning algorithms [2–7]; in this review, we provide more pathology-oriented point of view.", "Since the overwhelming victory of the team using deep learning at ImageNet Large Scale Visual Recognition Competition (ILSVRC) 2012 [8], most of the image recognition techniques have been replaced by deep learning.", "This is also true for pathological image analysis [9–11].", "Therefore, even though many techniques introduced in this review are related to deep learning, most of them are also applicable for other machine learning algorithms.", "Fig. 1 shows typical steps for histopathological image analysis using machine learning.", "Prior to applying machine learning algorithms, some pre-processing should be performed.", "For example, when cancer regions are detected in WSI, local mini patches around 256 × 256 are sampled from large WSI.", "Then feature extraction and classification between cancer and non-cancer are performed in each local patch.", "The goal of feature extraction is to extract useful information for machine learning tasks.", "Various local features such as gray level co-occurrence Matrix (GLCM) and local binary pattern (LBP) have been used for histopathological image analysis, but deep learning algorithms such as convolutional neural network [9,10,12–14] starts the analysis from feature extraction.", "Features and classifiers are simultaneously optimized in deep learning and features learned in deep learning often outperforms other traditional features in histopathological image analysis.", "Machine learning techniques often used in digital pathology image analysis are divided into supervised learning and unsupervised learning.", "The goal of supervised learning is to infer a function that can map the input images to their appropriate labels (e.g. cancer) well using training data.", "Labels are associated with a WSI or an object in WSIs.", "The algorithms for supervised learning include support vector machines, random forest and convolutional neural networks.", "On the other hand, the goal of unsupervised learning is to infer a function that can describe hidden structures from unlabeled images.", "The tasks include clustering, anomaly detection and dimensionality reduction.", "The algorithms for unsupervised learning include k-means, autoencoders and principal component analysis.", "There are derivatives from these two learning such as semi-supervised learning and multiple instance learning, which are described in Section 4.2.2.", "The most actively researched task in digital pathological image analysis is computer-assisted diagnosis (CAD), which is the basic task of the pathologist.", "Diagnostic process contains the task to map a WSI or multiple WSIs to one of the disease categories, meaning that it is essentially a supervised learning task.", "Since the errors made by a machine learning system reportedly differ from those made by a human pathologist [14], classification accuracy could be improved using CAD system.", "CAD may also lead to the reduce variability in interpretations and prevent overlooking by investigating all pixels within WSIs.", "Other diagnosis-related tasks include detection or segmentation of Region of Interest (ROI) such as tumor region in WSI [15,16], scoring of immunostaining [11,17], cancer staging [14,18], mitosis detection [19,20], gland segmentation [21–23], and detection and quantification of vascular invasion [24].", "Content Based Image Retrieval (CBIR) retrieves similar images to a query image.", "In digital pathology, CBIR systems are useful in many situations, particularly in diagnosis, education, and research [25–32].", "For example, CBIR systems can be used for educational purposes by students and beginner pathologists to retrieve relevant cases or histopathological images of tissues.", "In addition, such systems are also helpful to professional pathologists, particularly when diagnosing of rare cases.", "Since CBIR does not necessarily require label information, unsupervised learning can be used [29].", "When label information is available, supervised learning approaches could learn better similarity measure than unsupervised learning approaches [27,28] since the similarity between histopathological images may differ by definition.", "However, preparing sufficient number of labeled data can be a serious problem as will be described later.", "In CBIR, not only accuracy but also high-speed search of similar images from numerous images are required.", "Therefore, various techniques for dimensionality reduction of image features such as principal component analysis, and fast approximate nearest neighbor search such as kd-tree and hashing [31] are utilized for high speed search.", "Historically, many important discoveries concerning diseases such as tumor and infectious diseases have been made by pathologists and researchers who have carefully and closely observed pathological specimens.", "For example, H. pylori was discovered by a pathologist who was examining the gastric mucosa of patients with gastritis [32].", "Attempts have also been made to correlate the morphological features of cancers with their clinical behavior.", "For example, tumor grading is important in planning treatment and determining a patient's prognosis for certain types of cancer, such as soft tissue sarcoma, primary brain tumors, and breast and prostate cancer.", "Meanwhile, thanks to the progress in digitization of medical information and advance in genome analysis technology in recent years, large amount of digital information such as genome information, digital pathological images, MRI and CT images has become available [33].", "By analyzing the relationship between these data, new clinicopathological relationships, for example, the relationship between the morphological characteristic and the somatic mutation of the cancer, can be found [34,35].", "However, since the amount of data is enormous, it is not realistic for pathologists and researchers to analyze all the relationships manually by looking at the specimens.", "This is where the machine learning technology comes in.", "For example, Beck et al. extracted texture information from pathological images of breast cancer and analyzed with L1 - regularized logistic regression, and indicated that the histology of stroma correlates with prognosis in breast cancer [36].", "Other researches include prognosis predictions from histopathological image of cancer [37], prediction of somatic mutation [13], and discovery of new gene variants related to autoimmune thyroiditis based on image QTL [38].", "In this section, we describe unique characteristics of pathological image analysis and computational methods to treat them.", "Table 1 presents an overview of papers dealing with the problems and the solutions.", "When images such as dogs or houses are classified using deep learning, small sized image such as 256 × 256 pixels is often used as an input.", "Images with large size often need to be resized into smaller size which is enough for sufficient distinction, as increase in the size of the input image results in the increase in the parameter to be estimated, the required computational power, and memory.", "In contrast, WSI contains many cells and the image could consist of as many as tens of billions of pixels, which is usually hard to analyzed as is.", "However, resizing the entire image to a smaller size such as 256 × 256 would lead to the loss of information at cellular level, resulting in marked decrease of the identification accuracy.", "Therefore, the entire WSI is commonly divided into partial regions of about 256 × 256 pixels (“patches”), and each patch is analyzed independently, such as detection of ROIs.", "Thanks to the advances in computational power and memory, patch size is increasing (e.g. 960 × 960), which is expected to contribute to better accuracy.", "There is still a room for improvement in the method of integrating the result from each patch.", "For example, as the entire WSI could contain hundreds of thousands of patches, false positives are highly likely to appear even if individual patches are accurately classified.", "One possible solution for this is regional averaging of each decision, such that the regions is classified as ROI only when the ROI extends over multiple patches.", "However, this approach may suffer from false negatives, resulting in missing small ROIs such as isolated tumor cells [39].", "In some applications such as IHC scoring, staging of lymph node metastasis of specimens or patients, and staging of prostate cancer diagnosed by Glisson score of multiple regions within one slide, more sophisticated algorithms to integrate patch-level or object-level decisions are required [14,17,18,39,40,75].", "For example, for pN-staging of metastatic breast cancer, which was one of the tasks in Camelyon 17, multiple participating teams including us applied random forest classifiers of pixel or patch-level probabilities estimated by deep learning using various features such as estimated tumor size [39].", "Probably the biggest problem in pathological image analysis using machine learning is that only a small number of training data is available.", "A key to the success of deep learning in general image recognition task is that training data is extremely abundant.", "Although label information at patch-level or pixel-level (e.g. inside/outside boundary of cancerous regions) is required in most tasks in digital pathology such as diagnosis, most labels of WSIs are at case-level (e.g. diagnosis) at most.", "Label information in general image analysis can be easily retrieved from the internet and it is also possible to use crowdsource labeling because anyone can identify objects and perform labeled work.", "However, only pathologists can label the pathological image accurately, and labeling at the regional level in a huge WSIs requires a lot of labor.", "It is possible to reuse public ready-to-analyze data as training data in machine learning, such as ImageNet [76] in natural images and International Skin Imaging Collaboration [77] in macroscopic diagnosis of skin.", "In the field of digital pathology, there are some public datasets that contain hand-annotated histopathological images as summarized in Tables 2 and 3.", "They could be useful if the purpose of the analysis, slide condition (e.g. stain), and image condition (e.g. magnification level and image resolution) are similar.", "However, because each of these datasets focuses on specific disease or cell types, many tasks are not covered by these datasets.", "There are also several large-scale histopathological image databases that contain high-resolution WSIs: The Cancer Genome Atlas (TCGA) [78] contains over 10,000 WSIs from various cancer types, and Genotype-Tissue Expression (GTEx) [79,80] contains over 20,000 WSIs from various tissues.", "These databases may serve as potential training data for various tasks.", "Furthermore, both TCGA and GTEx also provide genomic profiles, which could be used to investigate relationships between genotype and morphology.", "The problem is that the WSIs in these repositories contain labels at the case-level, and in order to be able to use them for training data, some preprocessing or specialized machine learning algorithm for treating case-level labels is required.", "Many researches have attempted to solve the problem.", "Most of the approaches fall into one of the following categories: 1) efficient increase of label data, 2) utilization of weak label or unlabeled information, or 3) utilization of models/parameters for other tasks.", "One way to increase training data is to reduce the working time of pathologists to specify ROIs in the WSI.", "Easy-to-use GUI tools helps pathologists efficiently label more samples in shorter periods of time [41,42].", "For example, Cytomine [41] not only allows pathologists to surround ROIs in WSIs with ellipses, rectangles, polygons or freehand drawings, but also applies content-based image retrieval algorithms to speed up annotation.", "Another interesting idea to reduce working time is to automatically localize ROIs during diagnosis, which uses the usual working time for diagnosis as labeling by tracking pathologists' behavior.", "This approach tracks pathologists' eye movement [43], mouse cursor positions [44] and change in viewport [45].", "However, localizing ROIs accurately from these tracking data is not always easy since pathologist's do not always spend time looking at ROIs, and boundary information obtained by these approaches tends to be less clear.", "Another approach that utilizes a machine learning method is active learning [42,46–49,104,105].", "This is generally effective when the acquisition cost of label data is large (i.e. pathological images).", "Active learning is a method used in supervised learning, and it automatically chooses the most valuable unlabeled sample (i.e. the one that is expected to improve the identification performance of classifiers when labeled correctly and used as a training data) and display it for labeling by pathologists.", "Since this approach is likely to increase discrimination performance with smaller number of labeled images, the total labeling time to obtain the same discrimination performance will be shortened [46].", "Many criteria such as uncertainty sampling [42], Query-by-Committee [46], variance reduction [47], and hypothesis space reduction [48] have been applied for selecting valuable unlabeled samples.", "Even if the exact position of the ROI in a WSI is not known, it is possible that the information regarding the presence/absence of the ROI in the WSI is available from the pathological diagnosis assigned to the WSI or WSI-level labels.", "These so-called weak labels are easy to obtain compared to patch-level labels even when the WSIs have no further information, and in this regard, WSIs is considered as a “bag” made with many patches (instances) in machine learning settings.", "When diagnosing cancer, WSI is labeled as cancer if at least one patch contains cancerous tissue, or normal if none of the patches contain cancerous tissue.", "This setting is a problem of multiple instance learning [50,106] or weakly-supervised learning [49,51].", "In a typical multiple instance learning problem, positive bags contain at least one positive instance and negative bags do not contain any positive instances.", "The aim of multiple instance learning is to predict bag or instance label based on training data that contains only bag labels.", "Various methods in multiple instance learning have been applied to histopathological image analysis including boosting-based approach [49], support vector machine-based approach [52] and deep learning-based approach [51].", "In contrast, semi-supervised learning [30,53,107,108] utilizes both labeled and unlabeled data.", "Unlabeled data is used to estimate the true distribution of labeled data.", "For example, as shown in Fig. 1, decision boundary which takes only the labeled samples into account would form a vertical line, but that considering both labeled and unlabeled samples would form a slanting line, which could be more accurate.", "Since semi-supervised learning is considered particularly effective when samples in the same class form a well-discriminative cluster, relatively easy problem could be a good target.", "Performing supervised learning using too few training data would only result in insufficient generalization performance.", "This is true especially in deep learning, where the number of parameters to be learned is very large.", "In such a case, instead of learning the entire model from scratch, learning often starts by using (a part of) parameters of a pre-trained model optimized in another similar task.", "Such a learning method is called transfer learning.", "In CNN, layers before the last (typically three) fully-connected layers are regarded as feature extractors.", "The fully-connected layers are often replaced by a new network suitable for the target task.", "The parameters in earlier layers can be used as is [54], or as initial parameters and then the network is learned partially or fully from the training data of the target task [16,55,56] (so-called fine-tuning).", "In pathological images, no network learned from tasks using other pathological images are available, and thus networks learned using ImageNet, which is a database containing vast number of general images, are often used [16,54–56].", "For example, Xu et al., performed classification and segmentation tasks on brain and colon pathological images using features extracted from CNN trained on ImageNet, and achieved state-of-the-art performance [54].", "Although the pathological image itself looks very different to the general images (e.g. cats and dogs), they share common basic image structures such as lines and arcs.", "Since earlier layers in deep learning capture these basic image structures, such pre-trained models using general images work well in histopathological image analysis.", "Nevertheless, if models pre-trained on sufficient number of diverse tissue pathology images are available, they may outperform the ImageNet pre-trained models.", "Tissues are usually composed of cells, and different tissues show distinct cellular features.", "Information regarding cell shape is well captured in high-power field microscopic images, but structural information such as a glandular structure made of many cells are better captured in a lower-power field (Fig. 2).", "Because cancerous tissues have both cellular and structural atypia, images taken at multiple magnifications would each contain important information.", "Pathologists diagnose diseases by acquiring different kinds of information from the cellular level to the tissue level by changing magnifications of a microscope.", "Even in machine learning, researches utilizing images at different magnifications exist [57–59].", "As mentioned above, it is difficult to handle the images at its original resolution directly, images are often resized to correspond to various magnifications and used as input for analysis.", "Regarding diagnosis, the most informative magnification is still controversial [14,39,109], but improvement in accuracy is sometimes achieved by inputting both high and low magnification images simultaneously, probably depending on the types of diseases and tissues, and machine learning algorithms.", "Pathological image is different from cats and dogs in nature, in a sense that it shows repetitive pattern of minimum components (usually cells).", "Therefore, it is rather closer to texture than object.", "CNN acquires shift invariance to a certain extent by pooling operations.", "In addition, even normal CNN can learn texture-like structure by data augmentation by shifting the tissue image with a small stride.", "Meanwhile, there has been methods which utilize texture structure more intensively, such as gray level co-occurrence matrix [110], local binary pattern [111], Gabor filter bank, and recently developed deep texture representations using a CNN [64,112].", "Deep texture representations are computed using a correlation matrix of feature maps in a CNN layer.", "Converting the CNN features to texture representations would lead to the acquisition of invariance regarding cell position, while utilizing good representations learned by CNN.", "Another advantage of deep texture representation is that there are no constraints on the size of input image, which is very suitable for large image size of WSI.", "The boundary between texture and non-texture is unclear, but a single cell or a single structure is obviously not a texture.", "Better approach would thus depend on the object to be analyzed.", "WSIs are created through multiple processes: pathology specimens are sliced and placed on a slide glass, stained with hematoxylin and eosin, and then scanned.", "At each step undesirable effects, which are unrelated to the underlying biological factors, could be introduced.", "For example, when tissue slices are being placed onto the slides, they may be bent and wrinkled; dust may contaminate the slides during scanning; blur attributable to different thickness of tissue sections may occur (Fig. 3); and sometimes tissue regions are marked by color markers.", "Since these artifacts could adversely affect the interpretation, specific algorithms to detect artifacts such as blur [71] and tissue-folds [73] have been proposed.", "Such algorithms may be used for preprocessing WSIs.", "Another serious artifact is color variation as shown in Fig. 4.", "The sources of variation include different lots or manufacturers of staining reagents, thickness of tissue sections, staining conditions and scanner models.", "Learning without considering the color variation could worsen the performance of machine learning algorithm.", "If sufficient data on every stained tissue acquired by every scanner can be incorporated, the influence of color variation on classification accuracy may become negligible; however, that seems very unlikely at the moment.", "To address this issue, various methods have been proposed so far including conversion to gray scale, color normalization [65–68], and color augmentation [69,70].", "Conversion to grayscale is the easiest way, but it ignores the important information regarding the color representation used routinely by pathologists.", "In contrast, color normalization tries to adjust the color values of an image on a pixel-by-pixel basis so that the color distribution of the source image matches to that of a reference image.", "However, as the components and composition ratios of cells or tissues in target and reference images differ in general, preprocessing such as nuclear detection using a dedicated algorithm to adjust the component is often required.", "For this reason, color normalization seems to be suitable when WSIs analyzed in the tasks contain, at least partially, similar compositions of cells or tissues.", "On the other hand, color augmentation is a kind of data augmentation performed by applying random hue, saturation, brightness, and contrast.", "The advantage of color augmentation lies in the easy implementation regardless of the object being analyzed.", "Color augmentation seems to be suitable for WSIs with smaller color variation, since excessive color change in color augmentation could lead to the loss of color information in the final classifier.", "As color normalization and color augmentation could be complementary, combination of both approaches may be better.", "Digital histopathological image recognition is a very suitable problem for machine learning since the images themselves contain information sufficient for diagnosis.", "In this review, we brought up problems in digital histopathological image analysis using machine learning.", "Due to great efforts made so far, these problems are becoming tractable, but there is still room for improvement.", "Most of these problems are likely to be solved once a large number of well-annotated WSIs become available.", "Gathering WSIs from various institutes to collaboratively annotate them with the same criteria and making these data public will be sufficient to boost the development of more sophisticated digital histopathological image analysis.", "Finally, we suggest some potential future research topics that have not been well studied so far.", "In actual diagnostic situations, unexpected objects such as aberrant organization, rare tumor (thus not included in training data) and foreign bodies could exist.", "However, discrimination model including Convolutional Neural Networks forcibly categorizes such objects into one of the pre-defined categories.", "To solve the problem, outlier detection algorithms, such as one-class kernel principal component analysis [113], have been applied to the digital pathological images but only a few researches have addressed the problem so far.", "More recently, some deep learning-based methods utilizing reconstruction error [114] have been proposed for outlier detection in other domains, but they are not yet applied in the histopathological image analysis.", "Deep learning is often criticized because its decision-making process is not understandable to humans and therefore often described as being a black box.", "Although decision-making process of human is not a complete white box either, people want to know the decision process or decision basis.", "This could lead to a new discovery in the pathology field.", "Although this problem has not been completely solved so far, some research has attempted to provide solutions, such as joint learning of pathological images and its diagnostic reports integrated with attention mechanism [115].", "In other domains, decision basis can be inferred indirectly represented by visualizing the response of a deep neural network [115,116], or presenting the most helpful training image using influence functions [117].", "Pathological diagnosis during surgery influences intraoperative decision making, and thus could be another important application in histopathological image analysis.", "As diagnostic time in intraoperative diagnosis is very limited, rapid classification while keeping accuracy is of importance.", "Due to the time constraint, rapid frozen section is used instead of Formalin-fixed paraffin-embedded (FFPE) section which takes longer time to prepare.", "Therefore, for this purpose training of classifiers should be performed using frozen section slides.", "Few research has analyzed frozen sections [118] so far partly because the number of WSIs suitable for the analysis is not sufficient, and task is more challenging compared to FFPE slides.", "Because of the success of tumor immunotherapy, especially immune-checkpoint blockade therapies including anti-PD-1 and anti-CTLA-4 antibodies, immune cells in tumor microenvironment have gained substantial attention in recent years.", "Therefore, quantitative analysis of tumor infiltrating immune cells in slides using machine learning techniques will be one of the emerging themes in digital histopathological image analysis.", "Tasks related to this analysis include detection of immune cells from H&E stained image [119,120] and detection of more specific type of immune cells using immunohistochemistry [102].", "Additionally, the pattern of immune cell infiltration and proximity of each immune cells are reportedly related to cancer prognosis [121], analysis of spatial relationships between tumor cells and immune cells, and the relationships between these data and prognosis or response to immunotherapy using specialized algorithms such as graph-based algorithms [62,122] will also be of great importance." ]	[]
Incidence of urinary tract infections and antimicrobial susceptibility pattern among pregnant women in Akure, Nigeria	Background: Urinary tract infection (UTI) is the most common medical complications of pregnancy together with anemia and hypertension and it occurs approximately in 5–10% of all pregnancies. Objectives: This study was carried out to evaluate the incidence and antimicrobial susceptibility pattern of isolated microbes among pregnant women attending ante natal clinics in Akure, Nigeria. Methods: A total of 300 clean midstream urine samples were collected in a clean universal bottle. The urine samples were cultured, Microbact identification system was used to identify the presence of bacteria. Results: Out of the 300 urine samples cultured, 183(61.0%) showed significant growth of urinary pathogens while 117(39.0%) showed lack of growth and insignificant growth of urinary pathogens. Escherichia coli 58(31.7%) was the dominant pathogen isolated, followed by Klebsiella pneumoniae 32(17.5%), Staphylococcus aureus 27(14.8%), and the least isolated were Citrobacter freudii and Providencia retgerri 2(1.1%) and 2(1.1%), respectively. Gram negative bacteria showed 77.6%, 67.9% and 61.2% susceptibility to Ciprofloxacin, Augmentin and Chloramphenicol, respectively. Resistance against Septrin, Chloramphenicol, Sparfloxacin, Amoxicillin, Augmentin, Gentamycin, Pefloxacin, Trivid and Streptomycin were observed in the range 23.1% to 70.1%. Gram positive uropathogens showed high resistance to Amoxicillin (68.4%) and high susceptibility to the other antibiotics in the range 65.8% to 89.5%. Conclusion: There is need to treat pregnant women with UTIs with proven antibiotics that microbes are susceptible to in order to combat the evolution of resistant pathogens. Routine microbiological analysis and antibiotic sensitivity test of urine samples of pregnant women should be carried out so as to enhance the treatment and management of UTIs.	[ "MULT" ]	[ "Antimicrobial resistance", "Microbact", "Susceptibility", "UTIs", "Urine" ]	[ "1000" ]	[ "Urinary tract infections (UTIs) describe the microbial invasion and subsequent multiplication of these microbes on the entire urinary tract of the infected individual.", "Urine is a fluid with a variety of molecules and salts, some of which are waste products but does not usually have bacteria as a normal component [1].", "UTIs that occur in a normal genitourinary tract are considered as uncomplicated, complicated infections are diagnosed with structural or functional abnormalities with urethral catheters [2,3].", "Urinary tract infections are the most frequent bacterial infection in women [4].", "They occur most frequently between the ages of 16 and 35 years, with 10% of women getting an infection yearly and more than 40–60% having an infection at some point in their lives [5].", "These infections cause about 20% complication during pregnancy and are responsible for the majority of antepartum admissions into the maternal–foetal unit in hospitals [6].", "Urinary tract infection is a common health problem among pregnant women.", "This usually begins in the first six weeks of conception and reach the peak at 22nd to 24th week of the pregnancy due to many factors such as urethral dilatation, increased bladder volume and decreased bladder tone which contributes to increased urinary stasis and ureterovesical reflux.", "With this situation, about 70% of pregnant women develop glycosuria, which encourages bacterial growth in the urine [7].", "Some common clinical symptoms are dysuria, polyuria, suprapubic discomfort, and in some cases hematuria [8].", "Although dysuria and polyuria may suggest UTIs, these symptoms may concomitantly be present in pregnant women with other conditions, such as bacterial vaginosis [9].", "According to the American College of Obstetricians and Gynecologists, screening of asymptomatic bacteriuria (ASB) is recommended in all pregnant women [10].", "Urine cultures are recommended early in pregnancy in order to detect ASB.", "The treatment of UTIs can vary depending upon the physician and patient.", "The Infectious Disease Society of America [11] recommended a five-day course of nitrofurantoin as a first line treatment for acute, uncomplicated cystitis and other antibiotics as well [12].", "The safest antibiotics to use during pregnancy for the treatment of ASB include nitrofurantoin, amoxicillin, with clavulanate, and cephalosporin [13].", "Worldwide data shows that there is an increasing resistance in uropathogens to conventional drugs.", "Resistance has emerged even to latest and more potent antimicrobial drugs.", "The development of resistance to amoxicillin is common among uropathogens and should be monitored [14].", "In the U.S., up to 33% of the uropathogens that cause UTIs are resistant to amoxicillin [10].", "The study was conducted at the antenatal clinics in four primary health care centers; Basic Health Center, Ogbese, Comprehensive Health Center, Algon Iju, Comprehensive Health Center, Ala Ajagbusi and Comprehensive Health Center, Oba-Ile located in Akure.", "A descriptive cross-sectional study was conducted at the antenatal clinics for pregnant women attending the selected primary health care centers in Akure between February and June 2017.", "A total of 300 pregnant women comprising 35 symptomatic and 265 asymptomatic individuals attending antenatal clinics in the study sites were randomly selected for the study.", "The purpose of using random sampling techniques was to ensure that each of the pregnant women who was willing and available at the time of study and satisfied the inclusion and exclusion criteria.", "Inclusion criteria: Pregnant women with or without symptoms of UTI, of varied gestational age and with no history of being treated with antibiotics for more than two weeks prior to the day of specimen collection.", "Exclusion criteria: Pregnant women who had taken antibiotics in the last one week or two weeks before sample collection.", "The sample size was computed using the sample size determination Table of [15].", "Permission was obtained from Ondo State Ministry of Health, Akure and Ondo State Primary Health Care Board with number OSHREC/09/01/2017/063.", "Informed consent was requested and granted by the pregnant women to be sampled after the concept of the study was explained to them.", "A total of 300 clean catch midstream urine samples were collected in sterile screw capped universal container from consenting pregnant women of varied gestational age from the selected antenatal clinics.", "The sample bottles were labeled accordingly and transported to FUTA Biology laboratory in an iced pack for processing within two hours of collection, to ensure maximum recovery of the microbes.", "A loop full (0.001 mL) of well mixed un-centrifuged urine was streaked on to the surface of a dried Cysteine Lactose Electrolyte Deficient (CLED) medium and nutrient agar.", "These plates were incubated aerobically at 35 °C for 24 h. Colony counts were determined at the end of incubation period and expressed as colony forming units (cfu) per milliliter (ml).", "Each urine sample with over 105 CFU per milliliter was followed up as significant bacteria growth [16] and isolated colonies were sub-cultured on freshly prepared CLED agar incubated 35 °C for 24 h.", "The purified cultures were stored on nutrients agar slants and identified base on their cultural morphology, Gram stain and biochemical reaction according to [17,18]", "The isolated colonies were examined and recorded based on the type of growth, elevation, size, color, margin, edge, consistency, opacity, and change in medium [17].", "A thin smear of each isolate was made on clean grease free glass slide, air dried and heat fixed by passing it gently over flame and then Gram stained.", "Gram positive cells stained purple while the Gram-negative cells stained pink [17].", "Catalase test was done on the Gram-positive cocci to differentiate Staphylococcus spp from Streptococcus spp.", "Coagulase test was done to identify S. aureus which produces the enzyme coagulase.", "Oxidase test was done on the Gram-negative bacilli (GNB) to identify Psuedomonas spp from other Gram-negative bacilli.", "Microbact Biochemical Identification system was used to identify the species of the oxidase negative GNB.", "Microbact™ Gram-negative system 12A was used.", "It is a range of simple, standardized micro-substrate systems designed to simulate conventional biochemical substrates used for the rapid identification of Enterobacteriaceae and common miscellaneous Gram-negative bacilli (MGNB).", "Broth dilution technique described by Clinical Laboratory Standards Institute [19] guide lines was used for the antibiotic susceptibility tests on the urine isolates.", "The test was carried out by using conventional antibiotic disk.", "The antibiotic disk was placed gently on solidified agar plate already inoculated with the test organism.", "Isolates were considered sensitive after incubation for 24 h at 35 °C by measuring zone of inhibition with meter rule which was then compared with zone diameter interpretative to Clinical and Laboratory Standard Institute 2015.", "Data were analyzed using SPSS version 22 window based program.", "Discrete variables were expressed as percentages and proportions were compared using the Chi-square test.", "Statistically significant differences were considered at value of P ≤ 0.05.", "A total of three hundred (300) urine samples of pregnant women attending antenatal care were collected.", "Out of which 40 (41.9%), 46 (25.1%), 45 (24.6%) and 52 (28.4%) of the antenatal patients showed significant bacteriuria, respectively, with overall prevalence of 183 (61.0%) while 117 (39.0%) of the patients were negative with a significant difference (Fig. 1).", "A total of thirteen (13) bacteria species were isolated and identified.", "Ten (10) of the bacteria isolates were Gram-negative bacteria and three (3) were Gram-positive bacteria.", "There were mixed culture of two (2) species of bacteria in some samples.", "The results showed that the dominant bacteria isolates were Escherichia coli 58 (31.7%), followed by Klebsiella pneumoniae 32 (17.5%), Staphylococcus aureus 27 (14.8%), Proteus mirabilis 14 (7.7%), Klebsiella oxytoca and coagulase negative Staphylococci (CoNS) 8 (4.4%).", "Pseudomonas aeruginosa and mixed culture of S. aureus and K. pneumoniae 6 (3.3%), Citrobacter freudii and Providencia retgerri 2 (1.1%), respectively, were minor (Fig. 2).", "A total of 183 samples that showed significant growth, 101 representing 55.2% were in the age group 25–34, followed by 43 (23.5%) in age group ≤ 24 while the lowest number of isolates 39 (21.3%) was found in the age group 35 and above (Table 1).", "With 183 samples of the pregnant women with significant growth, 94 (51.4%) were in their 3rd trimester, 72 (39.3%) in their 2nd trimester while the least 17 (9.3%) in their 1st trimester.", "It was observed that number of isolates increases as the trimester stage increases (Table 2).", "The frequency of bacterial isolates in relation to the gravidity of the pregnant women revealed that majority of the samples with significant growth were multigravida 128 (69.9%) (Table 3).", "The Gram-negative bacteria species showed varying susceptibility patterns to ten (10) of the antimicrobial drugs.", "E. coli which constituted 43.3% of the Gram-negative bacteria showed high resistance against Sparfloxacin (60.3%), Amoxicillin (84.5%), Pefloxacin (77.6%), Trivid (79.3%) and Streptomycin (70.1%) but was highly susceptible to Chloramphenicol (70.1%), Ciprofloxacin (87.9%) and Augmentin (91.4%).", "K. pneumoneae which accounted for (23.9%) of the Gram-negative bacterial isolates was highly susceptible to Chloramphenicol (78.1%), Sparfloxacin (62.5%) and Ciprofloxacin (96.9%) (Table 4).", "Gram positive bacteria uropathogens isolated were S. aureus, Streptococcus spp. and Coagulase negative Staphylococci.", "Gram positive uropathogens isolated showed high resistance to amoxicillin (68.4%) and high susceptibility to the remaining 9 antibiotics in the range 65.8% to 89.5%.", "S. aureus which constituted 71.1% of the gram positive uropathogens showed 100.0% susceptibility to Ampiclox and high susceptibility to other antibiotic except for Amoxicillin where it showed 66.7% resistance.", "Coagulase negative Staphylococci (CoNS) showed 100.0% resistance to Gentamycin, and was susceptible to Pefloxacin (87.5%), Ampiclox (75.0%), Zinnacef (87.5%) and Ciprofloxacin (87.5%).", "S. spp. which constituted the least of the Gram positive uropathogens isolated (7.9%), showed 100.0% resistance against Pefloxacin and 100.0% susceptibility to Septrin (Table 5).", "This study revealed a prevalence of 61% UTI in pregnant women and the predominant pathogen was pathogenic E. coli.", "The isolated pathogens in this study were coliforms which show the index of organism for safety, qualitative sanitation and good hygiene.", "Some of the uropathogens showed resistance against many of the antibiotics tested.", "One Hundred and Eighty-three (183) urine samples were found to contain heavy and appreciable bacterial growth representing 61% prevalence in the study area.", "This study agrees with the reports of [20,1] and [21] where they reported high prevalence of 58, 55 and 61.5%, respectively, but lower than the prevalence rates of 77.9 and 85% reported by Theodore [22] and Turay et al. [23], respectively.", "The different prevalence recorded could be attributed to UTI perception, mode of screening and compounding risk factors, such as age, gravidity, type of toilet used, socio economic status of the pregnant women.", "However, the high prevalence of UTI in this study is probably due to the prevailing poor housing and drainage systems, lack of proper personal and environmental hygiene, population susceptibility, low socio-economic status and sexual intercourse among the pregnant women [24].", "It could also be due to hormonal effects observed in pregnancy, which reduces the tone of the ureteric musculature aided perhaps by mechanical pressure from the gravid uterus resulting into urinary stasis which results to bacterial proliferation in urine, serving as an excellent culture media [25].", "The isolated organisms in this study showed that 172 (94%) were of monomicrobial (single isolate) while 11 (6%) were of mixed culture.", "This was in line with [26] in South west, Nigeria who recorded seventy-seven (77) mixed culture representing 17.3% and [27] who also recorded mixed culture of Candida albicans and Staphylococcus epidermis of 7.5%.", "Escherichia coli was the most frequently isolated common UTI pathogens (31.7%).", "This agrees with previous report of [28] and [1] with incidences of 54 and 42%, respectively.", "The high prevalence of E. coli could be that, it is the most common commensal organism.", "Incidence value of E. coli in this work is lower compared to previous works, which may be as a result of the Microbact identification system used and emergence of new strains of pathogens [18].", "K. pneumoneae which was the second most common uropathogen isolated in this study is an indication that the organism is achieving more prominence as etiological agents of UTI than previously reported by Okonko et al. [28].", "S. aureus with prevalence rate of 14.8% constituted a recognizable percentage, Stanley and Kayode [1] reported it as the most frequent pathogen among pregnant women in Nigeria.", "The results of Gram-negative antibiotic susceptibility test revealed varied susceptibility ranging from high, moderate to mild.", "They are bacilli which are normal flora of the intestinal tract accounting for more than 80% of UTIs cases.", "All the Gram-negative organisms were variably susceptible to Chloramphenicol, Sparfloxacin, Ciprofloxacin, Augmentin and Trivid.", "Some of the quinolones were highly active but unsafe to be used in pregnancy.", "The gram-positive drug susceptibility pattern showed high susceptibility except for Amoxicillin which showed high resistant to Streptococcus species.", "Purchase of drugs over the counter, administration of false drugs in treating cases when no prior test is carried out and misused of drugs which could be termed self-medication are common and could be attributed to resistance pattern recorded in some of the organisms.", "The present study shows that overall prevalence of urinary tract infection among pregnant women in the study area is high.", "Predominantly isolated uropathogens were E. coli, K. pneumoniae and P. mirabilis from gram negative and S. aureus from gram positive bacteria.", "Increasing rate of resistance and multi-drug resistance to the commonly used antimicrobial drugs has been noticed for gram negative isolates.", "Routine microbiological analysis and antibiotic sensitivity test of mid-stream urine samples of pregnant women should be carried out to enhance administration of drugs for the treatment and management of UTIs.", "Health education with regular antenatal and personal hygiene is recommended as precautionary measures to UTI.", "I.A. Simon-Oke: Conceptualization, Formal analysis, Writing - original draft, Project administration.", "O. Odeyemi: Conceptualization, Formal analysis, Writing - original draft, Writing - review & editing.", "O.J. Afolabi: Writing - original draft, Project administration, Writing - review & editing." ]	[]
Post-wildfire landscape change and erosional processes from repeat terrestrial lidar in a steep headwater catchment, Chiricahua Mountains, Arizona, USA	Flooding and erosion after wildfires present increasing hazard as climate warms, semi-arid lands become drier, population increases, and the urban interface encroaches farther into wildlands. We quantify post-wildfire erosion in a steep, initially unchannelized, 7.5 ha headwater catchment following the 2011 Horseshoe 2 Fire in the Chiricahua Mountains of southeastern Arizona. Using time-lapse cameras, rain gauges, and repeat surveys by terrestrial laser scanner, we quantify the response of a burned landscape to subsequent precipitation events. Repeat surveys provide detailed pre-and post-rainfall measurements of landscape form associated with a range of weather events. The first post-fire precipitation led to sediment delivery equivalent to 0.017 m of erosion from hillslopes and 0.12 m of erosion from colluvial hollows. Volumetrically, 69% of sediment yield was generated from hillslope erosion and 31% was generated from gully channel establishment in colluvial hollows. Processes on hillslopes included erosion by extensive shallow overland flow, formation of rills and gullies, and generation of sediment-laden flows and possibly debris flows. Subsequent smaller rain events caused ongoing hillslope erosion and local deposition and erosion in gullies. Winter freeze-thaw led to soil expansion, likely related to frost-heaving, causing a net centimeter-scale elevation increase across soil-mantled slopes. By characterizing landscape form, the properties of near-surface materials, and measuring both precipitation and landscape change, we can improve our empirical understanding of landscape response to environmental forcing. This detailed approach to studying landscape response to wildfires may be useful in the improvement of predictive models of flood, debris flow and sedimentation hazards used in post-wildfire response assessments and land management, and may help improve process-based models of landscape evolution.	[ "EART" ]	[ "Erosion", "Hillslope", "Lidar", "Wildfire" ]	[ "1904" ]	[ "Intense rainfall on recently burned landscapes often results in increased flood magnitude, widespread erosion, initiation of debris flows, and sedimentation in stream valleys and on piedmonts in volumes that greatly exceed what is expected from rainfall on unburned landscapes.", "Post-fire sediment yields have been demonstrated to increase by as much as three orders of magnitude over background erosion rates (Wagenbrenner and Robichaud, 2014).", "In the western United States, a history of fire suppression and periodic drought has led to increased frequency, extent, and severity of wildfires over the past several years (Stephens, 2005; Littell et al., 2009).", "Additionally, as human development at urban-wildland interfaces continues, the risk of wildfires and associated hazards increases (Stein et al., 2013).", "This requires that we improve our understanding of the range of responses that arise from the complex functional relationships among basin morphology, mesoscale precipitation, burn severity, soil characteristics, and the thresholds for runoff and debris-flow generation in order to improve conceptual and physics-based predictive models (Moody et al., 2013).", "After a high-severity burn the primary modes of sediment transport on steep, burned slopes prior to the first rainfall include wind erosion and dry ravel (Florsheim et al., 1991; Gabet, 2003; Shakesby and Doerr, 2006; Lamb et al., 2011).", "However, the most dramatic post-wildfire sediment transport results from intense rain events.", "Rainsplash and runoff erosion is exacerbated by a combination of the loss of plants and organic ground cover that previously intercepted precipitation and dampened raindrop energy, the decrease in soil shear strength from the burning of roots, and changes in soil hydrologic properties, particularly decreased infiltration rates (soil water repellency) caused by high temperatures and hyper-dry soils (Robichaud and Hungerford, 2000; Martin and Moody, 2001; Larsen et al., 2009; Moody and Ebel, 2012; Beatty and Smith, 2013; Ebel and Moody, 2013; Nyman et al., 2014).", "Post-wildfire hydrologic response can vary in relation to many factors.", "Erosional patterns range from (1) extensive overland flow erosion in less steep watersheds or in response to lower intensity precipitation, to (2) rilling (defined as local erosion by running water that is shallow enough to be considered an ephemeral landscape feature) and gullying (defined as erosion that forms features with coherent vertical or sloped banks deep enough to persist over seasonal or annual timescales) on hillslopes and along channels where slopes and/or precipitation are moderate, and to (3) extensive mass movement, sediment laden flows, and generation of debris flows in steep landscapes in response to intense precipitation.", "The generation of damaging debris flows is an unresolved research topic; they can occur by several initiation methods including sediment transfer to channels by mass failures, initiation by concentrated water flow (the “firehose” effect) or by runoff generation (Coe et al., 2008; Kean et al., 2013).", "Several key questions regarding the response of landscapes to wildfire require ongoing research effort (e.g., Shakesby and Doerr, 2006).", "These include: (1) How do landscape form, underlying geological materials, and environmental forcing, especially precipitation amount and intensity, relate to the occurrence and vigor of specific landscape-altering geomorphic processes?", "(2) What is the relative role of sediment production from shallow hillslope erosion, rill erosion, and gully erosion, and how might these affect downstream processes such as debris flow generation?", "(3) Can collection of observational data related to precipitation, topography, and sediment delivery lead to improved ability to predict the geomorphic effects of wildfires?", "(4) If so, what measurements on what temporal and spatial scales are required and under what range of forcing conditions?", "We propose that these questions are best addressed by observational studies that span a wide range of landscape steepness, precipitation intensity, and geologic settings.", "Expanding this observational database, even with limited studies such as this, will provide the information required to improve empirical and process-based models of landscape response to wildfire.", "More specifically, it is increasingly common to measure post-fire landscape change with lidar (also referred to as LiDAR, light detection and ranging), because the accuracy and resolution of topographic measurement made with lidar is sufficient to quantify the magnitude of landscape change.", "Both airborne lidar with decimeter-scale resolution and accuracy and terrestrial lidar (also referred to as terrestrial laser scanning - TLS) with sub-centimeter resolution and accuracy allow for mapping of landscape change and the quantification of volumetric sediment yields.", "Schmidt et al. (2011), Wester et al. (2014), and Staley et al. (2014) provide centimeter-scale resolution of landscape change from steep shrubland sites in southern California that are susceptible to debris flows.", "Staley et al. (2014) also report sediment budgets partitioned by landscape morphological unit and by apparent surface process domains over a time interval that includes significant pre-rain dry ravel and one rain event.", "The studied rain event exceeded the rainfall-intensity threshold for post-fire debris-flow initiation, resulting in a series of debris flows reaching the catchment outlet.", "Rengers et al. (2016) provide centimeter-scale change detection and sediment budgeting from repeat terrestrial lidar following a wildfire in the Colorado Front Range.", "They highlight the complex patterns that arise from overland flow in a landscape with a range of slopes and surface roughnesses, and conclude that hillslopes, rather than convergent channels, generate the majority of sediment at their study site.", "McCoy et al. (2010) also report mapping of fine-scale landscape change in a channel subject to debris flows in an unburned drainage basin using laser scanning.", "Pelletier and Orem (2014) report on sediment yields from a larger area (19,700 ha) than this study, and note that they have confidence in integrating hillslope, hollow, and valley bottom changes that exceed the uncertainty of airborne lidar digital elevation models (DEMs).", "By comparing sediment yield from maps of change that include hillslopes and channels, and maps that include only change in channels, they propose that sediment yield from hillslopes and hollows exceeds the more reliably measurable sediment yield from valley bottoms.", "Orem and Pelletier (2015) use repeat airborne lidar, pre- and post-Las Conchas Fire in northern New Mexico, to make an approximation of upland post-fire sediment yield from two small drainage basins.", "They limit their analyses to changes in the valley bottoms in this study.", "Orem and Pelletier (2015) also report on TLS-derived measurements of sediment yield from repeat post-fire scans of piedmonts adjacent to burned basins, and find significant sediment production from the basin.", "This study highlights the potential for high accuracy change detection using multitemporal TLS and provides measures of basin sediment yields that appear to be highly dependent on rainfall characteristics.", "Other studies, including those synthesized in Santi et al. (2008), emphasize the role of sediment entrainment from channel bed erosion and scour and conclude that channels are the dominant source of debris following wildfire.", "Channel change and bed scour are visually impressive, and sediment derived from channel beds may dominate sediment delivery especially in situations in which debris flows initiate in many parts of the channel network and proceed downstream and entrain progressively more sediment.", "Development of predictive models of landscape change, flooding, debris flows, and sedimentation following wildfires is important for land managers and those living downstream of burned landscapes.", "We compare our results of measured landscape change to two empirical predictive models of sediment yield following wildfire (Cannon et al., 2010; Wagenbrenner and Robichaud, 2014).", "We do this in part to evaluate those models, but also to illustrate the range of parameter space and measurement techniques that need to be considered when evaluating risk from wildfire-induced flooding and sediment transport.", "We were motivated to assess the impact of wildfire on landscapes that were subject to dedicated fire suppression over the twentieth century.", "We aimed to map and quantify the diverse surface process regimes that may occur following a wildfire in steep terrain in a spatially extensive manner, and to attempt to use those maps to quantify sediment production.", "This required ultra-high resolution topographic change detection to allow for determination of occurrence or non-occurrence of processes such as dry ravel, overland flow, rill formation, gullying, debris flows, deposition, soil expansion, and channel incision.", "This allows us to test the hypothesis that in coupled hillslope-channel systems, the majority of sediment production comes from overland flow and rilling across extensive hillslope areas, and to address current debate regarding the relative importance of hillslopes and hollows and valley bottoms as sediment sources.", "We also aimed to test the straightforward hypothesis that monsoon rains following high severity wildfire on steep, formerly vegetated slopes would lead to dramatic landscape changes, dominated by widespread hillslope runoff and concentrated sediment-laden flows in convergent parts of the landscape.", "These data can address fundamental uncertainties in how landscapes respond to wildfire including measuring the relative amount of sediment that is produced via overland flow and rill formation on hillslopes versus debris generated by channel incision and bank erosion as a result of gullying in valley bottoms.", "We studied the 2011 Horseshoe 2 Fire in the Chiricahua Mountains of the Basin and Range physiographic province of western North America.", "Over the past few decades, the topographically isolated “Sky Island” massifs of the Basin and Range and the adjacent Mogollon Rim in the southwestern United States have experienced an increased number of wildfires, including many near developed areas (Fig. 1A) (Williams et al., 2015).", "The mountain ranges in this region have low rates of tectonic activity but fairly steep slopes and dense vegetation at high elevations.", "More than a century of fire suppression has allowed fuel loads to increase, and the drought that began in the mid-1990s and high temperatures have lowered moisture levels to the point that when fires do occur, they can leave extensive areas of high burn severity.", "These high severity fires can extend across significant portions of the isolated massifs characteristic of the Basin and Range or across broad portions of the forested Mogollon Rim (Westerling et al., 2006; Williams et al., 2015).", "This study focuses on the Horseshoe 2 Fire, which burned 90,226 ha of the Chiricahua Mountains in southeastern Arizona between May 8 and June 25, 2011 (Fig. 1B) (Youberg et al., 2013).", "This fire burned approximately 70% of the mountain range, and currently stands as the fourth largest wildfire in state history.", "The soil burn severity mapping indicates 30% of the Horseshoe 2 Fire burned at moderate severity and 12% at high severity (Youberg et al., 2013).", "The fire burned between elevations of approximately 1300 m at the base of the range to 2975 m at Chiricahua Peak, and consumed grasslands, Madrean woodlands, and oak, piñon-juniper, pine, and conifer forest.", "Underlying bedrock includes a diverse suite of Proterozoic, Paleozoic, and Cretaceous metamorphic, sedimentary, and volcanic rocks, Oligocene volcanic rocks, and Quaternary basalts and surficial deposits (du Bray and Pallister, 1994; Pallister et al., 1994; du Bray, 1996).", "The Horseshoe 2 Fire did not result in loss of human lives, but it did cause widespread landscape change including rill formation, gullying, debris flows, and downstream sedimentation.", "The fire and subsequent sediment transport caused significant damage to roads, campgrounds, summer homes, a fire lookout, and other forest infrastructure (Youberg et al., 2013).", "Following initial assessment by a U.S Forest Service-organized Burned Area Emergency Response (BAER) team (Youberg et al., 2013), we identified a steep, severely burned headwater catchment beneath Buena Vista Peak to study in detail (Fig. 1).", "The study site is entirely underlain by Oligocene (26–27 Ma) dacite porphyry lava flows emanating from a resurgent caldera (Pallister et al., 1994).", "At the study site regolith formed from this rock was characterized by angular to subangular blocks, up to one meter in diameter.", "Soils in the study area are rocky, gravelly, and sandy but contain organic accumulation and a dense root network.", "The studied catchment is ~ 75,000 m2 in total, but owing to limited time available to perform surveys while the fire was still active, we were able to study the steep ~ 30,000 m2 lower portion of the catchment that we judged to have the highest likelihood of significant landscape change (Fig. 2).", "The upper unsurveyed portion of the catchment rolls gently to a low-slope saddle between two rocky promontories.", "The studied drainage basin has an overall relief of ~ 185 m, though the intensely studied lower portion of the drainage basin has total relief of ~ 130 m.", "The basin has a length of 400 m and a width of just over 300 m. All but the very lowest and highest parts of the studied drainage basin has slopes in excess of 30° and an overall drainage basin slope in excess of 40°.", "The study basin is characterized by steep, soil-and-regolith-mantled slopes with bedrock only exposed at the highest elevations.", "The basin is convergent, but the convergent areas are best characterized (pre-fire) as colluvial hollows rather than channels.", "No evidence for channelized flow was visible prior to the first significant rainfall in this study, indicating that colluvial processes dominated for an unknown period of time before this study, leading to colluvial accumulation in valley bottoms with clear slope breaks to the steeper adjacent hillslopes.", "Numerous stumps and fallen logs left behind after the fire indicate the study area was forested under dense coniferous canopy prior to the wildfire, though the density and preponderance of conifer (likely ponderosa pine) trunks having diameter less than ~ 40 cm indicates the forest was second growth following logging rather than old growth.", "A sawmill was operational in Barfoot Park, just over the ridge from this study site, and extensive logging occurred to support nearby copper mines from 1878 until at least 1902 (Bahre, 1995).", "However, a 1902 map of the forest (Potter, 1902) indicated that even though the study site was adjacent to the mill, logging had not yet affected the site so it is therefore not clear when the site was logged.", "A fire history from nearby Chiricahua National Monument indicates cessation of wildfires in the 1880s as military and logging presence began (Swetnam et al., 1992).", "The relationship between the history of land use and the pre-fire landscape configuration is not clear, but it is possible that some of the colluvial material in the hollows was generated after logging.", "The study area is perturbed by two roads, one is the modern forest road that forms an artificial barrier, penetrated by a culvert, at the downstream end of the study area, and the other is an abandoned road that climbs the lowest part of the east-facing hillslope, crosses the main colluvial hollow, forming a low slope area above a steep reach, and crosses the adjacent hillslope as it climbs across the drainage basin.", "This road may have forced some of the accumulation of colluvium (and perhaps alluvium) in the valley floor immediately above where it crosses the study area.", "We installed one time-stamped 0.2 mm resolution tipping bucket rain gauge on July 1, 2011, in a location near the studied basin that was not blocked by tree canopy.", "This location is in the East Turkey Creek drainage just below Buena Vista Peak (Gauge 1, Fig. 1C).", "A temporary National Weather Service weather station was installed on the drainage divide between East Turkey Creek and Pine Canyon, south of Rustler Park, on July 26, 2011 (Gauge 2, Fig. 1C).", "We also deployed two digital cameras set to take hourly photographs at the locations indicated in Fig. 2.", "To capture high-resolution topography, we repeatedly surveyed the study site with a Leica ScanStation C10 high-definition laser scanner, first on June 21, 2011 (epoch 1) while the fire was still active in the mountain range.", "This instrument has a practical range of around 100 m in natural landscapes, and is able to make 50,000 measurements per second with a distance accuracy of 6 mm, but in practice it creates modeled surfaces with noise of < 2 mm, based on manufacturer specifications and our experience.", "We collected approximately 8 to 10 million points at each scan location.", "To co-register scans from multiple positions into a single coordinate system, we created 11 fixed monuments by hammering rebar into what were judged to be the most stable surfaces (generally divergent ridges and roads) and placed targets on survey poles supported by tripods and set into dimples drilled into the rebar ends.", "These reflective targets are specifically designed to be modeled in the Leica scanner firmware to identify a single center point.", "Three of the rebar monuments were surveyed using a Leica Viva GS15 RTK-GPS (Real-time kinematic - global positioning system) with multiple (~ 4) occupations of each location, providing 3D position accuracy of 4 cm or better.", "Approximately 8 h of GPS base data were post-processed using the NGS OPUS service (http://www.ngs.noaa.gov/OPUS/, accessed July 2011).", "We successfully reoccupied the rebar monuments during each repeat scan and did not re-measure their locations with GPS, so GPS error did not affect the accuracy of the temporal comparisons.", "Collecting GPS data simply allows for reoccupation of the study coordinate system in the future if monuments are lost or disturbed and for placing the survey into a geodetic reference frame.", "The site was rescanned on July 18 (epoch 2), August 1 (epoch 3), August 23, 2011 (epoch 4), and March 14, 2012 (epoch 5).", "During each scan, at least three fixed monuments were reoccupied.", "Each scan epoch utilized somewhat different geometry of scanner positions, which has the effect of creating some variability in presence of occlusions caused primarily by boulders, fallen trees, and standing trees.", "However, the dense network of scan positions and the relatively open nature of the severely burned slopes allowed for production of spatially dense point clouds from each survey epoch.", "Fig. 2 indicates the survey configuration and resulting shaded relief map from the June 21, 2011 survey.", "The data from disparate scanner set-up locations were aligned into a common reference frame and then brought into the Universal Transverse Mercator projected coordinate system.", "These aligned and georeferenced point clouds with point densities exceeding 1000 pt/m2 included measurements of ground but also of trees and other above-ground objects, and error points such as false returns from the sun.", "To isolate only the ground and near-ground environment for change detection, we utilized Terrasolid software to identify ground points from the full point clouds, and gridded these to 2.5 cm resolution using an inverse distance weighted mean algorithm with a search radius of 0.1 m.", "This avoids interpolating across data gaps and provides realistic maps of change displaying data only where data from two scans overlap.", "For scan epochs 1 and 2, we generated elevation models from ground points only as the other scans, and we also created terrain models from ground points and all points within 1 m of the ground using a triangulation with linear interpolation algorithm.", "In areas with high data density, triangulation most accurately represents the actual topography.", "The terrain models that include the points within a meter of the ground incorporate roughness elements such as boulders that were not included in the software-identified ground model.", "This had the effect of including the lowest part of standing trees and any fallen trees into our terrain model.", "We propose that this best defines the area of geomorphic interest; fallen and standing trees are an important part of the geomorphic system (e.g., DiBiase and Lamb, 2013), and including mobile boulders is critical to understanding landscape changes.", "We use these more richly featured data to investigate change between epoch 1 and 2.", "Because of vegetation regrowth, we did not use above-ground points for elevation model construction after scan epoch 2.", "The DEMs from epochs 3–5 have more limited spatial extent than the first two scans and more gaps in data coverage.", "Because of this, in order to create conservative and accurate maps of landscape change, we performed change detection using the DEMs made using an inverse weighted mean algorithm with a search radius of 0.1 m.", "To analyze changes in the landscape through the post-wildfire rainy season, we subtracted the DEMs from one another.", "For the first survey interval (epochs 1 and 2), change detection indicated a magnitude of change sufficient to perform flux calculations.", "For later epochs, we report changes in elevation and hence sediment volume but caution that vegetation regrowth, incomplete data coverage, soil expansion and contraction, and very small net elevation changes increases the uncertainty regarding calculated sediment yields.", "Change detection also provides maps of landscape change that can be interpreted for signatures of specific surface processes.", "The uncertainty inherent in using subtraction of DEMs generated from dense but heterogonous 3D laser scan data to quantify sediment flux can be spatially variable, and is dependent on many factors.", "It has been proposed that areas with increased roughness and slope might be most susceptible to uncertainty (e.g., Wheaton et al., 2010).", "In this study, the steep slopes were at higher angles to the scanner and therefore well resolved and the high density of our scan positions minimized occlusions, so for the majority of our study area, the uncertainty in change detection is minimal.", "Vertical and/or horizontal misalignment between scan epochs is a common source of uncertainty in change detection studies.", "Shilpakar et al. (2016) report that their TLS methods lead to measurement repeatability on stable geomorphic surfaces as poor as several centimeters.", "This suggests a relatively large minimum level of detection is appropriate for TLS-derived landscape change analyses.", "However, this test uses older scanners and a somewhat limited methodology, and, in our view, overstates the uncertainty minimization possible in studies of landscape change with advanced TLS data collection and point cloud processing methods (e.g., DeLong et al., 2015).", "Because we reoccupied monuments in this study, misalignment error only results from variation in setup of reflective targets over monuments, monument instability, and scanner measurement uncertainty.", "The mean elevation difference on the gravel road in the study area from the first to second surveys is 0.002 m with a standard deviation of 0.0038 m.", "The root mean square error (RMSE) calculated between the first two DEMs covering the road is 0.0043 m (number of DEM pixels analyzed n = 276,519).", "The mean elevation difference and RMSE between DEMs represents a combination of measurement repeatability error and actual change on the gravel road surface, which was subject to both rainfall, runoff and vehicle traffic.", "Thus, the elevation difference and RMSE is an overrepresentation of the actual uncertainty in repeatability of TLS scans on stable surfaces.", "This apparent stability indicates, at maximum, a very minor (< 0.002 m) systematic vertical error caused by misalignment of these scan epochs could exist.", "If a systematic error was found to be significant with any confidence it could be corrected, but we did not detect a particular bias in our measurements of topographic change.", "We also assessed horizontal offset by visual inspection of change maps in steep areas, as horizontally misaligned data have systematic bias in their vertical differences for slope of varying aspect (e.g., DeLong et al., 2012) and by modeling all tree trunks in each scan epoch as cylinders using a proprietary algorithm from Leica Geosystems.", "We detected no visual evidence of horizontal misalignment between elevation change maps, nor did we detect systemic bias in hundreds of tree-derived-cylinder positions.", "Furthermore, the lack of change evident at the locations of tree trunks in the DEM subtractions indicates good multitemporal alignment.", "These lines of evidence support the assertion that any horizontal offset is below a detectable level and that misalignment has a negligible effect on uncertainty budgets.", "Another possible source of uncertainty is in locations having low or disparate point density between scan epochs.", "The average rich-ground point density for scan epoch 1 is > 700 pts/m2 and for scan epoch 2 is > 900 pts/m2, but some data gaps near the margins of the scan area indicate lower point density, which can lead to spurious change detection.", "We mapped the point density and identified areas with point density < 50% of the mean in order to determine their influence on the sediment flux calculations.", "We found that these relatively low point-density areas make up 34% of the study area and contribute 7% of the volumetric sediment flux from the study area, so should not have undue influence on the overall sediment budgeting.", "The most widely used approach to error budgeting in survey-derived sediment budgets is to assign a minimum level of detection and discount the change that occurs within that level of detection using methods ranging from simple and spatially uniform to sophisticated and spatially variable (e.g., Wheaton et al., 2010; Pelletier and Orem, 2014; Staley et al., 2014).", "These methods are most appropriate if the source of error is uncertainty of individual points caused by measurement noise, interpolation effects, or occlusion effects.", "Though we do not detect systematic error in our analyses, we take a simple approach to assigning systematic uncertainty in our sediment budgeting.", "We use the lines of evidence we have: apparent repeatability of measurement of the road surface and the (lack of) apparent change of other stable surfaces and objects (e.g., trees) to assume that the uncertainty in any sediment budget derived from our data is approximately equal to the volume of a 0.005 m systematic offset across the calculation area in any two datasets.", "We propose 0.005 m is the level at which we would be able to visually identify a systematic offset, in accordance with our own experience and with the scanner manufacturer specifications, and assign this as the minimum level of detection.", "This value is also greater than our stable-ground RMSE and would likely be even lower in a study that did not include such widespread landscape change, which minimizes the availability of stable surfaces that provide confidence in assessment of systematic error.", "The effect of propagating such a seemingly small potential systematic error through the sediment budget is a reminder that quantifying the quality alignment of ultra-high resolution multitemporal data remains a topic for ongoing method development.", "The point clouds used for the topographic analyses are available in DeLong (2017).", "A summary of precipitation and GPS survey data can be found in the data supplement.", "On June 21, 2011, the study site appeared to have changed little since it burned.", "The fire advanced through the site on either June 2 or June 3, 2011 (http://inciweb.nwcg.gov/incident/map/2225/11/, accessed September 2015).", "It was apparent that much of the organic ash had been removed by particularly strong winds on June 19, 2011 (as observed by the second author as part of BAER team work).", "The landscape had a “mature” look – slopes were rocky but soil-mantled with rounded crests, and the convergent parts of the landscape had extensive colluvial deposits, no established channels, and no evidence for recent flowing water (Fig. 3A).", "The only vegetation present included live trees near the road at the bottom of the studied slopes, and hundreds of standing dead trees devoid of fine branches, leaves, or needles.", "The first and largest storm of the study period occurred on July 11, 2011 (Table 1).", "This storm was characterized by 54 mm of rain in 1.6 h and had a peak 10-min rainfall intensity of 82 mm h− 1.", "This and a subsequent smaller storm (16 mm rain total and peak 10-min intensity of 49 mm h− 1) were the only storms that occurred in this time interval.", "The larger storm peak intensity represents an approximately 10 yr recurrence interval for southeastern Arizona (Osborn and Renard, 1988).", "Time lapse photos (Figs. 4 and 5) make it clear that widespread overland flow led to channelized flow in the colluvial hollows and extensive landscape change, including the mobilization of large fallen trees and boulders.", "Erosion revealed that much of the soil root structure was left intact by the wildfire and loss of soil strength caused by the burning of roots may not be of primary importance in this study area.", "The change map (Fig. 6) provides a spatially extensive view of how this burned landscape changed.", "While it should be noted that the change map represents only part of the full headwater catchment, the pattern of change indicates that much of the area with > 0.005 m of change is likely included in this analysis area and captures many areas of shallow erosion, rilling, gullying, channelization, and deposition.", "As such, it is likely to be representative of the distribution of landscape responses across the full drainage basin and similar basins.", "The most obvious changes to the landscape were the result of widespread and significant sediment mobilization and erosion.", "The patterns of erosion (Fig. 7) indicate that nearly the entire ground surface experienced overland flow, which was focused into narrowly spaced rills that were parallel on planar slopes and convergent on convergent slopes.", "These coalesced to form extensive gullies that removed a significant amount of material from the formerly unchannelized colluvial hollows.", "Based on field observation and interpretation of patterns of landscape change, it is apparent that overland flow removed an average of 0.02 m of material from hillslope areas (Table 2).", "Rill formation, which appears to be narrow and shallow enough to be ephemeral over annual timescales, removed between 0.02 and 0.10 m of material.", "Gullying, which formed channels with banks that appeared cohesive enough to be preserved over annual time scales, eroded anywhere from 0.10 to > 1.0 m depth.", "There was also deposition that occurred in the study area during this time interval (Fig. 8).", "Most obvious is a large amount of sediment that was trapped upstream of the culvert that was designed to allow flow through the road embankment.", "Some apparent deposition at the margins of the study area is merely an artifact caused by heterogeneous data occlusions in the two datasets.", "However, significant gravel- to boulder-rich deposits were emplaced along the margins of the gullies formed in the lower reaches of the study basin (Fig. 9).", "These provide evidence for debris-flow occurrence, or at the least enough increased sediment concentration (bulking) in concentrated water flow to overwhelm the gully channels.", "Some of the largest boulders (up to ~ 1 m diameter) deposited at the margin of flows can be seen on the sub-horizontal surface (which is a remnant of a road) in the upper part of the lower panel of Fig. 5.", "The widespread landscape change that occurred in the first scan interval allows for calculation of volumetric changes in sediment from the study area (Table 2).", "We classify the study area in three morphologic units based on obvious slope breaks (Fig. 7): (1) hillslopes, with steep slopes averaging 30 degrees in the steepest areas (Fig. 3), (2) hollows, with slopes averaging 23 degrees that were initially unchannelized, filled with colluvium, and separated from hillslopes by distinct slope breaks, and (3) the depositional zone, which is an artificial basin formed upslope of the road berm.", "After the first post-fire rain event, it was apparent that the culvert intended to drain this catchment was not able to convey all water and sediment exported from hillslopes and hollows and the road berm acted as a sediment trap for some, but not all, sediment.", "Volumetric analysis (Table 2) reveals that the volume of sediment removed from the hillslopes (394 ± 125 m3) was more than double the volume of material removed from the hollow (184 ± 8 m3).", "However, the average erosion depth in the hollow (0.12 m), which was characterized by gully formation, was a factor of seven greater than the hillslopes (0.018) that experienced overland flow erosion, rilling and some gullying.", "The depositional area at the base of the catchment and upstream of the road berm captured less than half the volume of the sediment delivered from upstream.", "The missing sediment was transported farther downstream through the culvert, which clogged at some point during the flow event.", "It was clear that no flow occurred over the road surface.", "Some of the difference between the volume of eroded and deposited material could possibly be caused by changes in density between the depositional material and eroded soil.", "This volumetric balance also does not take into account material eroded from farther up in the catchment and transported through the study area.", "Our observations indicate that erosion depths farther up the drainage basin, above where we were able to scan, were minimal because of lower slope angles and smaller contributing area for runoff.", "However, the extent of the area is large enough that the volume contributed from above the scan area may be non-trivial.", "Four rainfall events of > 10 mm occurred between the second and third survey epochs, with peak 10-min rainfall intensities ranging between 6 and 41 mm h− 1 (See Table 1).", "Cumulatively, these caused modest hillslope erosion and sediment erosion and deposition in the gully bottoms (Fig. 10).", "The TLS survey completed on August 1, 2011 was less spatially extensive than earlier surveys, but captured patterns of change including limited hillslope erosion, widespread hillslope stability and observable deposition in some gully bottoms and continued incision in others.", "Over this survey interval sparse herbaceous vegetation (mostly ferns) began to regrow.", "This vegetation was concentrated in patches on the landscape, particularly in areas of minimal erosion high on slopes, as can be seen in Fig. 11.", "Point cloud classification removed much or all of this vegetation from the study area, except possibly in locations with very low laser pulse incidence angles and low vegetation that completely blocked laser returns from bare ground.", "From August 1 to August 23, 2011, three rain events totaling > 10 mm occurred with peak 10-min rainfall intensities ranging between 18 and 25 mm h− 1 (See Table 1).", "These events cumulatively led to modest landscape change (Fig. 12).", "This change included extensive but shallow hillslope erosion and deepening of rills.", "Changes on the hillslopes were mostly < 0.05 m of erosion.", "In the topographic hollows, extensive deepening of gullies occurred.", "Gully deepening included both removal of material deposited in previous survey interval and incision to unprecedented depths.", "Vegetation growth became quite significant during this scan interval, particularly on south-facing slopes.", "The late summer, fall, and winter months included three modest rainfall events and a number of precipitation events that were likely snow.", "Landscape change during this period was minimal (Fig. 13).", "Most emergent vegetation died and was flattened by snowpack.", "The most noticeable landscape change that occurred during this period was an overall increase in elevation of soil surfaces on the order of 0.036 ± 0.021 m (based on transects in Figs. 13 and 14).", "The road surface, by contrast was much less affected, with an average elevation increase of 0.007 ± 0.006 m over a linear transect (Figs. 13 and 14), and 0.001 ± 0.007 m over the entire road surface in the study area.", "During the warm months of 2012, thick vegetation covered the study basin, precluding the effective use of TLS (Fig. 15).", "This revegetation of the hillslopes likely increased soil stability and lowered rainfall impact energy.", "Our visual observations made during a visit to the study site in September of 2012 did not indicate significant landscape change occurred during the summer of 2012.", "The processes by which sediment is eroded, transported, and deposited following wildfire result from factors including rainfall characteristics, nature of surficial materials, state of vegetation and root structure, and the details of landscape form.", "Several specific processes should be considered when interpreting or anticipating landscape change from wildfires.", "These include changes in infiltration rate, presence of mass wasting including shallow landslides, generation of debris flows, and extensive overland flow and sediment entrainment.", "Because increased flood hazard is a primary concern following wildfire, the effect of wildfire on soil infiltration rates has been widely explored (Cannon et al., 2001; Martin and Moody, 2001; Cannon et al., 2003; Ebel et al., 2012; Parise and Cannon, 2012; Ebel and Moody, 2013).", "We were unable to make specific measurements of infiltration rates for this study, but did not observe evidence for phenomena that is commonly cited as drivers of radically increased runoff such as sealing by waxy buildup, extreme drying, macropore clogging with ash, or other chemical or physical changes that would affect soil infiltration (e.g., Robichaud and Hungerford, 2000; Nyman et al., 2014).", "We also did not observe evidence for mass wasting in the form of local soil failures, headscarps, or coherent masses of soil loss observable in the topographic change measurements.", "Further, we did not observe significant dry ravel manifest as debris cones or aprons at the base of steep slopes, nor did we note that walking on slopes revealed extensive loose sediment that was gravitationally unstable enough to ravel as dry avalanches when disturbed.", "Evidence for the occurrence of debris flows in the study area was ambiguous.", "In the first scan interval, coarse debris was transported through the convergent areas of the landscape and deposited both adjacent to apparent flow paths and in the artificial basin at the low end of the study area.", "However, it was not obvious that these were a result of flows with the rheology of debris flows; they may simply have been sediment-laden flows that overwhelmed gullies and expanded onto adjacent unincised parts of the landscape.", "While the deposits were unsorted, they were not obviously matrix-supported, and were not obviously in the form of levees that might provide stronger evidence for the occurrence of debris flows.", "In any case, the largest runoff event transported a wide array of coarse debris in a high-energy sediment transport event.", "The dominant surface-altering processes that we were able to infer from our field observations and our measurements of topographic change were erosion by flows that transitioned from shallow overland flow to slightly concentrated, rill-forming flow to channelized, gully-forming flow.", "Overland flow caused erosion over extensive areas of the landscape, upslope from convergent hollows.", "As this flow reached steeper slopes rill erosion occurred and this transitioned to deeper erosion in convergent parts of the landscape and formed gullies tens of centimeters deep in the colluvial hollows (Fig. 16).", "Subsequent to the largest gully-forming event on July 11, 2011, there was minimal change on hillslopes; however, there was localized erosion and deposition in the established gullies, indicating sediment transport by runoff.", "This relatively simple suite of processes generated significant landscape change, resulting in channelization of previously unchannelized colluvial hollows.", "This rapid increase in drainage density has the effect of changing hydrological behavior, and the extensive erosion may support the hypothesis that runoff can generate sediment-laden flows and perhaps even debris flows in absence of mass wasting (Cannon et al., 2003).", "We are able to map and quantify the processes first described in detail by Cannon et al. (2003) whereby sheetflow erosion transitions to rill erosion, then into rapid channelization of colluvial portions of the landscape.", "The significant erosion in the study area was a result of a storm that caused debris flows elsewhere in the mountain range (Youberg, 2014).", "The July 11, 2011 precipitation event exceeded all of the precipitation intensity thresholds proposed by Staley et al. (2017) for post-wildfire debris-flow initiation in southern Arizona, and indeed for any of the field areas studied.", "Results from the high resolution measurement of landscape change using the best available technology and methods at the time of the data collection represent accurate and spatially extensive landscape change in response to quantified environmental forcing.", "These fundamental observations provide the foundation for expanding our understanding of the physics and relevant processes that lead to landscape change in burned watersheds.", "Several key observations emerge from this study that corroborate and build upon previous studies of post-wildfire landscape change.", "These include the observation that in severely burned steep landscapes, nearly the entire land surface is subject to erosion by flowing water.", "Also, shallow but extensive erosion rapidly transitions to concentrated erosion, deep incision, and rapid expansion of the channel network into unchannelized convergent hollows and onto steep hillslopes.", "Furthermore, even in small headwater catchments, progressive sediment bulking leads to sediment-laden flows and possible debris flows capable of transporting remarkably coarse material (~ 1 m diameter).", "Collectively, these observations point to the likelihood that wildfires radically change the rates of landscape change from background rates.", "Though we are unaware of long-term erosion rates determined for the Chiricahua Mountains, millennial scale erosion rates from the nearby and morphologically similar Pinaleño Mountains range from 0.026 to 0.063 mm yr− 1 (Jungers and Heimsath, 2015).", "These long-term erosion rates are orders of magnitude lower than a rate derived from the 22 mm of average erosion that occurred from the year of monitoring following the Horseshoe 2 Fire in this steep basin.", "The notion that post-fire erosion and landscape change far outpace background rates and can involve relatively rare, highly energetic processes is well-accepted based on many previous studies (e.g., Moody et al., 2013 and references therein).", "More recent and detailed studies such as Cannon et al. (2010), Staley et al. (2014), Wagenbrenner and Robichaud (2014), and Rengers et al. (2016) improve our understanding of the specific relationships among landscape form, burn characteristics, and magnitude and intensity of precipitation.", "Staley et al. (2014) measured sediment delivery from distinct morphological units in relation to a single rain event in a steep, small basin in southern California.", "That study serves as an important contrast to this study because the pre-fire vegetation cover, frequency of wildfires, storm hyetograph, and types of surface processes are quite different between the two studies.", "In general, in the steep basins of the San Gabriel Mountains, the sequence of fires, floods and debris flows are considered to be an important part of long-term landscape evolution and have occurred with some frequency in modern times (e.g., Cannon and DeGraff, 2009).", "The Chiricahua Mountains, in contrast, had frequent, low-severity ground fires until the turn of the twentieth century, after which wildfires were exceedingly rare until the last few years (Seklecki et al., 1996; Swetnam, 2005; Fulé et al., 2012).", "Furthermore, Staley et al. (2014) studied the second of several debris-flow generating rain events, each of which were interceded by significant dry ravel transport from slope to channels.", "The studied rain event was much longer in duration (61 h) and lower in intensity than the storms that affected our study site.", "The peak rainfall intensity measured by Staley et al. (2014) was much lower (peak 15-min intensity of ~ 28 mm h− 1 and peak 30-min intensity of 23 mm h− 1) than the storms measured in this study (peak 10-min intensity of 82 mm h− 1, peak 15-min intensity of 79 mm h− 1, and peak 30-min intensity of 72 mm h− 1).", "The rain events in southern California led to pulsed debris-flow activity, highlighting the importance of precipitation and significant availability of sediment.", "Though Staley et al. (2014) perform finer-scale morphological unit classification than we did, they find that hillslope erosion with a typical depth of 0.01 to 0.02 m dominates the sediment budget.", "In contrast to our study they find that the channel network was a net sediment sink.", "This may be partially a result of the occurrence of dry ravel, and partially a result of filling antecedent topography (rills and gullies) created in the previous runoff event (Schmidt et al., 2011).", "In this case, channel sedimentation was a transient phenomenon that was captured over their survey interval but then was evacuated by later flows leading to bedrock-exposed channel beds by the end of the winter (D. Staley, personal communication, 2016).", "This channel deposition during lower intensity precipitation events and subsequent modification of aggraded channels and removal of channel sediment was also observed in the Chalk Cliffs of Colorado by McCoy et al. (2010).", "Rengers et al. (2016), in a study similar in scope and methodology to this one, measured erosion as a result of several storms on a burned area in the Colorado Front Range.", "They report change resulting from events including their largest one with shorter duration (22.5 min) but comparable intensity (peak 10-min intensity of 72 mm h−1) as the largest storm we measured.", "Several similarities, including evidence of extensive overland flow and widespread hillslope erosion at depths of 1–10 cm, exist between their study and ours.", "They do not, however, observe mobilization of large boulders, significant gully development, or coarse debris flows.", "The patterns of landscape erosion they measure are more spatially variable and less continuous, perhaps because of lower overall slopes, greater variation in roughness and lower overall rainfall magnitudes.", "The average slope of the hillslope portion of our field area is 30 degrees, whereas the Rengers et al. (2016) study site has average hillslope slopes ranging from 20 to 28°.", "The magnitude of erosion in their study is also notably lower than we measured.", "Following their largest storm event, they report a net erosion depth of between 0.0014 and 0.0089 m in hillslope regions as compared to 0.018 in the hillslope region in our study, and 0.021 m of erosion in convergent areas as compared to 0.12 m in our study.", "These differences highlight that the process transition to gullying and possible debris-flow generation leads to dramatically higher sediment yields from burned landscapes.", "The maximum 10- and 30-min precipitation intensities (72 and 82 mm h− 1) from the first post-fire storm in this study (Table 1) also sit at the high end of intensities reviewed in Moody and Martin (2009).", "They describe peak 30-min rainfall intensities > 52 mm h− 1 as “extreme”, and found only in the Great Plains and in southern Arizona in the United States.", "Wohl and Pearthree (1991) indicate catchment erosion magnitudes of 0.010–0.017 mm can be calculated from their estimates of sediment deposition volume following an intense storm totaling > 20 mm of rain on slopes with high antecedent moisture.", "These values integrate across entire 1.25 and 0.6 km2 catchments, making it possible that erosion magnitudes on the steepest sub-catchments similar to this study may have approached the > 20 mm of erosion that we measured in this study.", "Our measured sediment yields are similar to the highest values reviewed in Moody and Martin (2009), but our results (Table 2) indicate that sediment yield from hillslopes exceed those from hollows and valleys by a factor of two to one.", "This result contradicts Moody and Martin's (2009) findings, which indicate channels are the main source of post-wildfire sediment.", "Our results are, however, in general agreement with Rengers et al. (2016) findings that 87% of erosion occurred on hillslopes following the 2010 Fourmile Fire in Colorado.", "However, direct comparison among these studies is somewhat difficult because of the wide range of measurement techniques and the uncertainty of classification of landscape components from which other workers' data were generated.", "For example, in studies of larger drainage basins the ratio of channel area to severely burned hillslope and hollow area may be higher.", "When lidar is used to determine sediment production, the higher resolution of data available may do a better job of quantifying hillslope change than would a more traditional study of field-surveyed cross sections, for example, and the lidar studies may have a smaller footprint focused on the most changing part of a larger landscape.", "Empirical models such as that of Wagenbrenner and Robichaud (2014) and further studies reliant on hillslope plots and catchment-scale sediment traps may succeed at predicting sediment yield from low intensity rainfall events that are difficult to measure with TLS.", "However, TLS offers the advantage that observations regarding relevant surface processes can be made directly from the high-resolution change maps.", "For example, though total sediment yield was very low as a result of the low-precipitation events in our study area, we were able to map the locations from which sediment was liberated, and note the locations of increased sediment storage and ongoing channel incision.", "Sediment traps cannot provide such insights into specific hillslope and channel processes and even direct field observation does not easily reveal the spatial distribution of such minor landscape changes.", "The landscape changes that occurred over the winter of 2011–2012 were characterized by centimeter-scale elevation increase across soil-mantled hillslopes.", "We observed that the soil appeared to have lowered density as indicated by the impact of walking on it during the spring survey in contrast to the typically dense soil exposed in locations of recent overland flow erosion.", "We suspect that freeze-thaw and wet-dry cycles led to soil dilation, perhaps as a result of frost heaving (e.g., Taber, 1930).", "This dilation will affect soil hydrology, soil strength, vegetation regrowth, and the availability of fine particles for erosion in subsequent precipitation events.", "This phenomenon may demand consideration as a driver of landscape change in high-elevation or latitude post-fire research.", "Repeat terrestrial laser scanning, if initiated after a landscape burns, but prior to the first rainfall, offers the possibility of determining sediment yields and mapping surface processes that lead to dramatic landscape changes.", "When complemented by measurement of precipitation, the relationships between environmental forcing and landscape response can begin to be quantified.", "This approach, as well as more complete studies that integrate hydrological monitoring and detailed analysis of soil conditions, can lead to improvement of the sparse data available relating post-fire sediment yield and flood studies to basin characteristics and weather events.", "Improving our understanding of how landscapes respond to fire and subsequent storms is increasingly important as land managers and various stakeholders continue to cope with increased fire activity, drought, and human encroachment into wildlands.", "This study highlights the importance of precipitation intensity in driving post-fire landscape processes.", "The first rainfall following the wildfire was also the most intense, having a peak 30-min intensity of 72 mm h− 1 and causing the most dramatic landscape change during the study period, leading to widespread centimeter-scale hillslope erosion from overland flow, extensive centimeter- to decimeter-scale rilling, and establishment of decimeter- to meter-scale gullies in convergent hollows and valley bottoms.", "These processes generated an average erosion magnitude of 0.022 m across the study area, and a catchment sediment yield that likely exceeds 200 t/ha.", "The majority (68%) of the sediment was generated from relatively shallow hillslope erosion, and the remainder (32%) was generated from the more visually obvious erosion in colluvial hollows and previously unchannelized valley bottoms.", "Sediment yields from this event are largely consistent with predictions from an empirical model derived from debris-flow-dominated basins as well as with predictions from an empirical model derived from measurements from basins that experienced mainly lower-energy processes such as rainsplash, overland flow, and rill formation.", "These results highlight our increasing ability to predict landscape change following wildfires.", "However, the range of landscape conditions and weather events observed in previous studies is not comprehensive and the potential for further deviations from studied parameter space as fire and storm intensity increases owing to global climate change demands future study.", "Expanding direct measurement of rainfall and surface processes will help fill significant knowledge gaps regarding the details of post-wildfire response, as well as the range of flood and sediment discharges that occur in burned catchments under widely variable environmental and landscape conditions.", "Importantly, our study is representative of many of the tectonically quiescent ranges of the American Southwest that have experienced relatively few severe wildfires in the last century, and are characterized by long-term sediment accumulation in unchannelized headwater catchments and colluvial hollows.", "The results of this study may indicate that in such locations, wildfire can rapidly change the surface process regime, leading to rapid expansion of drainage networks, extensive overland flow erosion on hillslopes, and high sediment yields.", "It further suggests a need to study the longer-term landscape recovery following these rapid geomorphic changes, especially in terms of ecological recovery, hydrologic process, and the degree of ongoing hazard to a wide range of stakeholders." ]	[ "Wildfire leads to erosion following rain in a steep headwater catchment.", "Terrestrial laser scanning was used to characterize areas of erosion and deposition.", "Overland flow, riling and gullying were extensive following the first intense storm.", "More sediment was eroded from hillslope areas than channels and colluvial hollows." ]
Differences in the prospective association between individual plasma phospholipid saturated fatty acids and incident type 2 diabetes: The EPIC-InterAct case-cohort study	Background: Conflicting evidence exists regarding the association between saturated fatty acids (SFAs) and type 2 diabetes. In this longitudinal case-cohort study, we aimed to investigate the prospective associations between objectively measured individual plasma phospholipid SFAs and incident type 2 diabetes in EPIC-InterAct participants. Methods: The EPIC-InterAct case-cohort study includes 12 403 people with incident type 2 diabetes and a representative subcohort of 16 154 individuals who were selected from a cohort of 340 234 European participants with 3·99 million person-years of follow-up (the EPIC study). Incident type 2 diabetes was ascertained until Dec 31, 2007, by a review of several sources of evidence. Gas chromatography was used to measure the distribution of fatty acids in plasma phospholipids (mol%); samples from people with type 2 diabetes and subcohort participants were processed in a random order by centre, and laboratory staff were masked to participant characteristics. We estimated country-specific hazard ratios (HRs) for associations per SD of each SFA with incident type 2 diabetes using Prentice-weighted Cox regression, which is weighted for case-cohort sampling, and pooled our findings using random-effects meta-analysis. Findings: SFAs accounted for 46% of total plasma phospholipid fatty acids. In adjusted analyses, different individual SFAs were associated with incident type 2 diabetes in opposing directions. Even-chain SFAs that were measured (14:0 [myristic acid], 16:0 [palmitic acid], and 18:0 [stearic acid]) were positively associated with incident type 2 diabetes (HR [95% CI] per SD difference: myristic acid 1·15 [95% CI 1·09-1·22], palmitic acid 1·26 [1·15-1·37], and stearic acid 1·06 [1·00-1·13]). By contrast, measured odd-chain SFAs (15:0 [pentadecanoic acid] and 17:0 [heptadecanoic acid]) were inversely associated with incident type 2 diabetes (HR [95% CI] per 1 SD difference: 0·79 [0·73-0·85] for pentadecanoic acid and 0·67 [0·63-0·71] for heptadecanoic acid), as were measured longer-chain SFAs (20:0 [arachidic acid], 22:0 [behenic acid], 23:0 [tricosanoic acid], and 24:0 [lignoceric acid]), with HRs ranging from 0·72 to 0·81 (95% CIs ranging between 0·61 and 0·92). Our findings were robust to a range of sensitivity analyses. Interpretation: Different individual plasma phospholipid SFAs were associated with incident type 2 diabetes in opposite directions, which suggests that SFAs are not homogeneous in their effects. Our findings emphasise the importance of the recognition of subtypes of these fatty acids. An improved understanding of differences in sources of individual SFAs from dietary intake versus endogenous metabolism is needed.	[ "BIOC", "MEDI" ]	[]	[ "1310", "2712", "2724" ]	[ "Saturated fatty acids (SFAs) are generally thought to have detrimental effects on health, as represented by the widespread public health message advising a reduction in SFA intake to less than 10% or even 7% of total energy to benefit cardiometabolic health, including lowering of type 2 diabetes risk.1", "However, little evidence exists to support adverse effects of high SFA intake on risk of type 2 diabetes.2", "Indeed, the Women's Health Initiative Diet Modification Trial3 suggested no benefit of a reduction in SFA intake on the incidence of type 2 diabetes.", "Accumulating evidence suggests that intake of dairy products, which are typically high in SFA content, is inversely associated with type 2 diabetes,4,5 which, together with the null or inconsistent evidence about total SFA intake and risk of type 2 diabetes, has raised doubts about whether all SFA intake has adverse health effects.", "Previous studies of dietary SFA intake have had inconclusive results, limited by measurement error of dietary assessment, and have focused on total SFA intake without analysis of SFA intake varying by carbon chain lengths.", "The objective measurement of SFAs with different carbon chain lengths in blood fractions enables assessment of individual SFAs.6", "SFAs in blood can be directly interpreted as dietary SFAs for fatty acids that are good biomarkers of intake, such as 15:0 (pentadecanoic acid) and 17:0 (heptadecanoic acid), which are exogenously derived from dietary dairy fats.6–8", "However, interpretation is more complex for SFAs like palmitic acid (16:0) and stearic acid (18:0), which are synthesised endogenously through de-novo lipogenesis stimulated by increased intake of carbohydrates and alcohol,6,9–12 and which might only partly represent dietary intake.6,13", "The extent to which different dietary components can induce de-novo lipogenesis varies.11", "Additionally, uncertainties remain about the extent to which dietary SFAs are incorporated into blood SFAs, and the relative contribution of de-novo lipogenesis versus habitual diets to the amounts of SFAs circulating in the blood.14", "However, the varying effects of different blood SFAs on the risk of type 2 diabetes are of scientific and public health interest.", "Only a few small studies have assessed a range of circulating SFAs,15–21 and evidence supporting associations of different blood SFAs with the incidence of type 2 diabetes is scarce.", "In this large longitudinal study of the European Prospective Investigation into Cancer and Nutrition Study (EPIC)-InterAct study,22 we aimed to investigate the prospective associations between objectively measured individual SFAs in the plasma phospholipid fraction and incident type 2 diabetes.", "We also investigated associations of food consumption with circulating SFAs, and studied SFA metabolism indirectly by analysing ratios of relevant fatty acids.", "The methods of the InterAct project have previously been described in detail.22", "To summarise, we did a case-cohort study that combines the temporal sequence and power advantages of a large prospective cohort with the measurement efficiency of a case-control study.", "Additionally, since the random subcohort is selected from the entire cohort independent of the outcome, it can be used as a comparison cohort for various outcomes of interest.", "From 340 234 people with 3·99 million person-years of follow-up (1991–2007) in eight countries of the EPIC study (France, Italy, Spain, UK, Netherlands, Germany, Sweden, and Denmark), we verified 12 403 cases of type 2 diabetes.", "From the EPIC cohort, we also used a random number generator to randomly select 16 835 people with baseline plasma samples in a subcohort.", "After exclusions for prevalent diabetes and uncertain diabetes status, 16 154 individuals remained in the subcohort, including 778 with incident type 2 diabetes during follow-up (a feature of the case-cohort design).22", "From this case cohort of 27 779 participants, we excluded 483 for whom no fatty acid data were available, leaving 27 296 adults, among whom there were 12 132 cases of type 2 diabetes and 15 919 subcohort participants (including 755 incident cases of type 2 diabetes within the subcohort; figure 1).", "All participants provided written informed consent and the study was approved by all local ethics committees.", "Incident type 2 diabetes was ascertained up until Dec 31, 2007, through a review of several sources of evidence, reported previously:22 self-report, linkage to primary care registers, secondary care registers, medication use (drug registers), hospital admissions, and mortality data.", "No diabetes cases were ascertained solely by self-report and we sought further evidence for all cases with information about incident type 2 diabetes from fewer than two independent sources at a minimum, including a review of individual medical records in some centres.", "Cases in Denmark and Sweden were identified from local and national diabetes and pharmaceutical registers and were judged to be verified.", "Fatty acids were profiled at the Medical Research Council Human Nutrition Research (Cambridge, UK); profiling involved analysis of plasma samples stored at baseline at −196°C (or −150°C in Denmark)—a temperature at which fatty acids remain stable.6", "The assay methods have previously been described23 and included hydrolysis and methylation to convert phospholipid fatty acids into more volatile fatty acid methyl esters and separation of the different fatty acids by gas chromatography (J&W HP-88, 30 m length, 0·25 mm internal diameter [Agilent Technologies, CA, USA]) equipped with flame ionisation detection (7890N GC [Agilent Technologies]).", "Samples from people with type 2 diabetes and subcohort participants were processed in random order by centre, and laboratory staff were masked to all participant characteristics by the use of anonymised aliquots.", "We identified 37 different fatty acids by their retention times compared with those of commercial standards and expressed each level as percentage of total phospholipid fatty acids (mol%).", "These fatty acids included nine SFAs with relative concentrations higher than 0·05%: myristic acid (14:0), coefficient of variation 9·4%; pentadecanoic acid (15:0), 11·9%; palmitic acid (16:0), 1·6%; heptadecanoic acid (17:0), 4·2%; stearic acid (18:0), 2·0%; arachidic acid (20:0), 15·3%; behenic acid (22:0), 10·3%; tricosanoic acid (23:0), 18·9%; and lignoceric acid (24:0), 14·7%.", "We used human and equine plasma (Sera Laboratories International, West Sussex, UK) for quality control.23", "Weight and height were measured by trained professionals using standardised protocols and were used to calculate BMI (in kg/m2).", "Waist circumference was measured by staff, with the exception of French participants and a subset of participants from Oxford, UK, who self-reported their measurements, and in Umeå, Sweden, where this parameter was not recorded.", "We used lifestyle questionnaires to assess demographics, smoking status, medical history, and education level.", "Physical activity was assessed by a validated questionnaire from which a four-point ordinal category of activity was derived.", "We assessed habitual diet during the past 12 months at baseline using country-specific validated food frequency questionnaires or diet histories.", "Total energy and nutrient intakes were based on the standardised EPIC Nutrient Database.24", "HbA1C was measured in the erythrocyte fraction from samples stored at −196°C using the Tosoh-G8 HPLC analyser (Tosoh Bioscience, Japan) at Stichting Huisartsen Laboratorium (Etten-Leur, Netherlands).", "We analysed the distribution of individual plasma phospholipid fatty acids and expressed them as mol%.", "We estimated country-specific hazard ratios (HRs) and 95% CIs for associations per one standard deviation (SD, calculated in the overall subcohort) of each SFA with incident type 2 diabetes using Prentice-weighted Cox regression,22 which allows for over-representation of cases in a case-cohort design, and pooled our findings using random-effects meta-analysis.", "Heterogeneity between countries was expressed as I2 values, and we used meta-regression to assess whether the heterogeneity was explained by age, BMI, or sex.", "We adjusted for potential confounders as follows: model 1 included age (as the underlying timescale), study centre, sex, physical activity index, smoking status, and education level.", "Model 2 included these parameters plus total energy intake, alcohol intake, and BMI.", "After recording patterns of association for the nine individual SFAs, we made a post-hoc decision to create three additional exposures based on groupings of SFAs that fit with potential biological action: group 1 (sum of the even-chain SFAs 14:0, 16:0, and 18:0) since these represent both de-novo lipogenesis and dietary intake;9,11,14 group 2 (sum of the odd-chain SFAs 15:0 and 17:0) as potential sources of dairy fat;7,8 and group 3 (sum of the long- or very-long-chain SFAs 20:0, 22:0, 23:0, and 24:0) since these are under-researched SFAs that might undergo distinct peroxisomal fatty acid metabolism rather than mitochondrial metabolism.25", "In an additional analysis, we re-grouped 23:0 into group 2 because is it also an odd-chain SFA, and removed it from group 3.", "Since stearoyl-CoA desaturase-1 catalyses the desaturation of 16:0 to 16:1(n-7) and of 18:0 to 18:1(n-9) through the de-novo lipogenesis pathway, we also estimated stearoyl-CoA desaturase-1 activity using product-to-precursor ratios (ratio of 16:1[n-7] to 16:0 and of 18:1[n-9] to 18:0)6,15,26 and assessed each ratio for its association with incident type 2 diabetes.", "In a sensitivity analysis based on model 2, we analysed the effects of adjustment for dietary variables (intakes of meat, fruit and vegetables, soft drinks, total dairy products, and carbohydrates [g/day]).", "We also did an analysis that further accounted for baseline HbA1C value as a covariate.", "To minimise the possibility of reverse causality, we also excluded 2348 people with HbA1C of 6·5% or higher at baseline or those confirmed as cases of type 2 diabetes (n=1048) within the first 2 years after baseline.", "Further sensitivity analyses on model 2 included adjustment for: additional potential confounders (dietary carbohydrates intake [g/day] and waist circumference [cm]); comorbidity (prevalent myocardial infarction, stroke, or cancer); and the exclusion of 723 people who were probably dietary misreporters (those with a ratio of energy intake to energy requirement in the bottom or top 1% of the distribution).", "We also studied the association of SFA quintiles with type 2 diabetes incidence in models 1 and 2.", "We postulated that circulating SFAs would be derived from diet and through de-novo lipogenesis, and associated with carbohydrate and alcohol consumption.9,11,12", "Within the subcohort, we studied associations between each circulating SFA and food intakes, using Pearson correlation coefficients and 95% CI adjusted for age, sex, BMI, and energy intake.", "We used Stata, version 13.1 for all analyses.", "The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.", "The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.", "In the subcohort participants (mean age 52·3 years [SD 9·2]), SFAs comprised 46% (SD 1·2%) of the total phospholipid fatty acids, with the greatest contributors being the even-chain fatty acids 16:0 (mean 30·1% [SD 1·7%]) and 18:0 (14·1% [1·3%]), whereas 15:0, 17:0, and 20:0–24:0 were present in low relative concentrations (all <0·5%) (table 1).", "Older adults, those with higher BMI, and men had higher relative concentrations of even-chain SFAs (14:0, 16:0, and 18:0), whereas we noted the opposite for longer-chain SFAs (20:0, 22:0, 23:0, and 24:0) (table 1).", "Relative concentrations of odd-chain SFAs (15:0 and 17:0) were higher in people with a lower BMI and in women, but were similar across different age groups (table 1).", "Appendix p 2 shows SFA distribution by country.", "Model 2 in table 2 shows that even-chain SFAs were each positively associated with type 2 diabetes risk individually and when combined (SFA group 1 HR 1·43 [95% CI 1·29–1·58]).", "By contrast, odd-chain SFAs were each inversely associated with type 2 diabetes individually and when combined (SFA group 2 HR 0·70 [95% CI 0·66–0·74]).", "Longer-chain SFAs were each inversely associated with type 2 diabetes individually and when combined (SFA group-3 HR 0·70 [95% CI 0·59–0·84]).", "The results were similar when 23:0 was included in SFA group 2 and removed from group 3 (data not shown).", "Sensitivity analyses showed the same results (table 2 and appendix p 3).", "Analyses with quintiles of the fatty acid distributions led to similar conclusions in terms of direction and significance of associations (appendix p 5).", "For example, results of model 2 showed that the HR (95% CI) for a comparison of quintile 5 versus quintile 1 of even-chain SFAs (SFA group 1) was 3·66 (2·75– 4·87); for odd-chain SFAs (SFA group 2) 0·37 (0·32–0·42); and for longer-chain SFAs (SFA group 3) 0·47 (0·35–0·63); in all cases the p value for the trend was <0·0001.", "Country-specific associations were similar overall (figure 2).", "Although we recorded evidence of heterogeneity (I2=88·1%; p<0·0001), meta-regression analyses showed that age, BMI, and sex did not explain this effect (data not shown).", "Among estimated markers of stearoyl-CoA desaturase-1 activity, the ratio of 16:1(n-7) to 16:0 was significantly positively associated with type 2 diabetes, but the ratio of 18:1(n-9) to 18:0 was not (table 2; appendix pp 3–5).", "All three groups of SFAs were modestly or weakly correlated with foods, but with overall distinct patterns across fatty acid groups (figure 3).", "Even-chain SFAs were positively associated with alcohol, soft drinks, margarine, and potatoes, and negatively associated with fruit and vegetables, and both olive oil and vegetable oil (figure 3A).", "By contrast, odd-chain SFAs generally showed positive associations with dairy products, cakes and cookies, nuts and seeds, and fruit and vegetables, but negative associations with red and processed meat, soft drinks, alcohol, and margarine (figure 3B).", "The overall correlations for longer-chain SFAs were similar to, but weaker than, those for odd-chain SFAs (figure 3C).", "In this large prospective case-cohort study, we investigated the association of nine individual SFAs with the risk of type 2 diabetes, and recorded distinct patterns of association.", "Even-chain SFAs (14:0, 16:0, and 18:0) were positively associated with the incidence of type 2 diabetes, whereas odd-chain SFAs (15:0 and 17:0) and longer-chain SFAs (20:0, 22:0, 23:0, and 24:0) were inversely associated with type 2 diabetes.", "Our findings of the differential associations of individual circulating SFAs with the risk of type 2 diabetes are in line with the recent debate about the adverse effects of SFAs.2,27", "Our results indicate that different SFAs have differential associations with metabolic risk.", "Therefore, to classify all SFAs as having adverse health effects, as has conventionally been done, does not acknowledge their potentially heterogeneous associations.", "The existing evidence around this topic is inconclusive and is based on only a few studies with small sample size, in which the number of participants with type 2 diabetes ranged from 34 in a Finnish study21 to 673 in a German study.15", "Null associations have been recorded for the even-chain SFAs 14:0, 16:0, and 18:0,17–21 although positive associations have also been reported for 16:0 and 18:0.15,16,19", "Some studies reported an inverse association between type 2 diabetes risk and odd-chain SFAs 15:017,18 and 17:0,16,17 whereas one showed an inverse association only for the sum of 15:0 and 17:0,15 and one a non-significant association with 15:0.20", "Only one study15 reported a significant association with longer-chain SFAs, but showed a positive association for 24:0 (unlike the inverse association recorded in our study).", "Our findings with 12 132 cases of type 2 diabetes therefore provide the strongest evidence so far for differential associations between nine individual SFAs and type 2 diabetes (panel).", "Our pattern of findings for the correlations of SFA with food intakes are consistent with previous evidence from feeding intervention studies showing that even-chain SFAs (14:0, 16:0, and 18:0) are derived from de-novo lipogenesis, through which carbohydrates and alcohol are converted to fatty acids in the liver or adipose tissue.6,9,11,12", "Our finding of a positive association of type 2 diabetes with the product-to-precursor ratio of 16:1(n-7) to 16:0 but not with the 18:1(n-9) to 18:0 ratio (as markers of de-novo lipogenesis) is also consistent with previous observations in smaller samples.15,17", "However, studies with isotope-labelled dietary fatty acids show that some incorporation of these SFAs into phospholipids occurs,13 and evidence also indicates that de-novo lipogenesis is reduced when dietary starch is substituted for sugar, which suggests the importance of carbohydrate type.11", "The extent to which de-novo lipogenesis might also contribute to the circulating levels of these SFAs could be quite low under conditions of relatively high-fat habitual diets in free-living Western populations.14", "Because existing knowledge is insufficient, future research is needed to quantify the effects of habitual diets and metabolic state on blood concentrations of SFAs.", "Importantly, measurements of even-chain SFAs in the blood should not be used to directly interpret dietary SFAs.", "Given that de-novo lipogenesis is the underlying process for increased levels of even-chain SFAs, this pathway might increase the risk of type 2 diabetes through hepatic steatosis and related mechanisms.28", "Direct effects of even-chain SFAs could also promote the development of type 2 diabetes: biochemical studies indicate toxic effects specifically of 16:0, including activation of inflammatory cytokines and lipotoxicity to pancreatic β cells.29", "Epidemiological research cannot address biological mechanisms and should therefore be complemented by experimental studies.", "Our finding of positive correlations of dairy products with odd-chain SFAs (15:0 and 17:0) is consistent with previous evidence that these SFAs are markers of exogenous origin dairy fat intake.6–8", "Although residual confounding by other dairy components such as vitamin D or calcium cannot be ruled out, and nor can the possible effects of processes related to fermentation of dairy products,4,5,30 our strong findings for odd-chain SFAs in the blood lend support to accumulating evidence of an inverse association between dairy products and type 2 diabetes risk.4,5", "Our report of an inverse association between the very-long-chain SFAs and type 2 diabetes provides the largest appraisal so far of an as-yet under-researched group of SFAs that might undergo distinct fatty acid metabolism through peroxisomal fatty acid oxidation.25", "To our knowledge, the sources and metabolic effects of very-long-chain SFAs are largely unknown, and our new findings should provide the impetus for further research into this group of fatty acids.", "The main limitation of our study was that SFAs were measured at one timepoint only, and intra-individual variation over time is likely.", "However, we would not expect any errors to be differential with respect to case status.", "We could only assess relative—not absolute—concentrations of fatty acids (in mol%), but this valid approach is often used in epidemiological research and tends to provide a better interpretation of metabolic inter-relationships than do absolute measurements.6", "Fatty acids were measured in long-term stored samples, but stability is likely given that samples were stored at −196°C.6", "Plasma phospholipid SFAs might be indicative of both dietary and metabolic influences, affected by interplay of complex exogenous and genetic factors, which we were unable to tease out.", "This raises caution against inferring dietary consumption on the basis of measurements of SFAs that are not exclusively exogenously derived.", "Our indirect estimation of fatty acid desaturation ratios has limited use as a biomarker of stearoyl-CoA desaturase-1 activity,26 but direct measures involving liver biopsies or tracer techniques are not feasible in epidemiological studies.", "Our outcome ascertainment was complete for the entire cohort and therefore does not have the problem of ascertainment bias consequent on a requirement for attendance at a follow-up visit, but was limited by a reliance on clinically incident diabetes.", "However, we minimised false positives by applying rigorous verification criteria to ensure that no case was included unless verified by at least two independent sources.", "Although false negatives were also possible because of undiagnosed incident diabetes, such misclassification can be assumed to be non-differential with regard to the exposure and therefore any potential bias would be unlikely to change our conclusions based on a relative scale (HR) in our analysis.", "We also minimised the potential for reverse causation bias from undiagnosed prevalent diabetes by applying three separate sensitivity analyses to test the robustness of our results: adjustment for baseline HbA1C, exclusion of those with raised HbA1C at baseline, and exclusion of those with diabetes diagnosed within 2 years of baseline.", "The strengths of our study include the large sample size, prospective study design and hence the ability to study the temporality of association with type 2 diabetes, long follow-up, the inclusion of populations from eight European countries with diverse dietary intakes, comprehensive type 2 diabetes case ascertainment and verification, adjustment for many potential confounders, and a series of sensitivity analyses.", "We were able to analyse the associations of type 2 diabetes with a large number of objectively measured SFAs.", "In conclusion, our findings indicate that different individual plasma phospholipid SFAs are differentially associated with risk of type 2 diabetes, which supports the importance of recognising that individual blood SFAs exert heterogeneous effects.", "Further research into an increased understanding of dietary versus endogenous metabolic sources of circulating individual SFAs will help to inform updated public health messages about the dietary intake of saturated fats and their sources." ]	[]
Long-term trend of and correlation between vegetation greenness and climate variables in Asia based on satellite data	Satellite data has been used to ascertain trends and correlations between climate change and vegetation greenness in Asia. Our study utilized 33-year (1982–2014) AVHRR-GIMMS (Advanced Very High Resolution Radiometer–Global Inventory Modelling and Mapping Studies) NDVI3g and CRU TS (Climatic Research Unit Time Series) climate variable (temperature, rainfall, and potential evapotranspiration) time series. First, we estimated the overall trends for vegetation greenness and climate variables and analyzed trends during summer (April–October), winter (November–March), and the entire year. Second, we carried out correlation and regression analyses to detect correlations between vegetation greenness and climate variables. Our study revealed an increasing trend (0.05–0.28) in temperature in northeastern India (bordering Bhutan), Southeast Bhutan, Yunnan Province of China, Northern Myanmar, Central Cambodia, northern Laos, southern Vietnam, eastern Iran, southern Afghanistan, and southern Pakistan. However, a decreasing trend in temperature (0.00 to −0.04) was noted for specific areas in southern Asia including Central Myanmar and northwestern Thailand and the Guangxi, Southern Gansu, and Shandong provinces of China. The results also indicated an increasing trend for evapotranspiration and air temperature accompanied by a decreasing trend for vegetation greenness and rainfall. Increases in both the mean annual signal and annual cycle occurred in the forest, herbaceous, and cropland areas of India, Northwest China, and eastern Kazakhstan. The temperature was found to be the main driver of the changing vegetation greenness in Kazakhstan, northern Mongolia, Northeast and Central China, North Korea, South Korea, and northern Japan, showing an indirect relationship (R = 0.84–0.96). • Temperature is the main climatic variable affecting vegetation greenness.• A downward trend in vegetation greenness was observed during summer (April–October).• Temperature showed an upward trend across many areas of Asia during the study period.• In winter, rainfall showed downward and upward trends in different parts of Asia.	[ "BIOC", "HEAL" ]	[ "Correlation", "Evaporation", "Precipitation", "Statistical analysis", "Temperature", "Temporal trend analysis", "Trend", "Vegetation greenness" ]	[ "1308", "3607" ]	[ "The NDVI dataset is the newest version of the Global Inventory Modeling and Mapping Studies (GIMMS) NDVI3g.", "It is derived from the US National Oceanic and Atmospheric Administration’s (NOAA) Advanced Very High Resolution Radiometer (AVHRR) satellite record.", "It is the long-term global monthly time series of greenness index, being more than twice as long as those from newer sensors, such as the US National Aeronautics and Space Administration’s (NASA) Moderate Resolution Imaging Spectroradiometer (MODIS, February 2000 until present).", "The NDVI3g data were collected over a 33-year period, ranging from 1982 to 2014, with a spatial resolution of 8 km, calculated using the average monthly NDVI from January to December.", "The northern hemisphere latitudes (30° N–90° N) were analyzed in this study.", "Monthly average daily maximum temperature (°C), rainfall (mm per month), and potential evapotranspiration (mm per day) data were provided by the Climatic Research Unit Time Series (CRU TS).", "The current version of CRU TS3.2 was released in 2012.", "The dataset has a resolution of 0.5° × 0.5°, based on the analysis of over 4000 individual weather station records.", "A major portion of the input records have been homogenized.", "However, the dataset itself is not strictly homogeneous.", "We used the monthly climate data for the same period as the NDVI time series (1982–2014), including the average monthly temperature, rainfall, and evapotranspiration for all the seasons from January to December, totaling 1536 data points from 384 months.", "We changed the cell size to 0.083, which is the same as NDVI3g data (8 km), with 1319 columns and 598 rows covering all the climate data using Arc GIS.", "The monotonic trend (Mann–Kendall) option provides a nonlinear trend indicator that measures the degree to which a trend is consistently increasing or decreasing.", "It ranges from −1 to +1.", "A value of +1 demonstrates a trend that continuously increases and never decreases.", "The contrary is true when the value is −1 and 0 value is demonstrated no consistent trend.", "The trend of the time series data can be computed using a nonparametric technique that was introduced by Theil and developed by Sen. The Theil–Sen (TS) slope estimator is the median of the slopes computed for values observed at all pairwise time steps for a total of n(n−1)/2 slopes.", "This method has many advantages.", "The TS technique is robust against outliers and has the ability to reject anomalies without affecting the slope.", "The number of anomalies that this approach can reject without being affected (known as the breakdown bound) equals approximately 29% of the sample size.", "Temporal trends of the datasets were checked by applying linear regression and were used to model the series of monthly NDVI composites as a dependent variable versus the independent variables of potential evapotranspiration, rainfall, and temperature.", "The analyses were broken into periods covering 1982–2014, as determined by the temporal coverage of the climate and NDVI datasets.", "The outputs of the trend analyses include maps of relationship coefficients (R-values) and slopes, indicating the strengths and magnitudes of the computed trends.", "The relationship coefficient, R, is a measure of the strength of a linear relationship between two variables.", "The coefficient of definition, R2, is the proportion of the variation of a response variable that is explained by a fitted statistical model; R2 is mostly demonstrated as a percentage.", "The relationship is computed from the strength of the linear correlation between NDVI and temperature, rainfall, and potential evapotranspiration by computing the per-pixel relationship coefficient (R2) from 33-years of overlay monthly observations (January 1982–December 2014).", "The linear association between the NDVI and climatic variables and vegetation growth was determined using single correlation (R) and regression (R2) analyses.", "An annual time step (starting in January 1982) was used to examine the effects on the climatic driver and vegetation growth reported that local differences in the NDVI and rainfall relationship due to dominating soil and vegetation types and their individual rain use efficiencies were considered by calculating respective intercepts and slopes for each pixel.", "During the regression analysis, a causal correlation between the dependent NDVI values and independent temperature, rainfall, and potential evapotranspiration variables is calculated; the predicted high NDVI values for each month and each pixel are calculated based on the observed rainfall, temperature, rainfall, and potential evapotranspiration.", "The monotonic trend (Mann-Kendall) option provides a nonlinear trend indicator that measures the degree to which a trend is consistently moving upwards or downwards.", "It ranges from −1 to +1.", "A value of +1 indicates a trend that is continuously moving upward, with no downward movement.", "The contrary is true when the value is −1, while 0 indicates no consistent trend.", "The trend of the time series data can be computed using a nonparametric technique that was introduced by and developed by.", "The Theil-Sen (TS) slope estimator is the median of the slopes computed for values observed at all pairwise time steps for a total of n (n − 1)/2 slopes.", "This method has many advantages.", "The TS technique is robust against outliers, with the ability to reject anomalies without affecting the slope.", "The number of anomalies that this approach can reject without being affected (known as the breakdown bound) equals approximately 29% of the sample size (Fig. 1).", "The linear association of the NDVI and climatic variables with vegetation growth was determined using single correlation (R) and coefficient of determination (R2) of regression analyses.", "The relationship coefficient, R, is a measure of the strength of a linear relationship between two variables.", "The coefficient of determination, R2, is the proportion of the variation of a response variable that is explained by a fitted statistical model.", "R2 is mostly demonstrated as a percentage.", "The relationship is computed based on the strength of the linear correlation of NDVI with temperature, rainfall, and potential evapotranspiration, by computing the per-pixel relationship coefficient (R2) from 33 years of overlay monthly observations (January 1982–December 2014).", "For identifying the local differences more clearly, residual maps with the dependent variable of NDVI and independent variables of temperature, rainfall, and potential evapotranspiration were created (Fig. 2)." ]	[]
Configuration and evaluation of an integrated demand management process using a space-filling design and Kriging metamodeling	Objective: This research aims to develop a basic understanding of a demand management process integrating sales and operations planning (S&OP) and order promising in a Make-To-Stock environment and to compare different demand management policies with limited capacity. Contribution: Typical researches about demand management processes analyze few system specifications or vary few potential factors one at a time. Yet, additional insights can be obtained by employing a space-filling design and Kriging metamodeling for analysis. Methodology: We compare two configurations of the integrated demand management process. While the First-Come First-Served concept is used at the order promising level for the first configuration, the second configuration uses nested booking limits and gives advantage to profitable customers and attractive periods. Considering various order arrival sequences, we generate Kriging metamodels that best describe the nonlinear relationships between four environmental factors (demand intensity, demand forecast error, customer heterogeneity and coefficient of variation) and three performance measures (yearly profit margin, yearly sales and high-priority fill rate) for Canadian softwood lumber firms. Since our simulation experiments are time-consuming, we employ a Latin hypercube design to efficiently take into account different market situations. Results: Our analysis reveals the potential to improve the performance of the demand management process if we know high-priority customers needs before fulfilling low-priority orders and if we use nested booking limits concept.	[ "BUSI", "DECI", "MATH" ]	[ "Integrated demand management process", "Kriging", "Revenue management", "Sales and operations planning", "Softwood lumber industry", "Space-filling design" ]	[ "1408", "1803", "2606", "2613" ]	[ "The Canadian softwood lumber industry is struggling to cope with certain challenges.", "The industry difficulties are mainly due to the increased cost of woody supply and reduced demand during the last decade [1], coupled with the increased low-cost competition from emerging countries in Asia and Latin America [2].", "Moreover, sawmills’ profitability can be severely affected by fluctuations in the Canada–U.S. exchange rates and the numerous softwood lumber disputes between Canada and the United States [1].", "Canadian softwood lumber companies have employed cost-cutting strategies to maintain competitiveness and profit margins [2].", "However, they must be able to remain profitable in situations where markets experience disturbances.", "This requires a deepened understanding of the market side of the supply chain to take advantage of sales opportunities [3], and an improvement of existing processes by using real-time monitoring systems as well as integrated planning systems [4].", "This research is motivated by the need for Canadian softwood lumber firms operating in a supply-constrained environment and facing heterogeneous and seasonal market, to improve their demand management process and to anticipate how this process will perform in different situations.", "The dominant thinking currently in the Canadian lumber industry is to produce maximal volume from the available resource, which is constrained by raw material availability and complexity of divergent production processes.", "Although sawmills operate at full capacity most of the time, they do not take advantage of seasonal fluctuations of prices and of the willingness of some customers to pay more for better products and better services.", "To this end, an integrated demand management process (IDMP) has been proposed by Ben Ali et al. [5].", "They integrated sales and operations planning (S&OP) and order promising models, particularly those based on revenue management (RM) concepts.", "The integration between RM and S&OP is not well understood either in theory or in practice, particularly for Canadian softwood lumber firms.", "It is unclear how an IDMP, that can be configured differently as presented in [5], can perform facing various order arrival sequences and market disturbances.", "In fact, Canadian softwood lumber managers are confronted with different challenges such as a change of demand intensity, a rise of demand variability, poor accuracy of demand forecasts and increasingly heterogeneous customers.", "The simulation of the IDMP proposed by Ben Ali et al. [5] offers the possibility to experiment several demand management approaches and to measure the effect of these environmental factors on the IDMP performance.", "Searching for effects by varying factors one at a time is an ineffective means to estimate the factor effects [6–8] since it imposes restrictions on the number of factors and the number of values that these factors can take with a limited simulation budget, and so fails to consider nonlinear relationships.", "Using space-filling designs, and then Kriging metamodeling, is advantageous as an efficient tool with time-consuming simulation experiments to estimate factor effects on the IDMP performance in different situations.", "Our paper aims i) to develop a basic understanding of the IDMP proposed by Ben Ali et al. [5] facing various order arrival sequences and taking various market disturbances into account and ii) to compare different demand management policies.", "For these purposes, we have to identify: which factors are expected to have the most significant impacts on the IDMP?", "And how can they affect the performance (improvement or deterioration and in which situations)?", "Most multi-level decision processes and integrated decision-support systems in manufacturing context are too complex to be evaluated analytically and so have to be studied by means of simulation before implementation.", "This paper addresses the need to evaluate the ability of a multi-level decision process to face the different factors that could affect its performance.", "One of the main contributions of this paper is the novel procedure to experiment and to analyze the behavior of an integrated demand management process (IDMP) under a variety of scenarios: we employ a space-filling design and Kriging metamodeling to scan the effects of some relevant market factors on the IDMP performance.", "To the best of our knowledge, our study is among the few papers which use space-filling design and Kriging in a realistic supply chain setting, particularly to analyze factor effects and to compare different demand management approaches/practices.", "In addition, as motivated by an industrial problem, the paper discusses the potential implications of this analysis for firms operating in supply-constrained environments, such as Canadian softwood firms.", "The remainder of this paper is organized as follows.", "Section 2 presents the related literature.", "In Section 3, we describe the industrial context.", "Section 4 exposes the performance measures, the factors considered in the experimentation and the experimental design.", "While Section 5 explains the different steps for data generation and analysis, Section 6 presents the analysis results and discusses managerial implications.", "Finally, concluding remarks and further research opportunities are provided in Section 7.", "S&OP is a tactical process which supports cross-functional integration [9] and links company strategy and operational planning [10,11].", "In fact, it is important to create a specific leadership style and a culture in the organization to ensure integrated demand management and supply chain planning.", "This required the involvement of all functions in each stage through a continuous mechanism.", "However, the survey of Wagner et al. [12] shows that organizations’ current S&OP performance is underdeveloped and many improvements are indispensable to concretize the alignment process.", "The lack of participants’ commitment and information reliability, the absence of cross-functional integration and a siloed culture are the main barriers that jeopardize S&OP success [13].", "Although there are diverse researches available concerning S&OP implementation [13], the role of S&OP as a powerful tool for reaching business targets is mostly absent from the current literature [11].", "Moreover, systematic revues of Thom et al. [14] and Tuomikangas and Kaipia [11] show that there is still a need for more in-depth case studies with multiple perspectives to provide a deeper understanding and guidelines for companies to manage the S&OP implementation challenges.", "In this context, this paper aims to provide a better understanding of the link between the S&OP and the order promising function, particularly when the organization strategy focuses on customer heterogeneity.", "While S&OP makes mid-term decisions, order promising is a real-time problem which has impacts not only on company profitability and customer service level in the short, medium and long term, but also has significant influence on scheduling and execution of manufacturing and logistics activities [15].", "When all demand cannot be fulfilled, introducing RM in order promising activity can be considered as a powerful tool ensuring higher profitability and forging a stronger relationship with customers less sensitive to price [16]: Order promising concerns how to manage capacity allocation, aggregately set by tactical planning, to different customers and introducing RM in order promising activity consists in protecting capacity reserved for each customer segment by defining booking limits [17].", "Regarding application of RM concepts in manufacturing context, two research streams can be distinguished.", "Within the first stream, the focus is on the implantation of RM in Make-To-Stock (MTS) context [18–20].", "A second stream has evolved from more advanced work on Assemble-To-Order environment [21–23] and Make-To-Order environment [24,25].", "The relevance of integrating order promising with tactical planning tasks was exhibited in a built-to-order context by Volling and Spengler [26], who explicitly model order promising and master production scheduling as distinct and interdependent planning functions.", "Ben Ali et al. [5] have taken a further step forward by considering complex transformation processes with heterogeneous raw materials and divergent product structure, mid-term market seasonality and customer differentiation.", "Unlike existing studies which dealt separately with S&OP and RM in complex manufacturing situations (See Appendix A and Appendix B), Ben Ali et al. [5] proposed an IDMP including S&OP at the tactical level and real-time order promising based on RM concepts at the operational/execution level (see Fig. 1).", "This IDMP supports sales decisions in a way to maximize profits and to enhance the service level offered to high-priority customers: First, considering demand and prices forecasts, sales commitments made in previous periods and current inventories, S&OP is executed monthly over medium-term horizon to predetermine supply, production, transport and sales plans, taking into account demand and prices seasonality.", "Second, real-time sales decisions have to be taken for each received order based on RM concepts, which offers the possibility of prioritizing orders from customers less sensitive to price and for more profitable periods and to select the most profitable sourcing location.", "Our paper proposes going further by examining how such integrated process will perform using different demand management policies and facing different market situations.", "Among all researches dealing with S&OP and RM in manufacturing context (See Appendix A and Appendix B), analysis by running only a single system specification or by varying some potential factors one at a time were performed.", "Nonetheless, these tests can lead to different conclusions if we make some changes in the factor settings.", "Using a conventional Design Of Experiments (DOE), additional insights can be gleaned with the same simulation budget.", "Factorial designs (full or fractional) are the most popular DOEs used in supply chain settings (See Appendix C), but the disadvantage of these designs is that the number of scenarios grows exponentially when the number of factors or the number of factor levels increases.", "Taguchi’s [27] designs are also widely common to identify robust decision factor settings.", "These designs are limited to main effects, which is usually too restrictive for simulation environments [6].", "Employing finer grids (more than two or three levels) for some factors is important to view nonlinear relationships.", "Space-filling designs, including Latin Hypercube Designs (LHD), make the samples more uniformly spread in the experimental region [28].", "They can be employed for continuous factors or discrete factors with a potentially large number of levels [29].", "These designs are more interesting for time-consuming experiments like ours.", "On one hand, they are efficient and flexible for analysis.", "On the other hand, they use an attractive sampling technique to provide data with few restrictions on factors and to cover large design spaces [6].", "Metamodeling is usually employed to analyze time-consuming simulation experiments.", "The objective is to represent the Input/Output (I/O) function implied by the underlying simulation model, and so predict outputs for new factor combinations, other than those simulated.", "In particular, Kriging (also called Gaussian process modeling) is typically used to develop global metamodels [29]: “Kriging models are fitted to data that are obtained for larger experimental areas than the areas used in low-order polynomial regression metamodels” [30].", "Kriging has traditionally been used for deterministic computer models.", "However, during recent years, the application of Kriging to outputs from stochastic simulation models, as is our case, has been explored by Kleijnen [30].", "Simulation analysts often use LHD to generate the I/O simulation data to which they fit a Kriging metamodel [30].", "Confronting various trade and economic pressures, Canadian softwood lumber companies try hard to remain profitable and to maintain positive profit margins [31].", "In this context, our case study, illustrated by Fig. 2, is inspired from softwood lumber manufacturers located in Eastern Canada.", "In this region, lumber manufacturers principally offer their products to different markets such as the Canadian market, the Northeastern American market, etc.", "A large portfolio of products is offered to heterogeneous customers, having different attitudes and priorities.", "Home improvement warehouse companies and housing component manufacturers, for example, are willing to pay more for shorter lead times and personalized services.", "Other customers, such as dealers and distributors, are more sensitive to price.", "In this study, we deal with ten lumber commodity products.", "Demand for such products greatly exceeds supply offered by the company, as is usually the case for softwood lumber companies in Eastern Canada.", "In addition, prices are expected to move higher going into some periods of the year.", "Most of these seasonal fluctuations in softwood lumber prices can be explained by demand seasonality related to construction activities.", "Sawmills can be considered as a MTS environment as its activities are driven by forecasts.", "Unlike traditional manufacturing (i.e. assembly) which has a convergent product structure, sawmills have complex transformation processes (i.e. sawing, drawing, planning) with heterogeneous raw materials (great diversity in terms of wood quality, diameters, length, etc.), divergent product flows (generating many products at the same time) and radically different planning problems to be solved by each mill.", "Although sawmills operate most of the time at full capacity, products are not always available in stock at the right time to take advantage of price fluctuation for many reasons.", "First, there is little flexibility in raw material availability, depending on regulations of forestry activities and on the seasonal nature of harvesting operations, which limits the variation in the lumber sawing process.", "Second, production operations are complex since divergent processes induce the production of multiple products simultaneously.", "The studied network, illustrated by Fig. 2, is composed from three sawmills with the same capacity and dispersed over Quebec province.", "Sawmills can be supplied from two sources and sell to various markets (customers from different geographical regions and so with different transport costs) composed of differentiated segments (customers classified according to their willingness to pay).", "Whatever the market conditions, the dominant thinking of the Canadian lumber manufacturers is to produce the maximum volume from the available resource.", "Production is oriented towards large batches resulting in large inventories, low flexibility and low agility.", "Ben Ali et al. [5] have shown the potential profit that can be obtained by taking into account demand/price seasonality and by rejecting orders, not only if not enough resources are available, but in anticipation of more valuable ones from profitable customers and for more attractive periods.", "Based on multiple meetings with softwood lumber managers from the Eastern Canadian region, we identified that they have two principal preoccupations: To maximize the profit margin and the sales and to sell scarce products to the right customer (i.e. high-priority customers) at the right time.", "Therefore, in what follows, we will consider profit margin, sales and high-priority fill rate as performance measures.", "In this study, we follow the procedure recommended by Montgomery [7] for designing and analyzing experiments (see Fig. 3).", "We have already recognized the problem and identified the objectives of the experiments.", "Next, we have to define the performance measures which reflect the system/process performance.", "Then, we have to set the list of factors and the categories that they can take or the ranges over which these factors will be varied.", "Depending on the objectives of the experiments and the number and the nature of factors, we have to choose the type of experimental design.", "Based on sales managers objectives in softwood lumber industry, we choose to analyze results regarding three performance measures (see Fig. 4):", "The yearly profit margin (YPM) is calculated as the total selling price minus production, transportation and inventory costs.", "This output is measured over a year to take into account the benefits of tactical planning considering cyclical rises of demand/price.", "The yearly sales (YS) represent the total volume sold and delivered over a year.", "The HP fill rate (HPFR) measures the proportion of demand received from high-priority (HP) customers that has been fulfilled.", "While the two first indicators are oriented to evaluate global performance, the last one concerns the service level offered to HP customers.", "We have clustered the factors examined in this study into categorical decision factors and continuous environmental factors.", "In what follows, combinations of values for environmental factors are called environmental scenarios.", "Table 1 and Fig. 4 expose the factors and their associated categories or ranges.", "In this study, we assume that customer orders are treated individually and that the decision of accepting or refusing an order has to be instantaneous and definitive.", "However, order assignment to sourcing locations is temporary and may be changed.", "Partial fulfillment is not allowed, but an order can be fulfilled from different sourcing locations.", "Although the expected periodical demand is approximately known based on forecasts, the exact ordering quantity varies randomly.", "In this context, two categorical decision factors affecting the system performances are identified based on Ben Ali et al.’s [5] study:", "Order promising approach (A): reflects how orders have to be fulfilled.", "Quantities to sell for each customer segment at each period of the year are already set by the S&OP at the tactical level (see Fig. 1).", "Then, for each received order, real-time sales decisions have to be taken.", "For this purpose, different order promising approaches can be considered.", "First, we consider a First-Come First-Served approach (FCFS), which simply decides if we accept or refuse each order, based only on resources availability.", "FCFS approach will be compared to a second approach (NBL) based on RM concepts and using Nested Booking Limits.", "This approach can be applied in a manufacturing setting in order to take advantage of customer heterogeneity and profitability variation over time.", "According to Talluri and Van Ryzin [32], setting booking limits is a way to control the availability of capacity.", "NBL approach can support managers in a supply-constrained environment, such as in the softwood lumber case, to decide which orders should be rejected in anticipation of more valuable orders, not only if not enough resources are available.", "Further on, with nesting, capacities overlap in a hierarchical manner depending on the expected profit margin, so that capacities initially designated to a specific couple (customer segment, period) can be sold to other couples generating better profits.", "Order arrival sequence (S): reflects how orders arrive at order promising level.", "In this study, we consider three arrival sequences: a random sequence (RAND) where orders from different segments are randomly received, an ascendant sequence (ASC) where orders are received in an ascending order of priority i.e. low-priority orders arrive first, and finally a descendant sequence (DESC) where orders are received in a descending order of priority i.e. high-priority orders arrive first.", "S can be considered as a decision factor since, in our industrial context, sales managers can stimulate HP customers to express their needs before dealing with low-priority orders.", "Fig. 5 illustrates the experimental design.", "We consider the 6 combinations of the categorical decision factors.", "Each combination is simulated for m different environmental scenarios (i.e. combinations of the continuous environmental factors) generated using a Latin Hypercube Design (LHD).", "The Supplementary Material S1 visually schematizes the different steps for data generation and analysis.", "So, for each environmental scenario i and replication r, 3 final lists are obtained, sorted respectively by order of priority and by reception date.", "Common random numbers are used, so for each replication r, the same seed is used to generate data for the m different environmental scenarios.", "Our results are analyzed regarding three performance measures (i.e. outputs), as presented in Section 4.1: the yearly profit margin (YPM), the yearly sales (YS) and the HP fill rate (HPFR).", "As mentioned in Section 3, we consider ten lumber commodity products (we have a divergent product structure, so it is not possible to produce the different products independently).", "In what follows, we present performance measures for all products together since we are interested in the overall process performance of the company.", "In the following section, we analyze the impact of the decision factors and then the impact of the environmental factors using response surfaces, prediction profilers and the analysis of variance (ANOVA).", "Fig. 6 exhibits the performance measures of the two approaches A for different order arrival sequences S and the 95% confidence intervals on estimates over various environmental scenarios: Points in Fig. 6 represent average outputs (see Section 5.3) for the different (I, E, H,V) combinations.", "The yearly profit margin (YPM) and the yearly sales (YS) are respectively expressed in millions of Canadian dollars (million$) and in million board-feet measure (MMFBM).", "Less variation is seen for YS (the confidence intervals overlap in Fig. 6(b)), clearly due to the limited capacity of sawmills compared to the total demand.", "However, YPM and the HP fill rate (HPFR) are considerably sensitive to the approach A. Gaps between FCFS and NBL approaches are statistically significant regarding YPM and HPFR since the confidence intervals do not overlap when we pass from blue side to red side in Fig. 6(a) and (c) respectively.", "The gap is more pronounced if the HP orders arrived after low-priority orders (ASC sequence).", "Regarding the order arrival sequence S, gaps between the three sequences in Fig. 6(a) and (c) are statistically significant only for the FCFS approach.", "This means that, if we use FCFS, it is more interesting to receive HP orders early since we do not anticipate the arrival of HP orders, in contrast to NBL.", "Further, Fig. 6(a) and (c) exhibit that YPM and HPFR behaviors for the three sequences are too close to be significantly different if we use NBL: We can say that with this approach, no order arrival sequence is preferable.", "Response surfaces, prediction profiles and ANOVA tables are drawn by “Gaussian process platform” of JMP software, based on Kriging metamodels of the different performance measures for each decision factor combination (A, S).", "Response surfaces: We start our analysis by visualizing response surfaces to have an overview about the general trends of all performance measures throughout factor ranges.", "Fig. 7 shows examples of response surfaces for the effects of demand intensity (I) and demand forecast error (E) on the yearly profit margin (YPM) for FCFS and NBL approaches with H = 10%, V = 0.5 and random arrival sequence.", "As an example, Figs. 8 and 9 present, respectively for NBL approach and FCFS approach, the variation of sales and inventories over a year considering different demand forecast errors (E = 0% and E = 20%) with random arrival sequence, I = 1.5, H = 10% and V = 0.5.", "When E passes from 0 to 20%, the number of accepted orders in remunerative periods passes from 2537 to 2701 with NBL approach and from 2433 to 2572 with FCFS approach (equivalent to an increase of sales in remunerative periods by 8% for NBL approach and 6% for FCFS approach).", "The IDMP using FCFS approach anticipates remunerative periods and sets limits for sales only at the S&OP level.", "However, the IDMP using NBL additionally reserves quantities for HP orders since it sets limits for sales at the real-time level too.", "This explains the difference between NBL and FCFS curves respectively in Figs. 8 and 9.", "Prediction profilers: Prediction profilers offer the possibility to see how our prediction models change as we change settings of individual factors and to find optimal settings for your factors regarding all our performance measures at the same time.", "These two-dimensional multivariate profilers are interesting in order to interact with responses, which is another benefit of using multi-factor Kriging metamodels.", "Fig. 10 shows examples of the JMP profiler tool, which present the response for each performance measure as it relates to each factor, i.e. how the predicted response changes as one factor is changed while the others are held constant at the current values of factors.", "Current values of factors (I = 1.5, E = 0%, H = 10% and V = 0.5) and current predicted values of responses are presented in red respectively in the x-axis and the y-axis.", "The first thing to note is that the HP fill rate (HPFR) varies considerably when we change the order promising approach A (when we pass from left to right side in Fig. 10).", "Regarding the environmental factors, HPFR is almost only sensitive to the coefficient of variation V (see 3rd row of each profiler).", "Indeed, HPFR declines when V goes over 0.6 (the bottom right corner of each profiler).", "Even so, we can generalize that, no matter the environmental conditions, NBL has to be chosen if our objective is to improve HP customers’ satisfaction.", "Regarding the yearly profit margin (YPM) and the yearly sales (YS), the prediction profilers in Fig. 10 confirm the important effect of the decision factors (order promising approach A and order arrival sequence S), especially for YPM.", "They also lead us to believe that the most pertinent environmental factors affecting the YPM are the demand intensity I and the demand forecast error E (the four upper left squares of each profiler), and that YPM and YS increase if I and/or E increases.", "The yearly sales (YS) are however stabilizing when E goes over +5% due to the limited production capacity.", "We can see also that, for the ascendant sequence ASC and with NBL approach, the yearly profit margin (YPM) declines when I goes over 1.7, due to the rise in quantities stocked and reserved for HP customers (arriving last in sequence ASC).", "Finally, we note that the customer heterogeneity H significantly affects the YPM and YS if we use FCFS approach (the two upper squares in the 3rd column of each FCFS profiler).", "Analysis of variance (ANOVA): Kriging metamodels may also be analyzed through ANOVA [30], which allows us to quantify/measure the effects already shown by response surfaces and prediction profilers as trends, so we can identify the most pertinent factors in different situations: the objective for this analysis is to examine the contributions of environmental factors and interactions for each decision factor combination (A,S).", "We can assume normal distributions, and so use ANOVA, only for the yearly profit margin (YPM) and the HP fill rate (HPFR).5", "Considering a significant contribution if it exceeds 10%, ANOVA tables presented in Supplementary Material S3 give evidence that:", "Our study suggests implications for both supply chain management researchers and practitioners.", "For supply chain management researchers, our paper provides an evaluation of the value of integrating two common concepts in the demand management research, namely S&OP and revenue management (RM).", "In addition, we employ a space-filling design and Kriging metamodeling, which is a relatively new procedure for realistic supply chain management experiments.", "For practitioners, this paper provides a tool to evaluate the performance of an integrated demand management process (IDMP) in different industrial settings.", "The methodology proposed can support a sales manager to decide which configuration will be appropriate depending on his specific context and to identify the actions to be conducted in order to improve the performance of the demand management process.", "Our analysis demonstrates that in a supply-constrained environment such as the Canadian softwood lumber industry, managers can achieve better performances by integrating S&OP and RM: The IDMP makes an implicit trade-off between the objectives of the production team (dealing with divergent production challenges) and the incentives of sales team (fostering better relationships with profitable customers).", "Facing the potential market disturbances, production/sales managers need to be supported by a tactical plan such as S&OP in order to capture the possible revenue increase, rather than producing in a push mode.", "Our study demonstrates that sawmills should take advantage of any rise of demand intensity by allocating more for remunerative periods, which is possible within tactical planning and medium-term forecasting.", "In fact, forecasts are critical inputs to S&OP: Demand and price forecasting plays a determining role in the overall planning activities of a firm, especially in the forest industry [35] since forest product prices and demand are well known for their fluctuations.", "Moreover, our analysis has asserted that the performance of the IDMP is less affected by the forecasting error if we use an order promising approach considering nested booking limits (NBL).", "This study shows that NBL order promising approach is efficient to capture orders from profitable customers and for more remunerative periods, and so immunize the demand management process against different environmental disturbances.", "However, the use of NBL requires a deep understanding of the market.", "In fact, customer segmentation is needed to group the various types of customers and their behaviors and requirements, according to different criteria such as the willingness to pay, loyalty, etc.", "In this study, we assume that some customers are ready to pay more to have shorter transport lead-times.", "Potentially, other value-added services can be considered, like the stability of product quality and partnership agreements (see Lehoux et al.’s [36] study in the pulp and paper industry).", "Customer Relationship Management initiatives can be used to identify customer segments and to reach the customers who are most receptive to the products and services offered.", "Considering current practices and existing IT-systems, managers can face challenges to implement RM and S&OP.", "Our results are illuminating interesting managerial practices that can be easily introduced before RM and S&OP implementation.", "We demonstrate that the order arrival sequence should be taken into consideration: Even if orders are fulfilled on a FCFS basis, sales managers in softwood lumber industry should start by stimulating high-priority (HP) customers to express their needs before dealing with low-priority customers to improve the performance of the company.", "This paper aims to contribute to the research in demand management for MTS manufacturing systems and to analyze a process integrating S&OP and order promising, considering differentiated demand segments, divergent product structure and facing various market disturbances.", "For these purposes, we use relatively novel techniques–a space-filling design and Kriging metamodeling–in supply chain settings.", "We are also among the first who address the impact of decision and environmental factors on performances of an integrated demand management process.", "Our simulation results affirm that NBL approach can be a powerful tool to maximize revenues facing different environmental conditions.", "We also show how order arrival sequence can play a relevant role, especially with high customer heterogeneity.", "Therefore, sales managers in softwood lumber industry should, first of all, intensify their efforts to know, as early as possible, the needs of HP customers and to improve the performance of Customer Relationship Management, which might be simpler than implanting a new demand management platform.", "Then, they should focus on customer heterogeneity by using an integrated demand management process able to anticipate orders from profitable customers and for more remunerative periods.", "It is important to note that the validation of experiments was done only for a specific industrial case study.", "For generalizing, we provide the tool and the methodology needed to perform other simulation experiments with different settings and in other industry sectors, especially those dealing with stochastic behaviors in terms of supply, demand and manufacturing operations and divergent production processes.", "Future research efforts concerning the integrated demand management process validation may provide some new insights.", "First, in other contexts, it could be interesting to include other decision and environmental factors.", "Second, since in practice, prices offered for upcoming periods are uncertain at the order promising level, a scenario-based stochastic programming model could be considered at the tactical level.", "Third, other order promising options such as partial fulfillment and substitution could be investigated.", "This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC)." ]	[]
Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds	Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3–8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.	[ "BIOC", "CENG", "ENGI", "MATE" ]	[ "Epithelial cells", "Tissue engineering", "Tissue scaffolds", "Trachea", "Transplantation" ]	[ "1304", "1502", "2211", "2502", "2503" ]	[ "Tracheal tissue engineering has been at the forefront of efforts to generate bioengineered organs because of the lack of therapeutic options available for patients with severe tracheal disease [1–5].", "Both decellularized and synthetic scaffolds have been transplanted in compassionate-use cases [6–10] and subsequent work has turned to improving the functionality of these transplants.", "Techniques to improve the quality of cells for transplantation, either through adult stem cell isolation and expansion [11] or induced pluripotent stem (iPS) cell technology [12–16], are being investigated but the scaffolds onto which cells are seeded also need to be refined.", "There is currently little consensus on the optimal scaffold for tracheal bioengineering purposes with decellularized [6,8], synthetic [17] and hybrid stent-based [18] scaffolds at various stages of development.", "Clearly, a scaffold that closely mimics normal airway physiology in terms of structure and function is highly desirable [19].", "For this reason, decellularization of donor organs is an attractive approach in tracheal bioengineering because the full complexity of human tracheal architecture, extracellular matrix (ECM) and regional structural variation are not yet mimicked by synthetic scaffolds [20].", "Ideally, these properties should be retained whilst simultaneously removing all donor cells from the trachea to avoid complications involving immune reaction and rejection after transplantation [21,22].", "Achieving this delicate balance between cell removal and ECM ultrastructure disruption causes decellularization protocols to take longer than may be ideal for the clinical needs of some tracheal stenosis patients.", "Clinically applied protocols have required many weeks for decellularization [8,10,23], a timeframe that rules out the use of a decellularized scaffold in more urgent clinical scenarios such as tracheal agenesis or atresia where the entire trachea is absent and full replacement is required soon after birth [24,25].", "Methods to accelerate the decellularization process are being developed [26] but as yet no direct comparison of the scaffolds produced by different methods has been performed using human tracheae, particularly in terms of their in vivo responses [27].", "We developed an accelerated vacuum-assisted decellularization (VAD) protocol, which reduces the time required for decellularization from several weeks [8,10,28] to just nine days (Fig. 1).", "We hypothesized that VAD would effectively decellularize human tracheal scaffolds and that the resulting scaffolds would retain similar biomechanical properties and key extracellular matrix components to those obtained with the clinically applied detergent-enzymatic method (DEM).", "Further, we hypothesized that both VAD and DEM scaffolds would support the engraftment of primary human airway epithelial cells in vitro and would vascularize to similar extents following in vivo implantation.", "Human tracheae were obtained from cadaveric donors (30–80 years age) identified through the National Health Service Blood and Transplant (NHSBT) tissue retrieval team, by agreement and with appropriate consent, following the current clinical practice for non-living tissue retrieval in the U.K. Ethical approval was obtained through the National Research Ethics Committee (REC reference 11/LO/1522).", "Tracheae were retrieved less than 48 h post mortem and were removed in their entirety from cricoid to carina.", "Donor tracheae were immediately rinsed in 1 L 0.9% normal saline and surrounding tissue was dissected away.", "The tracheae were immersed in 20% chlorhexidine solution for 5 min followed by a further three rinses in 0.9% saline.", "Intact tracheae were divided into two equal halves and these matched donor pairs were processed for either the clinically applied standard protocol [23] of repeated cycles of detergent and enzyme (detergent-enzymatic method; DEM) or frozen and then decellularized using the vacuum-assisted decellularization (VAD) protocol [26] such that decellularization using both procotols finished simultaneously.", "A matched donor design was chosen due to the difficulties in obtaining large numbers of human donor organs for research purposes and to minimize the effect of inter-individual variability on results.", "Tracheae were subjected to 25 cycles of distilled water for 72 h at 4 °C, 4% sodium deoxycholate (Sigma) for 4 h and 2000 kU DNase I in 1 M sodium chloride (Sigma) for 3 h. Tracheae were continuously rotated on a roller throughout the decellularization process and were stored at 4 °C in PBS until use.", "Tracheae to be processed by VAD were aspirated dry and frozen at −80 °C.", "Tissue was thawed to room temperature for 24 h and rinsed in PBS.", "Decellularization was performed in an autoclaved stainless steel Ricordi chamber.", "Luer locks with 3-way ports and a pressure gauge were attached to the chamber such that a vacuum (1 Torr) could be generated.", "All decellularization steps were performed under vacuum on a tissue agitator at 140 rpm at 37 °C unless otherwise indicated.", "All solutions were supplemented with 1% penicillin/streptomycin (Sigma).", "Tracheae were incubated in detergent solution containing 0.25% sodium deoxycholate (Sigma) and 0.25% Triton X-100 (Sigma) in HBBS for 24 h at 37 °C.", "Tracheae were then rinsed twice in sterile water for 2 h and incubated for a further 44 h in sterile water at 4 °C on a tissue agitator at 20 rpm.", "Following the wash step, tracheae were incubated in DNase I (2 kU/ml) and RNase (4 U/ml) at 37 °C for 24 h, followed by another 24 h wash step in sterile water at 4 °C as described above.", "The DNase/RNase step and wash was repeated once prior to a final wash step of sterile water for 48 h at 4 °C as described above.", "Tracheae were then stored at 4 °C in PBS until use.", "Samples were fixed in 4% paraformaldehyde (PFA) overnight prior to standard processing for paraffin embedding.", "Serial paraffin sections were cut at 5 μm thickness.", "Haematoxylin and eosin, Periodic acid-Schiff, picrosirius red and Masson’s trichrome staining were performed on sections using an automated staining system (Tissue-Tek).", "For immunofluorescence staining, slides were dewaxed using an automated protocol and antigen retrieval was performed using citrate buffer (pH 6.0).", "Primary antibodies against cytokeratin 5 (CK5; Abcam; ab17130), collagen IV (COL-94; Abcam; ab6311) and laminin (Sigma; L9393) were used.", "Species-appropriate secondary antibodies conjugated to AlexaFluor dyes (Molecular Probes) were then used.", "Images were acquired using a Zeiss LSM700 confocal microscope.", "For immunohistochemistry, slides were dewaxed and antigen retrieved as for immunofluorescence.", "Primary antibodies against CD45 (Abcam; ab10558), endomucin (V.7C7.1; Abcam; ab106100) and F4/80 (Serotec; MCA497GA) were used.", "Sections were then incubated with species-appropriate biotinylated F(ab’)2 for 30 min, developed using ABC reagent and superDAB (Dako) and finally counterstained with haematoxylin.", "For whole-mount imaging, cell-seeded scaffolds were fixed in 4% PFA for 30 min and blocked for 1 h in blocking buffer (10% fetal bovine serum in PBS containing 0.01% Triton X-100).", "CK5 primary antibody (Abcam; ab17130) incubation was overnight at 4 °C in blocking buffer without Triton X-100.", "Scaffolds were washed three times in PBS for 5 min and species-appropriate secondary antibody (AlexaFluor dyes; Molecular Probes) was added for 2 h at room temperature.", "Scaffolds were washed three times in PBS and DAPI (1:10,000 in PBS) was applied to counterstain scaffolds before imaging.", "Scaffolds were oriented luminal-side down on a low-profile chamber-well slide (Ibidi; #80826) for image acquisition using a LSM700 confocal microscope (Zeiss).", "Isolation of DNA from tracheal samples was performed using a PureLink DNA extraction kit (Invitrogen) according to the manufacturer’s instructions.", "Briefly, samples were digested overnight in proteinase K and DNA was isolated using the supplied buffers and mini-columns.", "Quantification of eluted DNA was carried out by spectrophotometry using a NanoDrop 8000 (Thermo Scientific).", "All samples had a 260/280 ratio of between 1.8 and 2.0.", "Collagen, elastin and glycosoaminglycans were quantified as previously described [29].", "Collagen content was determined using a total collagen assay kit (QuickZyme Biosciences) according to the manufacturer’s instructions.", "Representative whole tracheal samples of native and decellularized trachea were weighed and directly hydrolyzed in 6 M HCl for 20 h at 95 °C.", "Samples were allowed to cool to room temperature and centrifuged to remove debris.", "To detect hydroxyproline residues, supernatants were mixed with a chromogen solution for 1 h at 60 °C.", "Absorbance for each sample was read using a colorimetric reader (555 nm).", "Standards were generated by serial dilution of rat tail collagen I (BD Biosciences) from 300 μg/ml.", "Elastin quantification was performed using an elastin assay kit (FASTIN, Biocolor).", "Representative whole tracheal samples of both native and decellularized tissue were homogenized and solubilized twice with 0.25 M oxalic acid incubated at 95 °C.", "Extracts were combined with FASTIN dye reagent (5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonate (TPPS)) and the absorbance was determined at 555 nm.", "Concentrations were determined using α-laminin standards.", "The sulphated glycosaminoglycan (sGAG) content was quantified according to the Blyscan GAG Assay Kit (Biocolor).", "Representative wet tissue samples were incubated in 1 ml of papain digestion buffer at 65 °C for 18 h. Aliquots of each sample were mixed with 1,9-dimethyl-methylene blue dye and the reagents from the GAG assay kit.", "The absorbance was read at 656 nm and quantified according to standards generated from bovine tracheal chondroitin-4-sulphate.", "Scanning electron microscopy (SEM) images show the luminal surface of decellularized human donor tracheal scaffolds.", "Scaffolds were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, washed with 0.1 M phosphate buffer (pH 7.4), post fixed with 1% osmium tetraoxide and 1.5% potassium ferrocyanine in 0.1 M phosphate buffer and rinsed with distilled water.", "Specimens were then dehydrated to 100% ethanol using a graded ethanol-water series and critical-point dried using carbon dioxide.", "Samples were mounted onto aluminum stubs with sticky carbon tabs.", "A Gatan ion beam coater was used to coat scaffolds with a layer of Au/Pd (approx. 2 nm thickness).", "Images were obtained using a 7401 FEG scanning electron microscope (Jeol, USA).", "All decellularized tracheal samples were equilibrated to room temperature prior to testing and kept hydrated in PBS for mechanical testing.", "Each trachea was cut into 3 cm sections and compressed with antero-posterior and lateral compressive forces.", "The load (N) exerted by the scaffold was calculated when the scaffold was compressed to 50% of the starting diameter.", "Further tracheal samples were cut into dumbbell sections with a template cutter such that vertical (axial) forces across tracheal rings and trachealis could be applied.", "A further dumbbell section was horizontally cut across a tracheal ring.", "The load required to pull apart the sample until breaking point was measured using an Instron 5565 tensile tester (Instron Ltd., UK).", "The tensile stress versus strain properties were assessed at a displacement rate of 50–100 mm/min.", "The Young’s modulus is a measure of material stiffness and was calculated using Bluehill software (Instron, U.K.) as the slope of the straight line portion between 0 and 5 mm.", "Primary human bronchial epithelial cells (HBECs) were obtained either by enzymatic digestion from donor human trachea or from explant biopsy outgrowth [30].", "Primary HBECs were maintained for up to three passages in bronchial epithelial growth medium (BEGM; Lonza, UK) as previously described [31].", "Decellularized human tracheal scaffolds were cut into 5 mm discs using a biopsy punch, such that they fitted into 96-well plate wells.", "Prior to epithelial seeding, scaffolds were oriented with luminal/mucosal-surface side up in a low-adhesion 96-well plate (Nunc) and incubated overnight in 200 μl of BEGM medium.", "Epithelial cells were seeded at 0.25 × 106 cells/cm2, 0.5 × 106 cells/cm2 or 1.0 × 106 cells/cm2 and allowed to adhere overnight.", "Scaffolds were transferred to new low-adhesion plates with the same orientation maintained.", "Subsequently, medium was changed every 48 h.", "Immortalized human bronchial epithelial cells (iHBECs; a gift from Dr. Gisli Jenkins, University of Nottingham, U.K.) were transduced with mStrawberry-Luc lentivirus to create a stable iHBEC cell line co-expressing mStrawberry fluorescent protein and luciferase.", "Lentivirus production was as previously described [32] but created with the pLV-LS1 transfer vector and the packaging and envelope vectors pCMV-dR8.74 and pMD.G2 (Plasmid Factory).", "Briefly, viral supernatants were created by co-transfecting 293T HEK cells with the above plasmids using JetPEI (Polyplus Transfection).", "Supernatant containing lentivirus was collected at 48 and 72 h and was concentrated by ultracentrifugation.", "Each collection underwent ultracentrifugation to concentrate virus.", "To generate a transduced cell line, iHBECs were grown in LHC9 medium (Gibco), seeded at 1 × 105 cells in a T25 flask and transduced at an MOI of 7.5 with 4 μg/ml polybrene (Sigma).", "After 8 h, medium was changed and cells were passaged when approximately 80% confluent.", "To obtain a pure luciferase-expressing population, mStrawberry fluorescent protein-expressing cells were sorted by fluorescence-activated cell sorting (FACS) using a BD Aria flow cytometer.", "IVIS imaging (Caliper Life Sciences) was used as a non-invasive in vitro method of tracking luciferase-tagged iHBECs on decellularized human tracheal scaffolds by detecting a luminescence signal from cells in the presence of the luciferase substrate luciferin.", "Scaffolds were fashioned into 5 mm discs and placed into 96-well plate wells with the luminal surface facing upwards.", "Scaffolds were incubated in LHC9 medium (Gibco) overnight at 37 °C and 5% CO2.", "Cells were seeded on the scaffolds and transferred to a new low adhesion plate prior to imaging.", "D-luciferin was added to the medium at 150 μ-g/ml and scaffolds were imaged within the plates at position ‘C’ using automatic acquisition times.", "Fertilized chicken eggs (Henry Stewart and Co., UK) were incubated at 37 °C.", "After 3 days of incubation, an approximately 3 cm diameter window was cut into the shell using dissecting scissors, revealing the embryo and CAM vasculature.", "Windows were sealed with tape and eggs were returned to the incubator for 5 days.", "At day 8 of incubation, decellularized human tracheal scaffolds (approximately 2–3 mm3) and pieces of absorbable gelatin sponge (Gelfoam; Pfizer; a negative control) were placed on the CAM between blood vessels.", "Scaffolds were checked daily for 6 days, when scaffolds were photographed in ovo with a stereomicroscope (Leica).", "Vessel ingrowth was quantified blindly as previously described [33].", "Human tracheal scaffolds were prepared as described above and cut into full thickness 5–7 mm2 pieces, irradiated with 10,000 Gy to ensure sterility and stored in PBS prior to implantation.", "Adult CD1 mice were used for subcutaneous implantation of scaffolds.", "Live animal work was ethically approved and carried out under Home Office Project Licence PPL 70/7504.", "Briefly, CD1 mice were anaesthetized with a 2–5% isoflorane:oxygen gas mix for induction and maintenance.", "Under aseptic conditions, the dorsum of the mouse was shaved and chlorhexidine was applied to the skin.", "5 mm transverse incisions over each dorsal flank were made and subcutaneous pockets created.", "One scaffold was inserted in each pocket and these were closed with buried 4/0 Vicryl interrupted sutures.", "Scaffolds were implanted for 56 days and the experiments were terminated accordingly.", "Scaffolds were retrieved en bloc for histopathological analysis.", "Results are presented as mean ± standard deviation.", "Data were analyzed in Microsoft Excel and GraphPad Prism 6.0.", "Statistical tests are indicated in figure legends and significance was accepted at the 5% level.", "To assess the efficacy of vacuum-assisted decellularization (VAD) we compared this protocol with the clinically applied detergent-enzymatic method (DEM).", "Histologically, VAD removed nuclei from the epithelium, the submucosal glands and the tracheal cartilage but cartilage lacunae remained intact in both scaffolds (Fig. 2A).", "Periodic acid-Schiff (PAS) staining demonstrated the widespread retention of proteoglycans and glycoproteins in cartilage and also in submucosal glands, which may reflect the protection of the glandular basement membrane during decellularization (Fig. 2A).", "Picrosirius red (PSR) staining demonstrated the widespread retention of collagen I and III fibers within tracheal scaffolds decellularized using both DEM and VAD, findings supported by Masson’s trichrome (MT) staining (Fig. 2A).", "Reflecting the extensive decellularization by both protocols, the DNA content of scaffolds was significantly reduced in comparison to native trachea (Fig. 2B).", "While there was a trend towards lower DNA content in VAD tracheae as compared to DEM tracheae, consistent with results following the application of vacuum pressure to porcine tracheal decellularization [26], this did not reach statistical significance.", "However, the abundance of structural components of scaffolds such as elastin, collagen and sulphated glycosaminoglycans (sGAGs) was not significantly affected by either DEM or VAD decellularization (Fig. 2B).", "Scanning electron microscopy (SEM) of decellularized scaffolds confirmed the absence of cells in both DEM and VAD scaffolds and showed the folded macroarchitecture characteristic of human trachea.", "At higher magnification, a meshwork of fibers was visible on the luminal surface of both scaffolds (Fig. 2C).", "However, the basement membrane proteins laminin and collagen IV were not found at the luminal surface using immunofluorescence but were retained deeper within scaffolds (Fig. 2D).", "An important aspect of tracheal bioengineering is the structural integrity of scaffolds post-transplantation.", "To assess whether VAD compromises scaffold mechanical properties, we compared these scaffolds with DEM-decellularized and native tracheae.", "We found that decellularization did not significantly affect a scaffold’s ability to resist compression in either the anterior-posterior (Fig. 3A) or lateral (Fig. 3B) axes.", "Further, the elastic properties of the cartilage rings (Fig. 3C), the tracheal wall (Fig. 3D) and the trachealis (Fig. 3E) were not significantly affected by VAD or DEM decellularization.", "For VAD-decellularized tracheae to be of use clinically, the scaffolds must retain the capacity to support cells in addition to retaining their mechanical properties.", "As airway epithelial cells represent a key functional aspect of bioengineered tracheae [34,35], we examined the adhesion of primary human bronchial epithelial cells (HBECs) to the luminal surface of DEM- and VAD-prepared scaffolds in vitro.", "By immunofluorescence staining 72 h post-seeding, we observed that HBECs adhered well to both scaffolds but that high seeding densities improved the retention of cells in both types of scaffold (Fig. 4A).", "Scanning electron microscopy (SEM) of scaffolds seeded at 1 × 106 cells per cm2 also demonstrated that cells had adhered to both grafts and revealed possible morphological differences between DEM- and VAD-prepared scaffolds with cells more rounded on DEM scaffolds and flatter on VAD scaffolds, a possible indication of more mature cellular adhesions to scaffolds in the VAD condition (Fig. 4B).", "In order to monitor cells in real time on decellularized scaffolds, we monitored the bioluminescence of luciferase-transduced immortalized human bronchial epithelial cells (iHBECs) using an in vivo imaging system (IVIS).", "Non-invasive monitoring of cells on the decellularized scaffolds using this system demonstrated a stable signal over the 5-day period.", "VAD scaffolds had higher signals throughout this experiment, apparently caused by higher initial engraftment of cells on VAD scaffolds compared to DEM scaffolds (Fig. 4C and D).", "However, we cannot rule out that differences between the decellularization protocols, such as enzyme concentration, affected these results as scaffolds were stored identically after decellularization and optimization of this step may be necessary for optimal cell seeding.", "For cells seeded ex vivo to survive following transplantation, there is a clear need for rapid integration and angiogenesis in implanted decellularized scaffolds.", "To assess the potential of DEM- and VAD-prepared scaffolds for vascular integration, we performed chick chorioallantoic membrane (CAM) assays [29,36] using fragments of either DEM- or VAD-decellularized human tracheae.", "Gelfoam® acted as a negative control.", "Macroscopically, we observed a spoke-wheel pattern of vessels four days after implantation, which suggested that vessels were attracted to both DEM- and VAD-prepared scaffolds (Fig. 5A).", "Quantification of the number of vessels converging on the decellularized scaffolds revealed that decellularized scaffolds stimulated significantly more vessel ingrowth than negative controls (Fig. 5B) and that the accelerated VAD protocol did not negatively influence vessel ingrowth (Fig. 5A).", "Finally, we compared the behavior of DEM- and VAD-prepared human tracheal scaffolds in a xenotransplantation assay.", "3 mm3 pieces of either DEM- or VAD-prepared tracheae were implanted subcutaneously into immunocompetent CD1 mice and retrieved after 8 weeks.", "Throughout the experiment the mice behaved normally without evidence of inflammation, extrusion of the implanted tissue or death.", "At the time of retrieval, tissue surrounding the implants appeared healthy, without signs of adverse reaction.", "Histological analyses revealed that DEM- and VAD-prepared tracheal scaffolds showed similar cellular infiltration around the graft (Fig. 6A) and that this was predominantly macrophages (Fig. 6B).", "Evidence of vascularization was also seen in both scaffold types as demonstrated by endomucin-stained vessels within the grafts (Fig. 6C and D).", "We show that a vacuum-assisted decellularization (VAD) protocol produces human tracheal scaffolds with comparable decellularization efficiency, extracellular matrix retention, mechanical properties, cytocompatibility and in vivo integration capacity to scaffolds decellularized using the clinically applied detergent-enzymatic method (DEM).", "However, VAD is an accelerated protocol requiring only 9 days from donor tissue procurement to final decellularized scaffold; by contrast, the DEM method takes 3–8 weeks [8,10,23].", "The efficacy of decellularization reported here is in line with previously published accepted standards for tissue decellularization [37].", "These results are an important step towards the use of decellularized human cadaveric donor tracheae as scaffolds for airway bioengineering because expedited preparation broadens the clinical scenarios in which decellularization is appropriate.", "The incorporation of a freezing step prior to decellularization is of particular importance as it suggests banking of donor tracheae as a clinically relevant strategy.", "An important consideration is that current protocols for storage of pre-decellularized tracheae mean their salient properties might deteriorate over time in storage [38] and the optimization of storage conditions for decellularized materials is a priority for the field.", "Production of decellularized scaffolds that share similar biomechanical properties to native tissue is an important aim as the tracheal cartilage maintains a patent airway in healthy individuals.", "Previous studies suggest that decellularization using some protocols could negatively affect the structural properties of porcine tracheae [39].", "Here, we comprehensively tested the biomechanical properties of VAD-decellularized human tracheae including lateral and antero-posterior compression finding that the resistance to compression and elastic properties remain similar to that of native human tracheae.", "It is noteworthy that donor tracheae have quite large variance between samples and this may contribute to non-significant differences found despite controlling for this with matched samples.", "In the future, particularly for clinical application, donor selection criteria may have to be more stringent than they have been for research purposes.", "Scaffold preparation represents the first step in the process of generating a transplantable tissue-engineered airway.", "Clinically applied protocols have recellularized scaffolds with autologous cells, including epithelial cells, mesenchymal stromal cells (MSCs) and MSC-derived chondrocytes [6,8–10].", "As such, it is important that, as well as maintaining the expected mechanical properties, scaffolds are able to support the adherence and survival of relevant cell types.", "Here, we demonstrated that human bronchial epithelial cells (HBECs) attach to and survive on both decellularized human scaffold types in vitro.", "However, neither the DEM or VAD decellularization method preserved the basement membrane that is normally present beneath airway epithelial cells; instead, a mesh of fibers ordinarily constrained to the stromal compartment was presented at the luminal surface [40].", "This may explain the high seeding densities of HBECs required for cell retention as many cells may undergo apoptosis before basement membrane production and/or remodeling occurs.", "We believe that future studies should investigate the importance of the basement membrane in airway bioengineering: basement membrane retention or reconstruction could improve the cytocompatibility of both decellularized and synthetic scaffolds, which presently require huge numbers of HBECs to re-epithelialize [11].", "Authors treating severe skin burns with cultured epidermal stem cells found that the application of these cells on a fibrin sheet greatly improved the ‘take rate’ and ease of application [41].", "Similar to other tissue- engineered epithelia, it may also be possible to transplant airway epithelium as a pre-prepared sheet product [42,43] or as organoids [44].", "Optimization of protocols for functional airway differentiation, to generate a ciliated epithelium on VAD human scaffolds, as has been demonstrated in rodent tracheal scaffolds [45], is also desirable.", "Pre-seeding and integration of stromal cells into VAD scaffolds could also be considered [46].", "The mechanism, time-course and quality of vascularization of tissue-engineered constructs is presently unknown and requires further research, as highlighted at a recent international consensus meeting [47].", "We believe that our data demonstrating comparable behavior of DEM and VAD scaffolds in terms of their ability to promote a rapid angiogenic response in CAM experiments and comparable vascular integration as subcutaneous implants provide reassurance that reducing the time taken to prepare scaffolds using a vacuum system does not have detrimental effects on scaffold performance.", "In the trachea, translation of these protocols into clinical practice will require the development of equipment capable of delivering vacuum pressure to GMP standards.", "While the reagents used in the protocol reported here have previously been adapted towards GMP manufacture, the development of standard operating procedures and quality control measures for VAD will be an important step towards this.", "Further, the concept of VAD might be useful in clinical protocols to produce decellularized scaffolds from other organs but protocols will likely require customization to each setting.", "This comparison between clinically used DEM decellularization and accelerated vacuum-assisted decellularization (VAD) of human tracheae shows that VAD can rapidly generate decellularized tracheal scaffolds that are comparable to those previously used for clinical transplantation in a number of key aspects.", "Importantly, this innovative procedure allows for banking of tracheae prior to decellularization for future applications.", "The similar properties of DEM and VAD scaffolds both ex vivo and in vivo provide a rationale for the use of VAD in clinical applications of tissue-engineered airway scaffolds." ]	[]
Low effective population sizes in Amblyomma variegatum, the tropical bont tick	Effective population sizes have rarely been estimated in ticks despite the importance of this parameter for evaluating the evolutionary and adaptive potential of tick populations. The present study was aimed at evaluating the effective population sizes of Amblyomma variegatum, the tropical bont tick, in three villages in Burkina Faso. For this purpose, microsatellites markers were developed. Eight out of 19 assessed markers provided good amplification results with 4 to 24 alleles recorded per marker on 216 genotyped ticks. The within-samples polymorphism was congruent with Hardy-Weinberg expectations at four markers while sex linkage and/or null alleles were observed at the others. As sampling involved two tick generations, effective population sizes were independently estimated by two methods insensitive to heterozygosity: the first one is based on linkage disequilibrium analysis within a single cohort while the second uses the changes in allele frequencies across generations. Both methods estimated the number of reproducing ticks ranging from two to a few tens reproductive adults per village and cohort. Such small estimates are congruent with the rarity of records of acaricide resistance in A. variegatum.	[ "AGRI", "IMMU", "MEDI" ]	[ "Amblyomma variegatum", "Effective population size", "Microsatellite markers" ]	[ "1109", "2404", "2405", "2725" ]	[ "While the past decades have seen the multiplication of successful applications of phylogenetic and population genetics analyses to many ticks for tracking their historical changes in distribution, identifying cryptic species as well as their mating patterns, evaluating their dispersal ranges and/or understanding how biotic and/or abiotic parameters jointly define their population genetic structure (see for review Araya-Anchetta et al., 2015), evaluation of effective population sizes (Ne) in ticks had so far been restricted to the Rhipicephalus microplus complex (Koffi et al., 2006a; De Meeûs et al., 2010; Biguezoton, 2016).", "This is curious given, on the one hand, the amount of the tick literature related to the efficacy of tick-control programs and/or acaricide resistance, and on the other hand, the key importance of Ne in the evolutionary potential of the studied populations.", "Indeed, the acquisition of new alleles (via migration or mutation) is proportional to Ne, the polymorphism lost by a population at each generation via genetic drift is proportional to 1/Ne and thus only the selection pressures s verifying s >>1/Ne overcome the effect of drift to actually impact the frequency of selected alleles (e.g. Hartl and Clark, 1997; Hedrick, 2000).", "The present study was aimed at evaluating the effective population sizes in Burkina Faso of a three-host tick species of major veterinary importance in West Africa: the tropical bont tick, Amblyomma variegatum.", "There, A. variegatum used to be the most abundant tick species found on livestock (Kabore et al., 1998; Farougou et al., 2006, 2007) until the last decade when R. microplus started to invade this area (Madder et al., 2011).", "Today, it remains the second (after R. microplus) most abundant tick on cattle throughout Benin and Burkina Faso (Biguezoton et al., 2016).", "Originated from sub-Saharan areas, A. variegatum has been reported in more than 30 African countries (Walker et al., 2003) and got out of Africa via the cattle trade to reach Indian Ocean islands by the 7th century (Stachurski et al., 2013) and the Caribbean’s in mid-18th century (Maillard and Maillard, 1998).", "The veterinary importance of A. variegatum arises from a combination of factors.", "Firstly, A. variegatum directly impairs animal growth and tend to heavily reduce milk production due to its preference as adults to attach on cattle udder (Stachurski, 2000a).", "Secondly, its saliva favors the development of severe acute dermatophilosis, a bacterial skin disease of animals (Barré et al., 1988).", "Thirdly and finally, A. variegatum vectors Ehrlichia ruminantium, the agent of heartwater (cowdriosis), that induces up to 80% mortality in susceptible sheep and goats (Uilenberg, 1983) and induces morbidity in the cattle breeds imported from free-heartwater areas (Uilenberg and Camus, 1993).", "E. ruminantium infection risks are quite high in West Africa: 10–16% of A. variegatum adult and nymphal stages are infected in Benin (Farougou et al., 2012) as well as 39–61% cattle and at least 51% sheep and goats in Ghana (Farougou et al., 2013; Koney et al., 2004).", "The only commercial vaccine available to protect against heartwater is based on the infectious challenge using a virulent E. ruminantium strain (Bezuidenhout, 1989) followed by antibiotic treatments.", "Meanwhile, selecting West African cattle breeds for poor attractiveness to A. variegatum was shown to be difficult (Stachurski, 2007).", "As a result, the usage of acaricide footbaths still seems as the most accurate way to protect cattle from all the deleterious effects of A. variegatum (Stachurski and Lancelot, 2006).", "As the durability of such protection would depend on the potential of A. variegatum to evolve acaricide resistance, we estimated the effective population sizes of A. variegatum in three neighbor villages from Burkina Faso.", "To this purpose, we sampled two successive tick generations within each village and characterized the polymorphism of newly developed microsatellite markers per village and tick-generation.", "Amblyomma variegatum ticks were collected in 2007 on herds merging cattle, sheep and goats which foraged communal pastures from either one of three neighbor villages (pairwise distances ∼ 10 km) from Burkina Faso: Bekuy (3.91 °W, 11.60 °N), Sara (3.83 °W, 11.71 °N) and Lamba (4.05 °W, 11.66 °N).", "There, A. variegatum reproduces once a year with adults attaching on hosts in the early rainy season (mid-May to July), freshly hatched larvae in late rainy season (September) and nymphs in the early dry season (November) (Stachurski, 2006).", "Three sampling campaigns were performed in May, July and November with the purpose to genotype ∼25 ticks per village and campaign.", "In May and July, the only available ticks were adults feeding on cattle.", "The next tick generation was collected as nymphs on sheep and goats in November.", "As nymphs cannot be sexed, they were allowed to molt under laboratory controlled conditions, so that sexing and genotyping were performed on adult stages.", "Microsatellite loci were isolated from two microsatellite enriched libraries according to Billotte et al. (1999).", "Total genomic DNA was extracted from a mix of adult males and females A. variegatum with the Qiamp DNA mini kit (Qiagen) following manufacturer’s instructions.", "DNA was restricted by HaeIII and fragments were ligated to Rsa21 and Rsa25 self-complementary primers (5′-CTCTTGCTTACGCGTGGACTA-3′ and 5′-TAGTCCACGCGTAAGCAAGAGCACA-3′) and amplified by polymerase chain reaction (PCR).", "Products were hybridized to biotin-labelled I5(GA)8 and I5(GT)8 probes and fragments containing microsatellite sequences were captured using Streptavidin MagneSphere Paramagnetic Particles (Promega).", "Enriched fragments were PCR amplified, cloned in pGEM-T (Promega) and transformed in XL1-Blue competent cells (Stratagene).", "Randomly picked recombinant colonies were PCR amplified with Rsa21 primer.", "PCR products were run on a 1.2% agarose gel and transferred onto a Hybond N + membrane (Amersham) which were hybridized with [γ32P]dATP end-labelled (GA)15 and (GT)15 probes to improve fragment selection.", "Among these clones 55% gave a satisfactory positive signal.", "A total of 192 positive clones were sent for sequencing (Genome Express).", "Sequences were analyzed and primers were designed using the primer 3 program (Rozen and Skaletsky, 2000).", "Nineteen loci were preliminary tested on DNA extracts from individual ticks obtained using the QIAamp DNA Mini Kit (Qiagen).", "PCR amplifications were performed following a M13 protocol where each forward primer is 5′-tagged with the M13-tail sequence (5′- CACGACGTTGTAAAACGAC-3′) and a 5′-dye (IR700 or IR800) labeled M13 is added to the reaction mix.", "PCR amplifications were run in a final volume of 20 μl containing 10-to-50 ng of genomic DNA, PCR buffer (Qiagen), 60 μM dNTPs (Invitrogen), 1.5–3.5 mM MgCl2, 0.2 μM of primer, 0.16 μM of M13 tailed primer, 0.2 μM M13 dye and 0.5 U Taq DNA polymerase (Qiagen).", "PCR amplifications were performed using a “touch down” procedure defined as followed: a denaturing step at 95 °C for 5 min; 10 cycles including a denaturing step at 95 °C for 1 min, a hybridization step at (Ta + 7) °C for 1 min with Ta decreasing by 0.7 °C at each cycle, and an elongation step at 72 °C for 1 min; 25 cycles including a denaturing step at 95 °C for 1 min, a hybridization step at Ta for 1 min, and elongation at 72 °C for 1 min; the final elongation step was operated for 5 min at 72 °C.", "An automated infrared fluorescence technology (4300; LI-COR Biosciences, www.licor.com) was used to detect each PCR product sample.", "The possible occurrence of genotyping errors due to the presence of null alleles, stutter bands and/or large-alleles dropout was assessed with MICROCHECKER 2.2.3 (van Oosterhout et al, 2004).", "Genotypic data were analyzed using GENEPOP 4.2 (Raymond and Rousset, 1995; Rousset, 2008) and FSTAT 2.9.4 (Goudet, 1995).", "Estimates of FIS were computed according to Weir and Cockerham (1984).", "Deviations from Hardy–Weinberg equilibrium (HWE) were tested using FIS statistic and performing 10 000 within-samples permutations of alleles among individuals.", "Assuming null alleles as the primary cause of heterozygote deficits, we estimated their frequency per sample using Brookfield’s (1996) method.", "We then tested, by an exact binomial test, the goodness of fit of the observed frequencies of blank genotypes (putative null homozygotes) to those expected under the hypothesis of panmixia (unilateral tests).", "Genotypic equilibrium among pairs of loci was tested over all samples with the randomization log-likelihood G-based test that is implemented in FSTAT software, and by performing 10 000 within samples permutations of bi-loci genotypes.", "This generates a distribution in P-values that was compared to an exact binomial test undertaken with R 2.15.2 (R Development Core Team, 2018) in order to detect cases where the multiplicity of tests resulted in a false rejection of the null hypothesis tested (de Meeûs et al., 2009).", "We tested whether the genotypic composition of tick samples clustered accordingly to the sampling geography and/or sampling dates using the principal component analyses adapted to genotypic data that is implemented in PCA-GEN v. 1.2 (http://www2.unil.ch/popgen/softwares/pcagen.htm).", "Permutation tests included in the program enabled testing the significance of individual axes inertia (10 000 genotype randomizations).", "Estimation of the effective population sizes (Ne) was performed using the two methods implemented in NEESTIMATOR v1.3 software (Peel et al., 2004), both insensitive to heterozygosity.", "The first is a temporal method based on sequential sampling of different populations (Waples, 1989) while the second is based on linkage disequilibrium (Bartley et al., 1992).", "The temporal method was run considering the tick samples collected in November 2007 and their parental generation sampled either in May or July.", "Complementarily, we also applied Balloux (2004)’s method, a method based on heterozygote excess; it is worthy to note that this last estimate Ne is sensitive to deviations from HWE since it is directly driven from the Weir and Cockerham (1984)’s FIS estimator so that Ne = 1/(-2FIS) + 1/2(1-FIS).", "Eight out of 19 developed markers were retained since they provided reproducible amplification results that were congruent with the expectations under the hypothesis of absence of duplication of the targeted loci.", "They displayed from 4 to 24 alleles each (Table 1) without any effect of large-alleles dropout.", "Possible occurrences of stutter bands were nonetheless detected at AvA02 and AvC08 markers.", "Only one pair (AvCl6; AvE04) out of the 28 possible pairs of markers showed linkage disequilibrium- i.e., non-random association of genotypes significant at 5% risk (P = 0.008).", "This was not an observation significantly different from that expected by chance under the null hypothesis of statistical independence of the genotypic composition across eight markers (Exact binomial test defined by k’ success = 1, k = 28 attempts and mean α = 0.05, unilateral test, P-value = 0.77).", "Therefore, the eight markers can be reasonably assumed to provide independent information on tick population structure.", "No deviation from Hardy Weinberg expectations (HWE) was detected at four markers (AvE04, AvG02, AvA05 and AvC01, Table 2).", "Significant heterozygote deficits were detected at the other four (AvA02, AvB03, AvCl6 and AvC08; P < 0.05, Table 2).", "The AvB03 marker appeared to be X-linked since restricting data to the males resulted in FIS≈1 while the FIS average drawn from females was FIS = 0.65 (range across samples: 0.5 to 1).", "Therefore, in male ticks, the genotype observed at AvB03 marker was coded either as homozygous when computing statistics insensitive to the correlation between alleles within individuals (i.e., effective population sizes) or as missing data otherwise (i.e., F-statistics).", "Assuming the presence of null alleles was enough to explain the observed heterozygote deficits at three markers (AvA02: P = 0.13; AvB03: P = 0.15; AvCl6: P = 0.12) but not at AvC08 (P = 0.004).", "The principle component analysis of multi-locus genotypes supported the existence of two distinct clusters (Fig. 1).", "The first axis captured a significant part (32.7%, P = 0.038) of the total genetic variance; this axis discriminated the samples from the parental generation collected in May and July (on the right in Fig. 1) from those of the offspring generation (collected in November; on the left in Fig. 1).", "The estimates in effective population sizes computed from the examination of two successive generations at the eight microsatellite markers ranged from 11 to 23 reproducing ticks per village and generation (see ‘temporal method’ in Table 3).", "The 95% confidence intervals (95% CI) of such estimates remained bounded by the extreme of six and 127 reproducing ticks per village (respectively observed at Lamba and Sara; Table 3).", "Congruent estimates in effective population sizes were provided by the linkage disequilibrium method.", "For seven of the nine samples, the average estimate in effective population size ranged from 2 to 17 reproducing adults per generation and village and the associated 95% CI remained bounded by finite estimates (2 ≤ minimum ≤ 11 and 4 ≤ maximum ≤ 29 ; Table 3).", "Two samples were different.", "The samples collected either in May at Lamba or in July at Sara provided average estimates in effective population sizes about 70 reproducing ticks per village and generation (without a finite upper bound for the 95% CI; Table 3).", "We tested the sensitivity of such estimates to the set of markers used by re-estimating the effective population sizes with the linkage disequilibrium method after having removed one pair of markers (8 tests performed with the following pair of markers successively removed: AvA02/AvC08, AvB03/AvC08, AvA05/AvG02, AvA02/AvCl6, AvC01/AvE04, AvA02/AvB03, AvC08/AvCl6, AvB03/AvCl6).", "No changes in the magnitude order of the estimates in effective population sizes was ever noticed (Table 3).", "Complementarily, the hypothesis of tick effective populations sizes below 20 reproducing adults per village was also supported by Balloux (2004)’s method: the estimates driven on the parental generation collected in either May or July were Ne = 5.4 and 13.4 in Bekuy, 3.0 and 16.9 in Lamba, and 3.5 and 3.6 in Sara; those driven on the offspring generation were Ne = 3.2, 7.3 and 7.4 in Bekuy, Lamba and Sara, respectively.", "Despite a relative abundance of microsatellite markers in A. variegatum, the main difficulty encountered was to identify loci that could be amplified specifically (i.e. without any suspicion of locus duplication) and unambiguously genotyped.", "Eight polymorphic microsatellite markers were characterized in the tropical bont tick A. variegatum.", "Half of them displayed, within cohorts and villages, polymorphism patterns congruent with Hardy-Weinberg expectations (HWE).", "A fifth marker was sex-linked while three others displayed heterozygote deficits relatively to HWE that looked mainly caused by the presence of null alleles.", "The presence of null alleles at microsatellite markers is common in Ixodidae (De Meeûs et al., 2004; Koffi et al., 2006b; Noel et al., 2012; Van Houtte et al., 2013).", "Relatively to the parental generation, the offspring generation collected displays a reduced genotypic diversity with corresponding samples apparently clustering together in the principle component analysis (PCA).", "Given the short distances between the villages involved in study (∼10 km), it is possible that domestic hosts from different villages shared some grazing areas at a time when tick larvae searched hosts to attach on.", "This would promote A. variegatum dispersal across villages, reducing thus the among-villages differences in tick genotypic compositions.", "As signals of host-associated genetic differentiation were reported in other tick-species (De Meeûs et al., 2010; Kempf et al., 2011; McCoy et al., 2005), one may wonder whether or not this could be involved in the apparent contrast along the PCA first axis between the genotypic composition of A. variegatum nymphs (collected on sheep and goats) and that of adults (collected on cattle).", "This hypothesis can easily be ruled out.", "For a signal of host-associated genetic divergence to emerge and persist, differences in host-preferences among adults and assortative mating with respect to the host-preferences are minimal requirements which are not fulfilled in A. variegatum.", "Indeed, A. variegatum adults usually avoid blood-feeding on sheep and goats.", "This avoidance is even more pronounced around Bobo-Dioulasso, Burkina Faso, where the local sheep race (Djallonké race) can host hundreds of A. variegatum nymphs without being infested by a single A. variegatum adult (Petney et al., 1987).", "Therefore, the most likely explanation to the contrast in genotypic composition defining the PCA first axis relies on the occurrence of a strong impact of genetic drift from parental to offspring generations (i.e., small effective population sizes).", "This conclusion was strongly supported by the congruency in the estimates of effective population sizes for A. variegatum driven by three estimation methods: in all cases, the estimates driven ranged from a few units to a few tens reproducing adults per village and generation.", "The Ne estimates presently driven for A. variegatum in Burkina Faso (2 ≤ Ne ≤ 90 per village) are much lower than those driven for the R. microplus ticks feeding on cattle in West Africa (Ne ∼150 per cattle herd; Biguezoton, 2016) or in the Pacific island of New Caledonia (Ne ∼1000 per cattle herd; Koffi et al., 2006a; De Meeûs et al., 2010).", "Let us investigate how the biology of A. variegatum can explain such a low estimate in effective population sizes.", "Could mating patterns provide an explanation?", "Indeed, one may a priori expect uneven reproduction success among A. variegatum males despite the field observation of a 1:1 sex-ratio among adults (Stachurski, 2000b).", "A. variegatum females do not attach on host until attached males have started to produce attachment pheromone, so that mating clusters could form around the most attractive males (Norval and Rechav, 1979).", "Moreover, as 50% males remain attached for at least 80 days (Barré, 1989), some males could easily mate with many more females than others (Norval et al., 1992).", "However, the absence of deviations from HWE within A. variegatum populations observed at four statistically independent markers does not support the hypothesis of a skewed distribution in male reproductive success.", "Low effective population sizes are not either due to low fertility since A. variegatum females lay up to 20 000 eggs (Barré, 1989; Nuttall, 1915).", "From then, the only explanation for these small effective population sizes has to be sought on weak survival rates somewhere along the A. variegatum life cycle.", "Such a life-cycle starts with mated and fully-engorged A. variegatum females dropping off their host to lay eggs on the ground.", "After egg-hatching, larvae wait for hosts to attach on by clustering in tight clumps at the tips of grass stems, so that many sibs could attach on the same individual-host.", "From then, the heterogeneous drop-off rhythm of ticks at nymphal and adult stages (Stachurski and Adakal, 2010) coupled with hosts movements ensure two successive rounds of sib re-distributions among the individual-hosts grazing on the same pastures i.e., among infrapopulations (Bush et al., 1997).", "According to this cycle, low survival rates of any stage present on vegetation (eggs, larvae, nymphs, adults) look as the most parsimonious explanation that will efficiently result in low effective population sizes.", "In addition, it is noteworthy that extinctions of some A. variegatum infrapopulations would also reduce the effective population sizes within communal herds (Criscione and Blouin, 2005), and so, without promoting deviations to HWE given the two rounds of ticks re-distributions among individual-hosts occurring at both nymphal and adult stages.", "What is the information provided by such low estimates in effective population sizes in A. variegatum regarding the sustainability of tick-control programs in Burkina Faso?", "The first information concerns the probability of apparition by mutation of a new allele conferring resistance to acaricides.", "The lowest the effective population size, the lowest is such a probability since the polymorphism resulting from mutation/drift balance is proportional to Ne.μ with μ the mutation rate (e.g. Hartl and Clark, 1997; Hedrick, 2000).", "The second information relies to the probability that a new allele conferring acaricide resistance may disappear from the tick population before having a chance to be selected for by tick-control programs.", "The lowest the effective population sizes, the highest the risk that the effect of genetic drift overcomes that of the positive selection resulting from acaricides use (Hartl and Clark, 1997; Hedrick, 2000).", "Today, the selection pressures caused by acaricide chemical remain rather low in Burkina Faso.", "Indeed, tick-control programs follow variable strategies (including manual removal and/or application of oil or dung on cattle) in the traditional and low input systems dominating the livestock production there, and, where occurring, chemical-based strategies are mainly achieved through empirical recycling of agricultural pesticides (Adakal et al., 2013).", "The low Ne estimates presently driven for the A. variegatum effective population sizes in Burkina Faso enhances thus the high probability for the genetic drift to actually overcome the effect of acaricide usages onto the fate of a newly arisen mutant conferring acaricide resistance.", "Regarding the evolution of acaricide situation, the situation of A. variegatum in Burkina Faso (with Ne ranging from a few unities to a few tens and s being weak when non-null) dramatically contrasts with that of the southern cattle tick R. microplus in the Pacific island of New Caledonia (with Ne ∼1 000 reproducers per cattle herd and s intense everywhere along the Western Coast; Koffi et al., 2006a).", "In the later case, such a high Ne estimate coupled with low gene flow across cattle herds and homogeneously high selection pressures applied along the West Coast were interpreted as favoring an evolutionary scenario of acaricide resistance due to recurrent events of local positive selection of diverse newly-arisen mutants conferring acaricide resistance rather than an alternative scenario involving the migration among tick populations of a very few resistance mutants (Koffi et al., 2006a).", "The characterization of the diversity of physiological changes involved in acaricide resistance in these populations allowed testing and confirming this prediction (Chevillon et al., 2007, 2013).", "The same rationale would indicate a very low probability of both apparition and selection for acaricide resistance mutants in A. variegatum, at least in West Africa.", "Interestingly enough, the absence of reports of acaricide resistance in the A. variegatum populations from Guadeloupe or Puerto Rico despite intensive eradication campaigns (Garris and Barré, 1991) may provide some support to such a prediction, even if population genetics studies performed in these New World areas are still required to settle this point.", "We characterized eight microsatellite markers from the tropical bont tick A. variegatum.", "These markers allowed highlighting very low effective population sizes of the tick populations from Burkina-Faso; a result suggesting that the evolution of acaricide resistance in A. variegatum is expected to be much slower than observed in its invasive competitor, R. microplus." ]	[]
Evaluation of antibodies for western blot analysis of frataxin protein isoforms	Frataxin is the protein that is down-regulated in Friedreich ataxia (FRDA), an autosomal recessive genetic disease caused by an intronic GAA repeat expansion in intron-1 of the FXN gene. The GAA repeats result in epigenetic silencing of the FXN gene and reduced expression of the cytosolic full-length frataxin (1−210) protein. Full length frataxin translocates to the mitochondria, leading to formation of mature frataxin (81–210) formed by cleavage of the mitochondrial targeting sequence at K-80 of the full-length protein. There are currently no approved treatments for FRDA, although experimental approaches involving up-regulation or replacement of mature frataxin protein through numerous approaches are being tested. Many of the pre-clinical studies of these experimental approaches are conducted in mouse and monkey models as well as in human cell lines. Consequently, well-validated antibodies are required for use in western blot analysis to determine whether levels of various forms of frataxin have been increased. Here we examined the specificity of five commercially available anti-frataxin antibodies and determined whether they detect mature frataxin in mouse heart tissue. Four protein standards of monkey, human, and mouse frataxin as well as mouse heart tissue were examined using polyacrylamide gel electrophoresis (PAGE) in combination with western blot analysis. One antibody failed to detect any of the frataxin standards or endogenous frataxin in mouse heart tissue. Three of the antibodies detected a protein in mouse heart tissue that ran slightly faster on PAGE (at 23.4 kDa) to that predicted for full-length frataxin (23.9 kDa). One antibody detected all four frataxin standards as well as endogenous mouse mature frataxin in mouse tissue. Significantly, this antibody, which will be useful for monitoring mature frataxin levels in monkey, human, and mouse tissues, did not detect a protein in mouse heart tissue at 23.4 kDa. Therefore, antibodies detecting the immunoreactive protein at 23.4 kDa could be misleading when testing for the up-regulation of frataxin in animal models.	[ "IMMU", "MEDI" ]	[ "Frataxin", "Friedreich's ataxia", "Gene therapy", "Mature frataxin", "Mitochondrial processing peptidase", "Mitochondrial protein" ]	[ "2403", "2723" ]	[ "Friedreich's ataxia (FRDA) is an autosomal recessive disease caused primarily by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of full-length frataxin protein (Lynch et al., 2002) and the mitochondrial processing peptidase (MPP)-derived mature frataxin (Condo et al., 2007; Schmucker et al., 2008b).", "A small number of patients are heterozygous for GAA repeats in one allele and an FXN mutation in the other (< 4%); these individuals also have reduced levels of mitochondrial mature frataxin (Campuzano et al., 1996; Cossee et al., 1999; Delatycki et al., 2000; Santos et al., 2010).", "FRDA is estimated to affect 1 in 50,000 in the US population with an average age of death at 37 years, most commonly from cardiac-related pathologies (Pousset et al., 2015).", "The FXN gene encodes a mitochondrial protein frataxin, a highly conserved protein found in both prokaryotes and eukaryotes (Bencze et al., 2006; Pastore and Puccio, 2013).", "There is evidence to suggest that the GAA repeats induce epigenetic changes and heterochromatin formation, thereby impeding gene transcription (Al-Mahdawi et al., 2008).", "For example, studies conducted using blood from FRDA patients and lymphoblastoid cell lines have detected increased DNA methylation of specific CpG sites upstream of the GAA repeat and found histone modifications are present in regions flanking the GAA repeats (Greene et al., 2007).", "Thus, FRDA alleles become associated with histones that are hypoacetylated and show more extensive DNA methylation in the region flanking the repeat.", "It has been suggested that the hypoacetylation and extensive DNA methylation result in the formation of a compact chromatin configuration, with reduced binding of transcription factors so that frataxin transcription initiation and elongation are reduced (Kumari and Usdin, 2012).", "Furthermore, the number of GAA repeats correlate inversely with disease onset and directly with progression of the disease (Friedman et al., 2010; Metz et al., 2013; Patel et al., 2016).", "Although the exact role of frataxin has not been completely delineated, compelling evidence indicates that it is involved in the creation of iron-sulfur clusters.", "It could serve as an iron donor or regulate cysteine-derived persulfide formation during the assembly of mitochondrial iron-sulfur complexes (Lill et al., 2012; Fox et al., 2015; Braymer and Lill, 2017).", "The deficiency in frataxin protein leads to decreased ATP production by the iron-sulfur cluster (Bürk, 2017; Chiang et al., 2018).", "The major FXN mRNA encodes the full-length 210-amino acid form of human frataxin with a molecular weight (MW) of 23,135 Da (Bencze et al., 2006).", "The corresponding cynomolgus monkey frataxin (1–210) and mouse frataxin (1–207) isoforms have MWs of 23,133 Da and 22,924 Da, respectively (Fig. 1).", "Upon being translated, the full-length human frataxin (1–210) is rapidly translocated from the cytosol to the mitochondria where a two-step process yields the mature biologically active form of human frataxin (Gakh et al., 2002a; Rufini et al., 2007; Schmucker et al., 2008a).", "MPP initially cleaves at the R-2 site between G41-L42 to give rise to an intermediate form of human frataxin (42–210), a 169-amino acid protein with a MW of 18,826 Da (Gakh et al., 2002b).", "The corresponding cynomolgus monkey intermediate frataxin (42–210) and mouse intermediate frataxin (41–207) isoforms have MWs of 18,741 Da and 18,574 Da, respectively (Fig. 1).", "The human intermediate frataxin then undergoes a second MPP-mediated cleavage at K80-S81 (a second R-2 site) to yield the mature active form of human frataxin (81–210), a 130-amino acid protein with a MW of 14,268 Da (Fig. 1) (Condo et al., 2007; Schmucker et al., 2008b).", "The corresponding cynomolgus monkey mature frataxin (81–210) and mouse mature frataxin (78–207) isoforms are also 130 amino acid proteins with MWs of 14,210 Da and 14,380 Da, respectively (Fig. 1).", "Additional minor human FXN alternative transcripts been reported (Campuzano et al., 1996; Pianese et al., 2002; Xia et al., 2012; Pérez-Luz et al., 2015), and we recently reported a novel frataxin protein of 135 amino acids (76–210, isoform E, MW 14,953 Da) found in human erythrocytes (Guo et al., 2018a).", "There are currently no approved approaches for treating FRDA or slowing the progression of its symptoms (Strawser et al., 2017).", "Numerous therapeutic approaches are currently being tested, with the majority involving up-regulation or replacement of frataxin protein (Vyas et al., 2012; Wilson, 2012; Evans-Galea et al., 2014; Aranca et al., 2016; Kearney et al., 2016; Lynch et al., 2019).", "As an alternative to classical pharmacologically-based approaches, gene therapy and CRISPR-based methods are continuing to progress rapidly to become a promising potential therapeutic approach to increase frataxin expression (Evans-Galea et al., 2014; Li et al., 2015; Ouellet et al., 2017; Strawser et al., 2017).", "Most of the pre-clinical gene therapy and CRISPR approaches have been conducted in mouse models and/or human cells.", "We anticipate that monkey models will also be used for pre-clinical gene therapy studies before such treatments are tested on human FRDA patients.", "The adeno-associated virus 9 (AAV9) has been reported efficiently delivers virus to a conditional mouse model with severe cardiomyopathy caused by complete frataxin deletion in cardiac and skeletal muscle.", "The injection of AAV9-frataxin reverses the functional features of cardiomyopathy (Perdomini et al., 2014).", "In addition, AAV9-frataxin increased frataxin protein expression and prolonged lifespan compared to typical FRDA mouse models (Gérard et al., 2014).", "To circumvent the possible toxic effect caused by overexpression of frataxin, other approaches such as CRISPR/Casp9 were also tested on mouse models (Li et al., 2015; Ouellet et al., 2017).", "The expression levels of full length frataxin protein in the gene therapy studies are usually monitored by quantifying the corresponding mRNA levels.", "However, this will not necessarily reflect the amount of mitochondrial mature frataxin that is expressed.", "Therefore, specific and sensitive methodology is required in order to reliably analyze expression of mature frataxin in tissue samples.", "Availability of this methodology would also enable surrogate tissues to be identified so that mature frataxin expression can be monitored in human subjects.", "In the present study, we report a screening of five antibodies in order to identify those that would be useful for conducting such studies using PAGE/western blot of tissue samples.", "Four protein standards were used in this study.", "Cynomolgus monkey mature frataxin (aa 81–210) was purchased from LifeSpan BioSciences (LS-G21788).", "Mouse recombinant intermediate form of frataxin (aa 41–207) was obtained from LifeSpan BioSciences (LS-G14956).", "Human and mouse mature frataxin were prepared using a protocol described previously (Guo et al.", "Anal.", "Chem.).", "Five antibodies were tested here, including AB113691, AB175402, AB124680 obtained from Abcam (Cambridge, MA), MAB1594 from Millipore-Sigma (Billerica, MA), and LS-C197243 from LifeSpan BioSciences (Seattle, WA).", "Anti-rabbit HRP and anti-mouse HRP were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX).", "Stainless steel beads for tissue homogenization were purchased from Next Advance (Troy, NY).", "NuPAGE™ LDS sample buffer was from Thermo Scientific (Waltham, MA) and chemiluminescence reagent (NEL103E001) was purchased from Perkin Elmer (Waltham, WA).", "Costar® Spin-X centrifuge tube filters (0.22 μm cellulose acetate) were obtained from Corning (Corning, NY).", "The heart samples were from WT C57BL/6 mouse, and were provided by Dr. Wellen (Associate Professor of Cancer Biology, Perelman School of Medicine, University of Pennsylvania).", "All surgery experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania.", "A piece of heart tissue sample was weighed (20–100 mg) and cut into 2–3 mm2 pieces.", "All the pieces were collected in an Eppendorf tube and immunoprecipitation (IP) lysis buffer [150 mM sodium chloride, 50 mM tris(hydroxymethyl)aminomethane (Tris).", "HCl pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X-100, 0.5% NP-40, 1 mM dithiothreitol (DTT)] with 0.5% SDS was added to make the concentration of 100 mg/mL.", "Approximately, 30–50 stainless steel beads (0.9–2.0 mm) were added to the mixture.", "The homogenization was carried out twice using Bullet Blender® Gold (Next Advance Inc., Troy, NY) at a speed of 6 for 4 min.", "If white fibers were still present in the samples, they were further homogenized using probe sonication at strength of 5 for 30 pulses.", "Following centrifugation at 17720 ×g for 5 min, a portion of the supernatant was withdrawn to mix with 1 × NuPAGE™ LDS sample buffer containing 2% β-mercaptoethanol and boiled at 95 °C for 5 min.", "After being briefly cooled down, the samples were filtered through Costar® Spin-X centrifuge tube filter (0.22 μm cellulose acetate) before being loaded on 12% SDS PAGE.", "A portion of tissue homogenate or protein standards including the intermediate form of mouse frataxin and the mature form of monkey frataxin, human frataxin, and mouse frataxin were mixed with 1 × NuPAGE™ LDS sample buffer containing 2% β-mercaptoethanol.", "The mixtures were boiled 95 °C for 5 min and cooled down to room temperature, the supernatants were loaded on 12% SDS PAGE.", "The gel was run at 100 V for about 80 min until the dye was at the bottom.", "Typically, 200–400 μg of tissue was analyzed for each western blot.", "There was 0.03–0.07 mg protein/mg tissue and so 6–28 μg of total protein was load in each lane.", "Intense signals were observed for the protein standards showing that the proteins were transferred well to the membrane.", "This was confirmed by Ponceau staining after transferring to the membrane, which showed that of all the high abundant protein bands in the tissue samples were present.", "Proteins were transferred to an Invitrogen nitrocellulose membrane with a pore size of 0.2 μm (Thermo Scientific, Waltham, MA).", "Membranes were blocked for 1 h at room temperature with 5% milk in Dulbecco's phosphate-buffered saline containing 0.1% Tween-20 (DPBST), then incubated with a range of anti-Frataxin antibodies that were diluted using 5% non-fat dry milk in DPBST as instructed by the manufacture at 4 °C for overnight.", "Ab113691 and Ab175402 were diluted 1:500, and Ab124680, MAB1594 and LS-C197243 were diluted 1:1000.", "The membranes were washed in DPBST for 15 min 3 times before being incubated with the secondary Ab, either anti-rabbit HRP 1:5000 or anti-mouse HRP 1:5000, as appropriate.", "Chemiluminescence was generated using NEL103E001 and captured by X-ray films that were developed using a Konica Minolta SRX-101A medical film processor (Browns Medical Imaging, Omaha, NE).", "The amount of sample loaded on the gel was adjusted so that bands could be observed with a similar intensity after a similar exposure time (30–45 s).", "Each western blot was performed at 3–5 times with very consistent results.", "Western blots using different antibodies were performed separately.", "The standards that were obtained commercially had defined concentrations so they served as loading controls.", "Control experiments were conducted without the primary antibody to show that the detected bands were specific to that antibody.", "Human and mouse mature frataxin, which were prepared as described with His-tags at their C-termini (for purification) (Guo et al., 2018b), had MWs of 15.3 Da and 15.4 kDa, respectively.", "The protein standards were subjected to SDS-PAGE followed by western blotting with all five selected antibodies.", "Four out of five tested antibodies detected human mature frataxin, but with different sensitivities (Figs. 24).", "The two Abs purchased from Abcam (AB113691 and AB175402) and the mAb from LifeSpan Bio (LS-C197243) had the highest sensitivity for human frataxin (Figs. 2A, B, and 3B; Table 2).", "Both of the Abcam Abs were prepared using recombinant full-length human frataxin as the immunogen (Table 1).", "The LifeSpan Bio mAb was raised against a peptide from human mature frataxin (aa 91–200) with GST tag (Table 1, Fig. 3B).", "Human mature frataxin showed low intensity when blotted with the mAb from Millipore-Sigma (MAB1594), even though a larger amount of the protein was loaded on the gel (Fig. 3A).", "Interestingly, this mAb was also raised against full length human frataxin, although fused to TrpE.", "The Ab (AB124680) prepared using a synthetic peptide corresponding to human frataxin aa 150 to the C-terminus as the immunogen (the full amino acid sequence was not divulged from the manufacture due to protection of intellectual property) did not recognize human mature frataxin at all (Fig. 4).", "Of the three Abs that were supposed to recognize mouse frataxin, AB175402 showed the best immunoreactivity (Fig. 2B), MAB1594 was less sensitive (Fig. 3A), and AB113691 barely recognized the mouse mature frataxin standard (Fig. 2A).", "The other two mAbs (AB124680 and LS-C197243) could not detect mouse mature frataxin standard, as expected from the supplier's description (Figs. 3B and 4).", "Cynomolgus monkey mature frataxin was expressed in E. coli, with a 4 kDa tag at the N-terminus and so the migration for this protein corresponded to 18.3 kDa.", "Two of the Abs that were tested (AB175402 and LS-C197243) recognized the monkey mature frataxin standard with good intensity (Figs. 2B and 3B).", "MAB1594 weakly detected it when a larger amount was loaded on the gel (Fig. 3A).", "AB113691, which detected human mature frataxin with good intensity, did not recognize mature monkey frataxin at all (Fig. 2A).", "This was very surprising in view of the sequence similarity between human and monkey frataxin (Fig. 1).", "Mouse heart tissue was homogenized and filtered before being analyzed by SDS-PAGE.", "A mouse intermediate frataxin form purchased from Lifespan BioSciences was also loaded on the side as a control.", "The intermediate standard was expressed with a 4 kDa His-tag according to the manufacture and so migrated at 22.4 kDa.", "Surprisingly, AB113691, which barely detected mouse mature frataxin, recognized the mouse intermediate standard with high sensitivity (Fig. 2A).", "It also detected two proteins in the mouse tissue samples, with one protein migrating the same as the mouse intermediate frataxin standard (22.4 kDa), the other migrating slower (27 kDa).", "Endogenous intermediate frataxin (41–207) is an 18.5 kDa protein whereas full-length mouse frataxin has a MW of 22.9 kDa.", "Therefore, the two proteins are most likely immunoreactive proteins detected by the Ab that are un-related to frataxin.", "This mAb also failed to detect either endogenous mouse mature frataxin or the mouse mature frataxin standard that was added to the tissue samples (Fig. 2A).", "AB175402 recognized both the endogenous mature mouse frataxin and the mouse mature frataxin standard that was spiked into the tissue samples (Fig. 2B).", "It also recognized the mouse intermediate standard but did not recognize either of immunoreactive proteins in the heart tissue at 22.4 and 27 kDa (Fig. 2B).", "Ab124680 detected numerous immunoreactive proteins in the mouse heart tissue but it was not clear whether any of them corresponded to a frataxin isoform (Fig. 4).", "Conversely, Ab124680 showed weak immunoreaction to mouse intermediate standard and no band was founded corresponding to endogenous mouse mature form (Fig. 4).", "MAB1594 weakly recognized endogenous mature frataxin in the mouse heart tissue and also weakly recognized the mouse mature frataxin standard that was spiked into the tissue (Fig. 3A).", "In contrast, MAB1594 recognized the mouse intermediate standard with high sensitivity and the protein that migrated with the same mobility as the mouse intermediate frataxin standard at 22.4 kDa as well as some higher MW proteins (Fig. 4A).", "LS-C197243 did not recognize endogenous mature frataxin in the mouse heart tissue or the mouse mature frataxin standard that was spiked into the tissue (Fig. 3B).", "However, it did recognize the mouse intermediate standard with high sensitivity and the protein that migrated with the same mobility as the mouse intermediate frataxin standard at 22.4 kDa as well as some higher MW proteins (Fig. 3B).", "Human, monkey and mouse frataxin sequences were down-loaded from Uniprot and aligned with the relevant Uniprot program.", "Full-length frataxin (1–210) from human and monkey frataxin share 91% sequence similarity while human full-length frataxin (1–210) and mouse full-length frataxin (1–207) share over 72% sequence similarity (Fig. 1).", "The sequence of intermediate frataxin (42–210) from human and monkey are over 92% similar whereas human intermediate frataxin (42–210) and mouse intermediate frataxin (41–207) share 78% sequence similarity.", "Human and monkey mature frataxin (81–210) differ by only 3 amino acids (E92D, W168R, A186G), while the human mature frataxin (81–210) and mouse mature frataxin (78–207) differ by 11 amino acids (Fig. 1).", "Of the five Abs that were tested, the suppliers indicated that five of them were immunoreactive with human frataxin; whereas, three suppliers indicated that their Abs were immunoreactive with both human and mouse frataxin (AB113691, AB175402 and MAB1594).", "Due to the high sequence similarity between monkey and human frataxin, it was highly likely that the selected Abs would also recognize monkey frataxin, although none of the suppliers claimed that they would.", "Clearly, the mAb obtained from LifeSpan Bio (LS-C197243) was the best for differentiating human and mouse mature frataxin (Fig. 3B).", "It recognized human mature frataxin but did not recognize endogenous mature frataxin in the mouse heart tissue or the mouse mature frataxin standard that was spiked into the tissue (Fig. 3B).", "Therefore, this mAb will be useful in mouse models such as the siRNA knockdown mouse (Chandran et al., 2017) to show that when treated with a human transgene, human frataxin protein is expressed.", "Curiously, this mAb detected a His-tagged mouse intermediate frataxin standard but not endogenous mouse mature frataxin (Fig. 3B).", "This suggests that the mAb could also be useful for confirming whether there is aberrant processing of the full-length mouse protein during pre-clinical gene therapy studies in mouse models.", "Abcam Ab175402, was undoubtedly the best for detecting both human and mouse frataxin (Fig. 2B).", "It detected both endogenous mouse mature frataxin in mouse heart tissue as well as the mature frataxin standard spiked into the tissue samples.", "There was no loss in sensitivity as a result of Ab binding to tissue components when compared with the standard alone (Fig. 2B).", "This contrast with MAB1594 where there was a significant loss of intensity in the presence of the tissue sample as well as a reduction in intensity of the signal for endogenous frataxin (Fig. 3A) when compared with Ab175402 (Fig. 2B).", "Four of the Abs bound strongly to the mouse intermediate frataxin (41–207) standard (Table 2).", "This means that they would be useful for determining whether there is aberrant processing of full-length frataxin in the animal models.", "AB113691 was the only Ab tested that could differentiate cynomolgus monkey mature frataxin form from human mature frataxin (Fig. 2A).", "However, it is not clear if the large tag that was present at the amino terminus could have caused this loss of immunoreactivity.", "Additional experiments with cynomolgus monkey tissue coupled with expression of monkey frataxin with a shorter His-tag will be required to clarify this issue.", "AB175402, MAB1594, and LS-C197243 were able to detect both human and monkey frataxin without clear preference (Figs. 2B, 3A, and B).", "This means that if frataxin is up-regulated in a monkey model after gene therapy, it will be necessary to confirm the presence of the human frataxin isoform using mass spectrometry-based procedures such as those we have described recently (Guo et al., 2018a; Guo et al., 2018b).", "Finally, AB124680 was unable to detect any of the frataxin standards or endogenous frataxin in mouse heart tissue because of the high level of non-specific binding (Fig. 4).", "In conclusion, an Ab has been identified that can distinguish human from mouse frataxin (LS-C197243), an Ab that binds to human, mouse and monkey frataxin (Ab175402), and an Ab that binds to human and mouse mature frataxin but not monkey mature frataxin (Ab113691).", "We anticipate that these Abs will be useful for analyzing frataxin expression in both pre-clinical animal and human cell models to determine whether human frataxin is up-regulated by novel therapeutic interventions such as gene therapy and CRISPR designed to correct the defect in frataxin production in Friedreich's ataxia." ]	[]
Daring metabolic designs for enhanced plant carbon fixation	Increasing agricultural productivity is one of the major challenges our society faces. While multiple strategies to enhance plant carbon fixation have been suggested, and partially implemented, most of them are restricted to relatively simple modifications of endogenous metabolism, i.e., “low hanging fruit”. Here, I portray the next generation of metabolic solutions to increase carbon fixation rate and yield. These strategies involve major rewiring of central metabolism, including dividing Rubisco's catalysis between several enzymes, replacing Rubisco with a different carboxylation reaction, substituting the Calvin Cycle with alternative carbon fixation pathways, and engineering photorespiration bypass routes that do not release carbon. While the barriers for implementing these elaborated metabolic architectures are quite significant, if we truly want to revolutionize carbon fixation, only daring engineering efforts will lead the way.	[ "AGRI", "BIOC" ]	[ "Calvin cycle", "Carboxylation", "Formate assimilation", "Metabolic engineering", "Photorespiration", "Rubisco", "Synthetic biology" ]	[ "1102", "1110", "1311" ]	[ "Global crop production needs to double by 2050 to meet the demands of a growing population.", "To achieve this goal, agricultural yield needs to increase by 2.4% per year, almost double the average annual yield increase in the past 40 years [1].", "As the efficiency with which plants intercept light and the partitioning of biomass into harvested parts are close to their theoretical maxima, major improvements in plant productivity could mainly arise from increasing the efficiency by which solar energy is converted into biomass [2–4].", "Averaged throughout the growing season, this efficiency is limited to a few percent, in spite of a theoretical maximum of ∼12% [2–4].", "Multiple factors, related to several key processes, were shown to limit the efficiency by which light is used to generate biomass; accordingly, numerous strategies for enhancing plant productivity have been proposed, and, at least some, have been tested in vivo.", "These strategies can be roughly divided into four categories, depending on the physiological process they are trying to optimize [3–11]: light reactions, CO2 diffusion and concentration, activity of Rubisco, and regeneration of ribulose 1,5-bisphosphate (RuBP).", "Table 1 summarizes these approaches and provides references to representative studies and recent reviews that exemplify and discuss them.", "This perspective aims to put forward (mostly) unconventional ideas to rewire carbon fixation metabolism for increased photosynthetic efficiency.", "As most current efforts center around “low hanging fruit”, i.e., minimal engineering interventions that maximize short-term gains, I decided to focus on long-term prospects to revolutionize carbon fixation using both enzyme and metabolic engineering [12], as summarized schematically in Fig. 1.", "First, I discuss the distribution of Rubisco’s catalytic steps across several consecutive enzymes.", "This is followed by a suggestion to replace Rubisco with a different carboxylating enzyme while maintaining the Calvin Cycle largely intact.", "Then, I analyze the possibility of substituting the entire Calvin Cycle with alternative carbon fixation pathways.", "A special emphasis is given to synthetic carbon fixation pathways that rely on carbon reduction to formate, followed by formate assimilation.", "Finally, replacement of the CO2-releasing photorespiration pathways with carbon-neutral or carbon-positive bypass routes is presented and analyzed.", "Throughout the discussion, it is important to remember that besides directly enhancing plant productivity, increased carbon fixation efficiency holds more benefits for plant growth.", "First, Rubisco accounts for 30–50% of the total soluble proteins in the leafs of C3 plant, and holds 10–30% of the total nitrogen [7].", "Increasing carbon fixation rate would enable a reduction in Rubisco levels, and therefore higher efficiency of nitrogen use, potentially alleviating a major growth constraint [4,7,13].", "Similarly, establishing a higher carbon fixation rate could permit photosynthesis to operate under a lower chloroplastic concentration of inorganic carbon, thus enabling the plant to close its stomata when necessary and maintain higher efficiency of water use [4,7].", "The maximal rate of Rubisco is an order of magnitude lower than the average of central metabolism enzymes [14].", "Its lack of specificity towards CO2 (see below section on oxygenation and photorespiration) further substantially reduces its effective carboxylation efficiency.", "Efforts to improve Rubisco’s kinetic parameters resulted in marginal success at best [15], mainly due to the inherent tradeoff between the enzyme’s rate and specificity [16].", "Instead of trying to pursue small improvements in the activity of an enzyme that was under immense selective pressure for eons, we should consider replacing it with other enzymes that together take over its catalytic activity.", "An advantage of this approach is that it keeps the Calvin Cycle largely intact, thus minimizing further disruption of the plant physiology.", "Rubisco’s reaction involves four consecutive steps: enolization, CO2/O2 addition, hydration, and cleavage (Fig. 2A) [17,18].", "These reaction steps could potentially be catalyzed by three enzymes, each operating via a well-established mechanism (Fig. 2B–D).", "The first would be an isomerase, transferring the carbonyl group from the second to the third carbon (Fig. 2B).", "This reaction is very similar to the one catalyzed by 6-phosphate-3-hexuloisomerase (EC 5.3.1.27) of the ribulose monophosphate (RuMP) cycle [19].", "The second reaction is a carboxylation (Fig. 2C) that could be catalyzed by a biotin-dependent enzyme, which does not suffer from the problem of oxygenation.", "The location of the carbonyl group – on the third carbon – is ideal to support the required CO2 addition reaction (Fig. 2C).", "Lastly, a third enzyme would catalyze a hydration-cleavage reaction, as shown in Fig. 2D, with a mechanism matching that of the enzyme oxaloacetase (EC 3.7.1.1) [20].", "This set of enzymes, once evolved and optimized, could break the kinetic constraints imposed by Rubisco’s mechanism, enabling unhindered improvement of catalytic rates.", "The extra ATP cost associated with the biotin dependent carboxylation is tolerable considering the complete suppression of oxygenation and photorespiration, and potentially improved kinetics that would substantially lower the protein burden associated with Rubisco biosynthesis.", "Moreover, biotin-dependent carboxylases accept bicarbonate instead of CO2.", "As the concentration of bicarbonate in the alkaline stroma is orders of magnitude higher than that of CO2, the availability of inorganic carbon is not expected to limit photosynthetic rate.", "It is conceivable to replace Rubisco with another carboxylase while keeping the basic operation of the Calvin Cycle mostly unhindered.", "6-phosphogluconate dehydrogenase (6PGDH) presents such an opportunity.", "6PGDH is a reversible reductive carboxylase (oxidative decarboxylase) operating at ΔrG’m ∼ +6 kJ/mol (Gibbs energy change at 7 < pH < 8, ionic strength of 0.25 M, and assuming reactant concentrations of 1 mM [21]).", "Reductive carboxylases, which also include the malic enzyme and isocitrate dehydrogenase, couple the favorable reduction of a carbonyl group to an unfavorable carboxylation [22].", "A recent review has compared the kinetics of different carboxylase enzymes, revealing that the best reductive carboxylases have kcat values higher than 100 s−1, almost two orders of magnitude higher than that of Rubisco [23].", "However, their maximal affinity towards CO2 is 1 mM, almost two orders of magnitude worse than that of Rubisco.", "Together, kcat/KM values of these reductive carboxylases can be as high as that of Rubisco [23].", "While the kinetics of 6PGDH in the reductive direction is mostly unexplored, it is plausible to assume that some of its variants − perhaps not plant ones but rather prokaryotic – have similar kinetics, making its rate comparable to that of Rubisco, without the problem of oxygenation.", "It is worth noting that if coupled to a carbon concentrating mechanism, 6PGDH kinetics would become even more favorable.", "As a carboxylation substrate, 6PGDH accepts ribulose 5-phosphate (Ru5P), the direct precursor of ribulose 1,5-bisphosphate (RuBP), Rubisco’s substrate.", "Hence, both carboxylation reactions share almost identical substrates.", "However, the products of the reactions are very different, leading to the question of how 6-phosphogluconate (6 PG) – the product of 6PGDH carboxylation – is going to be recycled back into the Calvin Cycle.", "Several options can be envisioned.", "Reducing 6 PG to glucose 6-phosphate – which is then isomerized to fructose 6-phosphate − would be the most straightforward approach (Fig. 3A).", "However, this is thermodynamically infeasible, unless the carboxyl group is activated – for example, as a phosphoanhydride group.", "Therefore, to enable the operation of this cycle it is vital to establish glucose 6-phosphate dehydrogenase (phosphorylating) and 6PG-1-kinase activities, which could potentially be evolved from glyceraldehyde 3-phosphate dehydrogenase (phosphorylating) and 3-phosphoglycerate kinase, respectively.", "Supporting this possibility, human glyceraldehyde 3-phosphate dehydrogenase was already shown to accept glucose [24].", "As the activity of the glucose 6-phosphate shunt [25] directly counteracts the reductive assimilation of 6 PG, it would probably have to be deleted (e.g., knocking out glucose 6-phosphate dehydrogenase, ZWF).", "Yet, this shunt is predicted to play an important role in stabilizing the activity of the Calvin Cycle [25] and its deletion might result in deleterious effects.", "Conversely, 6 PG can be reasssimilated using only existing enzymes (Fig. 3B): the enzymes of the Entner–Doudoroff pathway – 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase – can metabolize 6 PG to GAP and pyruvate.", "Pyruvate can then be reassimilated into the Calvin Cycle via gluconeogenesis.", "However, chloroplasts do not contain the enzymes of lower glycolysis – phosphoglycerate mutase and enolase [26–28] – and their introduction to this organelle could result in a major disruption of endogenous metabolism.", "Two other pathways could recycle 6 PG with little overlap with endogenous metabolism.", "In these pathways (Fig. 3C,D), a hydroxyl group of 6 PG is oxidized to give a carbonyl, followed by an aldolase reaction that produces a triose phosphate and glycerate, which can be directly fed into the Calvin Cycle.", "The 2-keto-6-phosphogluconate pathway (Fig. 3C) might be more approachable, as 6-phosphogluconate 2-dehydrogenase actually exists (EC 1.1.1.43), while 6-phosphogluconate 5-dehydrogenase (Fig. 3D) needs to be engineered.", "The aldol cleavage of 2-keto-6-phosphogluconate (Fig. 3C) and 5-keto-6-phosphogluconate (Fig. 3D) requires enzyme engineering, but enzymes that catalyze similar reactions are available.", "For example, 2-dehydro-3-deoxyglucarate aldolase (EC 4.1.2.20) catalyzes the cleavage of this metabolite to pyruvate and tartronate semialdehyde [29], demonstrating an aldolase reaction that generate this later glycerate-precursor.", "Six pathways are known to support carbon fixation in autotrophic organisms.", "Of these, the Calvin Cycle can be considered as the least efficient one.", "In all other pathways, ATP is invested only to energize unfavorable reactions that are essential for carbon fixation: carboxylation and carboxyl reduction [30].", "Moreover, many of the other pathways couple these unfavorable reactions to exergonic reactions other than ATP hydrolysis, thus reducing ATP consumption.", "On the other hand, the Calvin Cycle is the only carbon fixation pathway that hydrolyzes ATP for purposes other than activating unfavorable reactions.", "Specifically, the ATP consumed by phosphoribulokinase does not help to overcome any thermodynamic barrier [30].", "While this phosphorylation reaction plays a role in ensuring that all pathway intermediates are phosphorylated and further enables two irreversible steps – fructose 1,6-bisphophatase and sedoheptulose 1,7-bisphosphatase – that generate extra thermodynamic push and enable better flux control, these are not strictly necessary for pathway activity.", "Biochemically, it would have been possible to operate the cycle with non-phosphorylated sugars and without the irreversible bisphosphatases, achieving the same metabolic goal with a lower ATP cost.", "The extra ATP consumption of the Calvin Cycle reduces the efficiency of the pathway and decreases the biomass yield it can support (under energy-limited conditions).", "As such, the Calvin Cycle suffers from two major inefficiencies, the first related to the poor kinetics of its carboxylating enzyme and the second to its wasteful use of cellular resources.", "This leads to the question of whether the Calvin Cycle can be replaced with another pathway that could support higher carbon fixation rate and efficiency.", "Out of the known natural pathways, only two are oxygen-tolerant enough to potentially operate within the oxygen-rich chloroplast: the 3-hydroxypropionate bicycle and the 3-hydroxypropionate-4-hydroxybutyrate cycle [22,31].", "Both pathways are based on efficient biotin-dependent carboxylations [23], directly tapping into the extended reservoir of plastidic bicarbonate.", "However, both pathways are rather complex, in terms of enzymatic steps and overlap with central metabolism.", "For example, both pathways interact with fatty-acid biosynthesis (malonyl-CoA metabolism), which could distort this essential chloroplastic process.", "As an alternative, synthetic pathways can be designed to offer tailor-made metabolic solutions for carbon fixation.", "Two general approaches can be used towards this goal: (i) considering only existing enzymes, potentially from different sources, which are combined together in a “mix-and-match” approach to offer a novel carbon fixation route [32]; (ii) employing enzyme engineering techniques to evolve novel catalytic capabilities, which can be combined with existing enzymes to enable highly efficient carbon assimilation [12].", "While the first approach is more straightforward, the second could lead to the development of highly optimized metabolic structures, the efficiency of which cannot be reached using only known enzymatic components [12].", "Regardless of the approach taken, candidate synthetic pathways should be analyzed according to multiple physiochemical parameters in order to identify those that are most suitable to support in vivo carbon fixation.", "The central parameters to consider are summarized in Box 1.", "Synthetic carbon fixation pathways have yet to be implemented in living organisms.", "However, an in silico study has identified numerous candidate pathways that could potentially replace the Calvin Cycle [32].", "Among these, one group of pathways, termed the MOG (Malonyl-CoA-Oxaloacetate-Glyoxylate) cycles, was estimated to be especially promising in terms of efficiency and rate.", "As exemplified by one variant, shown in Fig. 4, the MOG cycles utilize a single carboxylating enzyme: PEP carboxylase (acetyl-CoA conversion to malonyl-CoA proceeds via a carboxyl-transferase enzyme [33]).", "PEP carboxylase is the best carboxylating enzyme in terms of kcat and kcat/KM [23,32], and could thus provide an optimal in vivo entry point of inorganic carbon.", "Recently, a synthetic carbon fixation cycle was established in vitro, demonstrating, for the first time, that this central process can be supported by a non-natural metabolic structure [34].", "The CETCH cycle, as shown in Fig. 5, utilizes reductive carboxylation to efficiently add CO2 onto metabolic intermediates and was shown to support a higher carbon fixation rate than the Calvin Cycle [34].", "However, both the MOG cycles and the CETCH cycle produce glyoxylate, the assimilation of which into central metabolism – via the bacterial glycerate pathway [35] – is rather inefficient as it involves a decarboxylation step.", "Moreover, these cycles are very long and highly complicated.", "While implementing these pathways in microbes might be possible, their integration into the plant metabolic network seems highly unlikely.", "If one considers also unnatural reactions, simpler, more direct pathways can be proposed.", "To give only one example, carboxylation of 3-hydroxypropionate – via a promiscuous a biotin-dependent carboxylase, e.g., [36] – followed by a mutase and epimerase reactions − as are normally operated on methylmalonyl-CoA [37] – would result in a very short pathway, shown in Fig. 6, which might be considerably easier to implement.", "Many efforts to improve carbon fixation have focused on redesigning, analyzing, and optimizing the activity of carboxylating enzymes.", "However, these can be, at least partially, replaced by CO2 reducing enzymes, followed by condensation of the reduced C1 compound with a metabolic intermediate [23].", "Reduction of CO2 results in one of two compounds: CO or formate.", "As the redox potential associated with CO is very low (E0′ < −500 mV [21]), the reduction of CO2 to CO using cellular electron carriers is very challenging.", "Furthermore, since the assimilation of CO can proceed only via the highly oxygen sensitive acetyl-CoA synthase, this compound is of little interest in the context of plant metabolism.", "Formate, on the other hand, is associated with a less negative reduction potential (E0′ ∼ −420 mV [21]), making its production from CO2 an easier task.", "Furthermore, multiple formate assimilation pathways can be envisioned and tailored to fit the host organisms [38].", "Therefore, an alternative carbon fixation strategy could be envisioned in which the low reduction potential of the chloroplastic electron carriers (e.g., ferredoxins) is used to reduce CO2 to formate, which is then assimilated to central metabolism.", "As formate assimilation can be considerably more ATP-efficient than the Calvin Cycle, this approach could enhance biomass yield [23,39].", "Reduction of CO2 to formate can be catalyzed by formate dehydrogenase (FDH).", "Some metal-dependent variants of this enzyme were shown to support high CO2-reduction activity [40].", "For example, FDH from Desulfovibrio desulfuricans catalyzes CO2 reduction with kcat ∼ 50 s−1 and KM ∼ 15 μM, making this enzyme a much more efficient entry point for inorganic carbon than Rubisco [41].", "However, this enzyme variant is highly oxygen sensitive.", "Other CO2-reducing FDHs operate under fully aerobic conditions, e.g., FDH from Rhodobacter capsulatus [42] and from Cupriavidus necator [43].", "The latter enzyme was shown to reduce CO2 with kcat > 10 s−1.", "The most challenging aspect of establishing FDH activity within the chloroplast is that the enzyme is molybdenum-dependent.", "This means that molybdenum needs to be transported to the chloroplast and then embedded within the enzyme active site using a molybdenum cofactor (MoCo).", "Yet, the plant MoCo is not present in the chloroplast and is different than the bacterial variant that is required for FDH activity [44].", "Hence, the entire biosynthesis pathway for the bacterial MoCo will have to be introduced into the chloroplast, together with an appropriate molybdenum transport system.", "Once formate is generated from CO2 reduction it needs to be assimilated.", "One promising formate assimilation route is the reductive glycine pathway, as shown in Fig. 7 [38,39,45].", "The production of glycerate 3-phosphate via this pathway requires the investment of three ATP molecules instead of the eight needed by the Calvin Cycle.", "The pathway utilizes only enzymes that are known to operate in plant cells: attachment of formate to THF, followed by its reduction to methylene-THF (CH2-THF) is supported by ubiquitous enzymes that exist in the chloroplast, cytosol and mitochondria [46]; the glycine cleavage system (GCV) and serine hydroxymethyltransferase operate in the mitochondira [47]; and conversion of serine to glycerate occurs in the peroxisome and cytosol [48].", "Taken together, the reductive glycine pathway overlaps with the native photorespiration pathway, but rewires it from being a carbon releasing process (see below) into a Calvin-Cycle-independent carbon fixation route.", "The GCV is known to be reversible both in vitro [49,50] and in vivo [51–53], and its overall thermodynamics (ΔrGm’ ∼ −5 kJ/mol in the reductive direction, pH 7–8, I = 0.25 M [21]) indicates that its flux direction is determined by the concentrations of the reactants (THF, CH2-THF, glycine, CO2, NH3, NAD+, NADH).", "Yet, the GCV from plants was never demonstrated to work in the reductive direction, suggesting that a foreign, well-established reversible system might be necessary to support glycine biosynthesis.", "Another interesting approach involves formate reduction to formaldehyde, which is then assimilated via the RuMP pathway [19], as shown in Fig. 8.", "Reduction of formate, via a formyl-CoA intermediate, has been previously demonstrated using promiscuous acetyl-CoA synthetase and acetaldehyde dehydrogenase [54].", "3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), the key enzymes of the RuMP pathway were already shown to be fully active when expressed in plant chloroplast, supporting the assimilation of exogenously supplied formaldehyde [55,56].", "As the affinity of HPS to formaldehyde is very high (KM as low as 59 μM) and the enzyme operates at very high rate (kcat as high as 97 s−1) [19], the steady-state concentration of formaldehyde can remain very low, avoiding the toxicity effects associated with its accumulation.", "The main advantage of the RuMP pathway is that it is much more efficient in the utilization of cellular resources than the Calvin Cycle: production of a triose phosphate from CO2 requires only 4 ATP molecules (assuming that formate activation with CoA requires a single ATP), which is less than half of the 9 ATP molecules hydrolyzed by the Calvin Cycle.", "Moreover, formaldehyde assimilation is metabolically contained within the Calvin Cycle, i.e., converting ribulose 5-phosphate to fructose 6-phosphate.", "This could minimize the disturbance to endogenous fluxes and reduce regulation complexity.", "In C3 plants Rubisco’s oxygenation accounts for 20–30% of catalytic turnovers [57–62], producing large amount of 2-phosphoglycolate (2 PG) that needs to be recycled into the Calvin Cycle in a process termed photorespiration.", "Photorespiration also plays a central role in protecting the cells from photoinhibitory effects by dissipating excess reducing power and energy which accumulate under carbon starvation conditions [63].", "Even organisms that use carbon concentrating mechanisms, such as C4 plants, algae, and cyanobacteria, are dependent on photorespiration [64,65].", "Here, the main role of the process is probably to detoxify 2-phophoglycolate – an inhibitor of triose phosphate isomerase and sedoheptulose 1,7-bisphosphate phosphatase [66,67] – as well as glycolate [65].", "Overall, photorespiration is an essential process that cannot be avoided, as was shown in numerous studies that tried to abolish or reduce the activity of photorespiratory enzymes [68].", "Yet, the essentiality of this process does not rule out that other pathways could perform the same metabolic tasks in a more efficient manner.", "Several drawbacks are associated with the endogenous photorespiration pathway.", "First, it involves multiple transport steps between several organelles, some probably requiring energy investment [69].", "In addition, it dissipates reducing power by transferring electrons from glycolate to O2.", "It further releases ammonia that needs to be reassimilated at the expense of ATP hydrolysis.", "Yet, the most wasteful aspect of photorespiration is that it releases carbon − one CO2 per two glycolate molecules assimilated – thus directly counteracting the activity of the Calvin Cycle and Rubisco.", "Replacing photorespiration with a synthetic alternative could tackle one or more of these drawbacks.", "Several CO2-releasing photorespiration bypass routes – some shown in Fig. 9 – were previously suggested and at least partially implemented [35,70–72].", "These were analyzed in multiple studies and reviews [5,73–76], and hence will not be discussed in detail here.", "Yet, it should be noted that the mechanism behind the beneficial effect of the most studied bypass, the glycerate pathway, remains somewhat vague.", "In this pathway, two glyoxylate molecules are directly condensed into tartronate semialdehyde (and CO2) which is subsequently reduced to glycerate, all within the chloroplast [35,72] (Fig. 9).", "However, it was found that the overexpression of glycolate dehydrogenase by itself – shown to increase glyoxylate content in chloroplasts – exerts a similar beneficial effect on photosynthetic productivity [35,72,77].", "It is therefore difficult to decipher whether the enhancement of carbon fixation by the chloroplastic glycerate pathway is truly related to the more efficient recycling of glycolate.", "As CO2 release is the main drawback of photorespiration, a metabolic bypass which would avoid this carbon loss could have a much more substantial effect on carbon fixation than the CO2-releasing pathways.", "Fig. 10 shows a possible general structure of such alternative photorespiration routes.", "In these pathways, glycolate is first reduced to glycolaldehyde, via a glycolyl-phosphate or glycolyl-CoA intermediate.", "Glycolaldehyde then serves either as an acceptor (Fig. 10A) or a donor (Fig. 10B) for an aldol condensation with a phosphosugar intermediate of the Calvin Cycle, resulting in the production of a longer chain phosphosugar than can be reintegrated into the cycle.", "As glycolaldehyde inhibits the Calvin Cycle [78–80], and, being a small aldehyde, is quite reactive [81–83], it is important to use an aldolase that can accept it with high affinity, thus preventing its accumulation and keeping its concentration below 1 mM.", "Recently, a similar metabolic structure was suggested as a photorespiration bypass, where 2 PG is reduced to 2-phosphoglycolaldehyde, which is then condensed with dihydroxyacetone phosphate (DHAP) to give xylulose 1,5-bisphosphate.", "Xylulose 1,5-bisphosphate is integrated to the Calvin Cycle via its dephosphorylation to xylulose 5-phosphate [3].", "However, direct reduction of 2 PG is not advisable as the concentration of this metabolite is probably very low, unlike the high levels of glycolate [84] that push forward its reduction both energetically and kinetically.", "An even more daring approach would be to transform photorespiration from a carbon-releasing process into a carbon-fixing one.", "By using this tactic, the burden of assimilating inorganic carbon would be shared between Rubisco and the photorespiratory carboxylating enzyme.", "In the extreme case, Rubisco could be evolved to catalyze only the oxygenation reaction – at very high rate – where carbon fixation is performed only via photorespiration.", "A previously suggested carbon-fixing photorespiration bypass converts glyoxylate to pyruvate via one of the cycles of the 3-hydroxypropionate bicycle [85], as shown in Fig. 11.", "While this provides a very interesting approach, the length of this bypass makes it very challenging to implement.", "Moreover, the cycle’s product, pyruvate, needs to be assimilated back into the Calvin Cycle.", "As discussed above, this involves introducing missing glycolytic enzymes to the chloroplast – phosphoglycerate mutase and enolase – which could result in a severe flux disruption.", "Identifying promising carbon-neutral and carbon-positive photorespiration bypass routes should follow the general guidelines presented in Box 1.", "While this manuscript is not the place to perform such a comprehensive analysis, there are two interesting routes that are worth a short discussion.", "First, CO2 reduction to formate, followed by reduction to CH2-THF and condensation with glycine to give serine, can transform photorespiration into a carbon-fixing process, as shown in Fig. 12.", "This pathway is similar to the reductive glycine pathway (Fig. 7), where the main difference is that the photorespiration bypass is not dependent on de novo glycine production via the glycine cleavage system and as such is easier to implement.", "Notably, assimilation of formate into serine, in line with the suggested flux of this pathway, was demonstrated before in planta [86,87].", "A more speculative route is shown in Fig. 13, where glyoxylate is cleaved into two formate molecules (ΔrGm’ < −45 kJ/mol [21]), which, upon activation and reduction to CH2-THF, can replace glycine cleavage and make photorespiration a carbon-neutral process.", "Yet, while the oxidation of glyoxylate to formate (releasing CO2) was suggested to support a similar alternative photorespiration [86,87], a non-oxidative cleavage of glyoxylate into two formate molecules was not reported.", "Although an enzymatic mechanism to support this reaction is challenging to identify, a previous report on the condensation of formate into glyoxylate in potato tubers – although thus far unverified by other groups – suggests that it might be possible [88].", "The metabolic solutions suggested here are by no means easy to execute.", "In fact, as I elaborated for each approach separately, considerable barriers await the researcher who will try to implement them in plants.", "Yet, in the long-run, and as our enzyme and metabolic engineering capabilities improve, especially in planta [3], some of the proposed strategies might prove feasible and could completely transform the way plants perform carbon fixation, enhancing productivity in an unparalleled way.", "It is further important to emphasize that the metabolic architectures suggested here do not necessarily have to replace the endogenous pathways, but can rather, at least initially, operate in parallel, providing support to the natural processes.", "For example, carbon fixation via CO2 reduction to formate can operate next to the Calvin Cycle, where formate is assimilated via the reductive glycine pathway or (followed by another reduction) the ribulose monophosphate cycle, both directly integrate into Calvin Cycle.", "While it is not clear yet which solution will show the greatest promise in vivo, the time is ripe to expand our horizons towards daring metabolic designs that could reshape the most central biochemical process and enable us to boldly tackle a prime challenge of our society." ]	[ "Rubisco’s catalytic steps could be distributed across several novel enzymes.", "Carboxylation via 6-phosphogluconate dehydrogenase could replace Rubisco.", "Synthetic carbon fixation pathways could enhance carbon fixation rate and yield.", "Carboxylation reactions could be replaced with CO2 reduction.", "Photorespiration bypass routes that do not release CO2 can boost carbon fixation." ]
Impact of diabetes on male sexual function in streptozotocin-induced diabetic rats: Protective role of soluble epoxide hydrolase inhibitor	Diabetes-induced male sexual dysfunction is associated with endothelial dysfunction. Inhibition of soluble epoxide hydrolase (sEH) is known to improve endothelial function in diabetes. Therefore, we hypothesized that sEH inhibitor (sEHI), [trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]cyclohexyloxy}benzoic acid] / t-TUCB can restore the male sexual function in diabetic rat. After one week of administration of diabetogenic agent STZ (52 mg/kg i.p) injection, diabetic rats were treated with t-TUCB (0.1 and 0.3 mg/kg, p.o) or vehicle for 8 weeks. The sexual behaviour parameters of the animals were evaluated at the end of dosing period. The levels of testosterone and glucose in serum, and sperm were quantified. Effect of treatment on weight of reproductive organs and histopathology of penile tissue was evaluated. Diabetes had a negative effect on male sexual function, weight of sexual organs and production of sperm with a parallel decrease in the level of testosterone. The sEHI, t-TUCB, significantly preserved the sexual function and minimized an increase in the level of blood glucose in diabetic rats. It also prevented a decrease in the level of testosterone and sperm in diabetic rats, in comparison to diabetic control rats. Further, diabetes induced distortion of corpus cavernosum was attenuated by t-TUCB. Based on our findings, sEHI may delay the development of sexual dysfunction in diabetes.	[ "PHAR" ]	[ "Corpus cavernosum", "Diabetes-induced sexual dysfunction", "Sperm count", "Testosterone", "sEH inhibitor t-TUCB" ]	[ "3004" ]	[ "Sexual dysfunction (SD) refers to a problem or a group of problems occurring during any phase of the sexual response cycle, including desire, physical pleasure, arousal or orgasm, which prevents the individual from experiencing satisfactory sexual activity [1].", "It can contribute to psychological problems such as feelings of inadequacy, frustration, loss of self-esteem, and despair.", "Erectile dysfunction (ED) is one of the sexual dysfunctions which, primarily, results from impairment of penile vascular and smooth muscle relaxation in multiple pathological conditions including diabetes.", "A threefold increase in the risk of ED was documented in diabetic men compared with non-diabetic men [2,3].", "One of the major pathophysiological mechanisms underlying diabetic ED is endothelial dysfunction [4].", "Endothelial dysfunction is manifested in diabetes due to alterations in many signal transduction pathways responsible for the stimulation of penile vascular and smooth muscle relaxation.", "An enhanced destruction of nitric oxide (NO) due to an increase in oxidative stress, and a decrease in the release of endothelium-derived hyperpolarizing factors (EDHFs) are few of the mechanisms for development of diabetic ED [5].", "In addition to NO, EDHFs contribute significantly to the dilation of penile arteries [6].", "Epoxyeicosatrienoic acids (EETs) derived from the arachidonic acid act as EDHFs [7,8].", "EETs are rapidly hydrolyzed to less bioactive diols by the soluble epoxide hydrolase (sEH).", "Data from animal studies have shown that inhibitors of sEH attenuate endothelial dysfunction in diabetes [9].", "Yousif et al reported that CDU, a sEH inhibitor, regulates the tone of corpus cavernosum smooth muscle [10].", "Earlier, we reported that Moringa oleifera (seed) extract inhibits sEH and augments sexual function in healthy rats [11].", "Based on these studies, we hypothesized that inhibition of sEH might have a positive impact on sexual function in diabetes.", "The soluble epoxide hydrolase inhibitor, t-TUCB, was synthesized as described previously [12].", "Streptozotocin/STZ (MP Biomedical Pvt. Ltd.), ketamine hydrochloride (Neon Laboratories Limited, India), xylazine (Indian Immunologicals Limited, India), diethyl stilbestrol (Penta Pharmaceuticals, India), progesterone (Sun Pharmaceutical Ind.", "Ltd, India) and assay kit for estimation of blood glucose (Autospan diagnostic, India) level were procured.", "Twentyfour, in-bred sexually active male Wistar rats weighing (250 ± 5.00) g and an equal number of female rats were obtained from the central animal house, Al-Ameen College of Pharmacy, Bangalore.", "The rats were harboured in polypropylene cages and housed in a well-ventilated animal house under following standard laboratory conditions: temperature, 22 ± 3 °C; 12 h light/12 h dark cycle; humidity, 45–50%.", "Ethical clearance was obtained from the institutional animal ethics committee and the procedures were performed according to the recommendations and guidelines of Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA), India.", "Sexually active male rats were divided into four groups.", "Six rats were considered as normal control and treated with vehicle for 8 weeks.", "Another 18 rats were induced diabetes.", "Diabetes was induced via a single i.p. dose of 52 mg/kg STZ that was freshly prepared in 0.1 mM citrate buffer, pH 4.5 and used within 10 min of preparation [13].", "All rats receiving STZ were given a 10% sucrose solution in the first 48 h after injection to prevent hypoglycaemia.", "After 72 h of STZ administration, rats with fasting serum glucose more than 230 mg/dL were considered as diabetic.", "Long-acting insulin (3 IU/kg, s.c) was administered twice in a week to diabetic rats to prevent mortality throughout the study.", "Six diabetic rats were treated with vehicle for 8 weeks and considered as diabetic control.", "Remaining 12 diabetic rats were equally divided and treated with t-TUCB either with 0.1 or 0.3 mg/kg, p.o for eight weeks.", "In our study, female rats were used to stimulate male rats for initiating a sexual function.", "Ovariectomized female rats were used to prevent pregnancy and minimize the number of female rats used in the study [14].", "Female rats were paired with multiple male rats on different days during training and actual study.", "The ovariectomized female rats were artificially brought into oestrus phase by the administration of diethylstilbestrol (1 mg/kg, p.o, administered 2 days before the study) and progesterone (5 mg/kg, s.c., administered 6 h prior to the study) to facilitate mating [15].", "It is shown that female rats in the estrous phase allow intromission by exhibiting lordosis posture [16].", "Intromission was considered as a marker of erectile function.", "The male and female rats were trained for sexual behaviour study before induction of diabetes and treatment [17].", "The male and female rats who did not involve in sexual activity even after 10 min of pairing during training were removed from the study.", "The male rats were allowed to acclimatize to the environment for 10 min in a wooden cage (45 × 50 × 35 cm) having glass covering and illuminated by red light and then a female rat was placed in the cage and following sexual behaviour was observed for 30 min [18]: Mount latency(ML), time from the introduction of female rat into the cage of the male rat up to the ﬁrst mount; Intromission latency (IL), time from the introduction of the female rat up to the ﬁrst intromission by the male rat; Mount frequency (MF), number of mounts before ejaculation; Intromission frequency (IF), number of intromission before ejaculation; Ejaculation latency (EL), time from the ﬁrst intromission of a series up to the ejaculation); Post-ejaculatory interval (PEI), time from the ﬁrst ejaculation up to the next intromission by the male rat.", "Blood was collected from the heart of an anesthetized rat and serum was separated.", "Levels of testosterone and glucose in serum were quantified using commercial diagnostic kits according to test procedures outlined in the manual of the kits [19].", "At the end of the study, male rats were sacriﬁced by an overdose of anaesthesia.", "Epididymis, testis and penile tissue from each rat were excised and weighted.", "Epididymis was used for sperm count and morphology studies, whereas penile tissue was stored in 10% formalin solution for histopathology.", "Sperm count and analysis were performed by the methods reported earlier [20].", "Abnormal sperms (coiled sperms, tailless sperms, sperms with a bent neck, midpiece and tails) were observed using an eosin staining method.", "Two hundred sperms per animal were examined microscopically at 40x magnification and numbers of abnormal sperms were counted to calculate the percentage of abnormal sperms.", "Excised shafts of penile tissues were ﬁxed in a 10% formalin-salines olution for 24 h followed by washing in distilled water for half an hour.", "Then tissues were stored in a centrifuge tube containing 70% ethanol till processing.", "Parafﬁn sections of tissue (5 μm thickness) were obtained and H&E staining was performed.", "The pathologist was blinded to the grouping [21].", "Values are expressed as mean ± S.E.M. Statistical signiﬁcance with respect to diabetic control was evaluated using one-way ANOVA followed by Dunnet’s test using Graph Pad Prism 5 (Version 5.0, GraphPad Software Inc., San Diego, CA).", "The chronic hyperglycaemia in sexually active male rats significantly reduced MF, IF, and EL, and increased ML, IL and PEI compared to normal rats.", "Treatment with t-TUCB restored the sexual function in diabetic rats as evident by an increasing in MF, IF, and EL and a decrease in ML, IL and PEI compared to diabetic control rats in a dose dependent manner (Fig. 1).", "A significant increase in the level of glucose in the serum was observed in diabetic rats compared to normal rats.", "Treatment with t-TUCB decreased serum glucose level compared to diabetic rats in a dose dependent manner (Fig. 2).", "A decrease in the weight of testes, and penis were observed in the diabetic rats compared to normal healthy rats.", "Treatment with t-TUCB in diabetic male rats prevented a significant decrease in the weight of testes, and penis compared to diabetic rats (Table 1).", "A significant reduction in sperm count was observed in the diabetic rats with more abnormality in sperm morphology compared to normal rats.", "Treatment with t-TUCB prevented a decrease in the total sperm count and abnormality in sperm morphology compared to diabetic control rats in a dose dependant manner (Fig. 3).", "The level of testosterone in serum was decreased in diabetic rats compared to normal rats.", "Treatment with t-TUCB minimizes a decrease in the level of testosterone compared to diabetic control rats in a dose dependant manner (Table 1).", "Histopathology of penile tissue of normal rat revealed the dilated corpus cavernosum, lined by endothelium and surrounded by smooth muscle, fibro-elastic connective tissue, enclosed by tunica albuginea.", "The penile tissue of diabetic rat was distorted anatomically with collapsed cavernous spaces and a prominent reduction in the endothelium.", "The penile tissue of diabetic rat treated with t-TUCB 0.1 mg/kg had fewer distortion of smooth muscle and connective tissue.", "The penile tissue of diabetic rat treated with t-TUCB 0.3 mg/kg had almost normal cavernous spaces lined by endothelium with intact smooth muscle and fibroelastic connective tissue (Fig. 4).", "With the increasing prevalence of diabetes, a large number of males suffer from diabetic ED which has a severe impact on health and life [22].", "Diabetic endothelial dysfunction is the common denominator leading to vascular ED.", "Interestingly, cytochrome P450 (P450) generates EETs in the endothelial cells which are known to relax endothelial cells via activation of calcium-activated and voltage-gated potassium ion channel (BKCa) through a cAMP and PKA mediated mechanism.", "EETs stabilized by sEH inhibitors are known to alleviate endothelial dysfunction [23–26].", "Jin et al reported that P450 2B, 2C and 2 J are present in rat corpus cavernosum.", "They further demonstrated that P450 2C which generates EETs efficiently than other P450 s are localized in cavernosal endothelial cells and reported that 11, 12-EET is one of the main EETs generated in the corpus cavernosa.", "Administration of EETs antagonist reduced major pelvic ganglion stimulation-induced increase in the intracavernous pressure, a marker of penile erection [27].", "Recently, several researchers have reported that chronic therapy with t-TUCB significantly decrease the incidence of cardiovascular disease and protects endothelial function in diabetic animals [28,29].", "Therefore, we hypothesized that t-TUCB treatment may have a beneficial effect against STZ-induced diabetic ED.", "STZ-induced diabetes in animals is an excellent model to study diabetic ED.", "The chronic hyperglycaemia clearly affected sexual function in male rats.", "Mounting behaviour represents sexual provocation, while intromission indicates the efficiency of erection and penile orientation [30].", "Increase in ML and decrease in MF of the diabetic rats in our study indicates a decrease in sexual desire, while an increase in IL and decrease in IF indicates an erectile dysfunction.", "A significant increase in MF and IF, and a decrease in ML and IL in diabetic rats by t-TUCB suggests that it has preserved the sexual performances in diabetic rats.", "Ejaculatory latency and post-ejaculatory interval reveal duration of erection and time taken for regaining an erection after first ejaculation, respectively [31,32].", "A decrease in ejaculatory latency and increase in post-ejaculatory interval in diabetic rat confirms impairment of penile erection.", "A reduction in PEI by t-TUCB suggests that copulatory efficiency of diabetic male rats increased with the treatment.", "The treatment with t-TUCB in diabetic rat restored the sexual excitement and desire during the period of sexual interaction with active female rats.", "Inhibition of sEH preserves vasodilator EETs which has a therapeutic benefit in improving endothelial function in diabetes and cardiometabolic diseases [28,33].", "Extract of the plant Moringa oleifera, a natural inhibitor of sEH, is reported to relax penile tissue ex vivo and increase intracavernous pressure in vivo [11].", "Therefore, inhibition of sEH is, at least, responsible for the alleviation of sexual dysfunction in diabetic rats.", "A decrease in the level of testosterone in diabetic rat could be considered as one of the contributing factors responsible for an overall decrease of sexual performance, because testosterone regulates nearly every component of erectile function, from sexual desire to penile erection involving co-ordinated activity of pelvic ganglions, smooth muscle and endothelial cells of the corpora cavernosa [19].", "Castration is known to decrease the nitric oxide synthase immunoreactivity in medial preoptic nucleus and nucleus of stria terminalis, suggesting a role of testosterone in controlling production of NO [34].", "Our results are in support with previously published report which revealed that induction of diabetes by high doses of STZ in male leads to a reduction in testosterone level [35].", "A decrease in the function of both Leydig (testosterone producing cell) and sertoli (spermatogenesis) is associated with a reduction in the secretion of insulin [36,37].", "The treatment with t-TUCB prevented a decrease in the level of testosterone in diabetic rats which could be due to the anti-hyperglycemic effect of sEH inhibitor as reported by us earlier [38].", "This study demonstrated an importance of sEHI in regulating the level of testosterone in diabetes.", "Earlier, we had reported that the level of testosterone was decreased in the male mice lacking sEH [39].", "Similarly, the activity of sEH in the kidney and liver of the castrated male mice was decreased.", "As expected, administration of testosterone to castrated mice increased the activity of sEH in kidney and liver in comparison to sham mice.", "Interestingly, testosterone also affects the spermatogenesis [40].", "Further, diabetic rat exhibited low sperm count and more abnormality in sperm morphology.", "The mechanism for impaired spermatogenesis is through hyperglycaemia-induced oxidative stress in testis [41].", "The increased oxidative stress in diabetes mellitus favours peroxidation of polyunsaturated fatty acids (PUFA) in the sperm cell membrane.", "This increased lipid peroxidation causes DNA damage in sperm cell [42].", "Treatment with antioxidant is reported to increase live sperm percentage and decrease sperm morphological damage [43].", "The sEH inhibitor, t-TUCB was reported to possess anti-oxidant property [29].", "Therefore, t-TUCB might have decreased sperm damage my reducing oxidative stress in diabetes.", "Even sexual dysfunction in diabetes is associated with oxidative stress [44,45].", "and inflammation [46].", "A reduction in oxidative stress [47] and inflammation [48] is known to alleviate sexual dysfunction.", "We have earlier demonstrated the anti-inflammatory and anti-oxidant effect associated with the inhibition of the sEH [49,50,38].", "Therefore, t-TUCB might have alleviated the sexual dysfunction via reducing oxidative stress and inflammation.", "Further, the damage to sperm may also be due to the decrease in the level of androgen or hyperglycaemia induced degeneration of seminiferous epithelium and epididymal function [51,52].", "The abnormal sperm may be the cause for the decreased fertility in diabetic rats [53].", "Administration of t-TUCB signiﬁcantly increased the epididymal sperm count and decreased abnormal sperm percentage, which clearly indicates the spermatogenic efﬁcacy of the sEH inhibitor.", "Further diabetes also alters the normal architect of penile tissue.", "In diabetic rats, corpora cavernosa were collapsed and were lined by depleted endothelium with either the absence or significantly reduced smooth muscle compared to normal rats.", "Treatment with t-TUCB at 0.3 mg/kg in diabetic rats restored the normal anatomical architecture of penile tissue which supports the protective effect of sEH inhibitor in diabetic ED.", "Though we demonstrated that t-TUCB ameliorates diabetic sexual dysfunction, many questions remain unanswered.", "Though the sEH inhibitors are known to have pharmacological activity due to an increase the level of EETs [54], the sEH inhibitors also have direct effect [55].", "The direct effect of sEHI on corpus cavernosum could not be determined.", "An Ex vivo study demonstrating the effect of sEHI on isolated corpus cavernosum from normal and diabetic rat could have provided conclusive evidence.", "EET-mimetics have been helpful in delineating the mechanism via which EET or sEHI exhibit protective effect in various disease conditions [56,57].", "Use of EET mimetic would have provided conclusive data about the erectogenic potential of t-TUCB independent of its effect on the level of blood sugar and testosterone.", "Evaluating the effect of oxidative stress in testes and corpus cavernosa of study animals could have provided additional information about the role of oxidative stress in diabetic sexual dysfunction and effect of t-TUCB in this pathological condition.", "The present study demonstrates that the sEH inhibitor t-TUCB ameliorates diabetic sexual dysfunction by minimizing a decrease in the level of testosterone and preserving penile tissue architecture.", "In addition, the ability of t-TUCB to minimize a decrease in sperm count and reduce sperm defects in diabetic rats may also help in increasing the overall reproductive function of diabetics.", "This inhibitor may be used to alleviate male sexual dysfunction arising due to diabetes.", "The University Of California, Davis holds patents on soluble epoxide hydrolase inhibitors.", "Professor Bruce D. Hammock is a co-founder of Eicosis LLC, a company for promoting the use of sEH inhibitors in humans and companion animals.", "Other authors have no conflict to declare" ]	[ "Administration of streptozotocin induces sexual dysfunction with a parallel decrease in the level of testosterone in rats.", "Treatment with the soluble epoxide hydrolase inhibitor, t-TUCB, attenuates sexual dysfunction in diabetic rat.", "Protective effect of t-TUCB on sexual function of diabetic rat was associated with a preservation of level of testosterone.", "Treatment with t-TUCB can be a novel approach for the management of diabetic sexual dysfunction." ]
Sand dune restoration experiments at Bei-Men Coast, Taiwan	The Bei-men barrier beach in southwestern Taiwan is a coastal plain with rivers, tidal channels and lagoons. Together with dunes and associated water bodies, the beach and tidal flats provide valuable habitat for many fauna and flora. However, in the last 3 decades, due to both natural and anthropogenic activities, the barrier beach and sand dunes eroded rapidly. As a result, not only is the habitat progressively destroyed, but also the lives and properties of nearby residents are threatened. It is well known that, sand dunes on barrier beaches can serve as a natural buffer against high waves in stormy weathers, preventing or delaying the intrusion of water and sediment into lagoon areas. As compared with other hard engineering measures, dune nourishments and/or reconstructions are more preferred in a more demanding society. These are considered as the so-called ecological solutions. To guarantee a sustainable development for the Bei-men barrier beach, experiments on sand dune restoration and nourishment were carried out. The main objective of these experiments is to find the best possible way to build sand dunes that should be both economically, as well as ecologically, feasible. To achieve this, all the materials applied are required to be either locally available, and/or biodegradable. It is shown that, the bamboo fences are the most efficient setup to nourish sand dunes. The newly nourished dunes are then covered with dried giant Miscanthus (Miscanthus floridulus) which serves as mulches, with Littoral Spinegrass (Spinifex littoreus) planted atop for further stabilization. It is shown that the proposed methods are rather satisfactory, and can be valuable aids for the restoration of barrier beaches in Taiwan.	[ "ENVI" ]	[ "Barrier beach", "Ecological engineering methods", "Sand dune restoration", "Sand fence" ]	[ "2305", "2308", "2309" ]	[ "Taiwan has a total of 82 wetlands, and 34 of them, covering an area of 49 thousand hectares, are distributed in the coastal region.", "These coastal wetlands consist of barrier islands, lagoons, low-lying plains, or estuaries.", "On one hand, they act as buffers against high-energy typhoon waves, preventing or delaying intrusion of water to the inland area (Nicholls et al., 2007).", "On the other hand, they are natural habitats rich in biodiversity.", "However, in the past few decades, all these regions are found to be in different degrees of deterioration.", "Studies have shown that the coastal construction works carried out in these years are the main reason for this (Hsu et al., 2006, 2007).", "Harbor entrance jetties, and/or estuarine training walls, in adjacent areas, for example, could intercept littoral drift.", "Furthermore, progressive advancing of the bulkheads seawards have reduced the littoral zone.", "In the meantime, sediment supplies from the rivers decreased with the constructions of reservoirs and bank revetments.", "As a result, there is a deficiency in sedimentary budget in the coastal area.", "Wetlands are important both in ecology, as well as in coastal defense.", "Recognizing this, the Construction and Planning Agency of the Ministry of the Interior, hereafter referred to as CPAMI, reacted accordingly.", "A draft of “The Wetland Restoration and Enhancement Bill” was completed by Construction and Planning Agency, Ministry of the Interior (CPAMI) (2010).", "The draft underlines both the legal and the technological aspects of the restoration of the wetlands, in which, their ecological and physical functions are stressed.", "According to the draft, ecosystem-based restoration approaches for the protection of barrier beaches, sand dunes, and lagoons are to be encouraged.", "For the case where engineering measures are necessary, their impacts on the existing wetland environments are to be minimized.", "Constructions of the hard engineering structures are, therefore, in principle, not to be carried out.", "Sand dunes on a barrier beach which are parallel to the shore play vital roles in coastal stability, for they act as barriers against waves and tides.", "It is also a reservoir of sands that functions to nourish eroding beaches, and to feed the nearshore sand bars during storms, keeping thereby the barrier beach in a dynamic equilibrium (O’Connell, 2008).", "This is the reason why sand dunes are treated as one of the framework for safeguarding against coastal flooding in the “integrated coastal zone management (ICZM)” plans of many countries (Mohan et al., 2003; van Wesenbeeck et al., 2014).", "Thus, dunes form important links in the coastal sediment budget and need to be considered in the scope of coastal management and engineering measures (Arens and Wiersma, 1994; Saye et al., 2005).", "In Taiwan, where 3 to 4 typhoons may invade annually, sand dunes can be seriously eroded by high-energy waves.", "It would take years for these sand dunes to rebuild through natural processes.", "Since hard engineering methods are to be avoided, the only option left is then dune enhancement and reconstruction.", "Not only it is feasible and efficient, but also it is the least expensive measure against shoreline erosion (Gregorio et al., 2002).", "Over the years, a variety of measures have been used effectively to build up, and to stabilize dunes, or to prevent the erosion of dunes and beaches.", "Devices that have been effectively implemented to trap the aeolian sand are fences, nylon nets, and rugged materials (Cong, 1991; Neves et al., 2004; Hotta and Harika, 2010).", "In the ‘Guidelines of Barrier Beach Management in Massachusetts’, snow fences and Christmas trees were used to build sand dunes and to minimize erosion (Massachusetts Barrier Beach Task Force, 1994).", "Sand fences in various configurations and their respective efficiencies in dune formation have also been studied and discussed in detail (see for example, Savage and Woodhouse, 1968; Waterways Experiment Station, 1984; Gravens et al., 2001 Miller et al., 2002; Williams, 2007).", "Sand-trapping fences have been used in Europe for some 700 years (Nordstrom, 2008).", "However, the design and employment of sand fences are still an approach based on trial-and-error.", "This is because the sand accumulation efficiency of a sand fence depends on a number of factors; most of them are locality related.", "Besides the characteristics of the sand fence itself, local meteorological and topographical features of the site can also be important as well.", "It is also noted that, once the fences were damaged, the fragments of them, or the wires used for joining the slabs could pose a potential threat to the coastal wildlife such a migrating birds, fish, and marine mammals.", "Sand dunes formed through fencing are unstable and can become deteriorated rapidly.", "The most common practice is to stabilize them by vegetation (Khalil, 2008).", "However, the dunes close to the coastline are in a very harsh environment for the plants.", "Limited amounts of fresh water, constant salt spray, sand blasting or burial caused by the wind, and additional disturbance due to human activities, all these can be hostile to the vegetation.", "Experiences have shown that, the selection of plant species and adequate maintenance such as irrigation or fertilization are the key factors of successful transplant vegetation (Nordstrom, 2008; NSW Department of Land and Water Conservation, 2001).", "Coarse netting and mats are useful auxiliaries in protecting dunes while the transplanted dune grasses are sowed (Dahl and Fall, 1975; Qu et al., 2013).", "In this way, not only can the dunes be stabilized by vegetation, they can also be attractive habitats for some shore-dwelling animals.", "In this paper, we describe our attempts to build up and stabilize sand dunes to turn the barrier beach of Bei-men more resistant against erosion.", "We will give some details of the ecological engineering setups, together with the results of their efficiencies.", "It is hoped that in this way, impact of the hard engineering can be avoided, in accordance with the spirit of sustainable use and management of wetland areas.", "Bei-men barrier beach is situated at the coast of Tainan City, a city in the southwestern of Taiwan.", "In early years, sediments carried by the Zeng-Wen River system were deposited at the river mouth.", "Gradually, barrier islands and lagoons along the coast were formed.", "Located on the route for water birds migration in Asia, tens of thousands of migratory birds fly southwards passing this area; some even stay there for the winter.", "According to the web page of the Taijiang National Park (2013), almost 200 species of birds have been sighted in this area.", "Among them, 21, including the endangered black-faced spoonbill (Platalea minor), are on the IUCN Red List.", "Not only the land, but also the marine ecological resources, as well as the variety of the landscape resulted in the unique dynamic barrier beach ecosystem.", "Due to its richness in biodiversity, this area has been declared as Coastal Nature Reserve by the CPAMI.", "However, similar as many places of the world, the barrier system suffers from erosion due to the natural, as well as the anthropogenic effects.", "In Fig. 1, we show the coastline composed of digitized results of satellite images, aerial photographs, and bathymetric surveys.", "The trend of recession of the barrier island over the last three decades can be clearly seen.", "Factors leading to the evolution of the Bei-men coastline have been proposed by many researchers (see for example, CECI Engineering Consultants Inc. (2006); National Cheng Kung University Research and Development Foundation (NCKU RDF) (2011); National Cheng Kung University Hydraulic and Ocean Engineering Research and Development Foundation (NCKU HOERDF) (2012); Sinotech Engineering Services (2013)).", "The main causes for the erosion of the barrier beach are now summarized as follows.", "High waves from typhoon are by far the most devastating factor for the recession.", "Normally, the sea state here is slight in summer seasons with significant wave height and period in the range of 0.93 m and 4.84 s, respectively.", "The tidal range is 1.13 m.", "This changes, however, when typhoon approaches.", "Waves up to 10 m have been measured occasionally.", "Large waves combined with storm surges, move the sand from the seaward side of the barrier island to the backside.", "Afterwards, the seaside is eroded, while the backside is accreted.", "In a sense, the barrier island rolls over toward the mainland.", "When this happens, even the most intensive coastal protection will not be able to keep these islands in place.", "In addition, human-related activities also tend to speed up the trend of erosion in this region.", "The exploitation of the salt evaporation ponds by the Taiyen Company in earlier years is believed to be the main cause of diminishing littoral sediments.", "Constructions of harbor entrance jetties and estuarine training walls in the 70’s and 80’s have also changed the balance of the littoral drift in this region.", "This so-called “groin effect”, i.e., the structures perpendicular to the coastline tend to cut off the longshore sediment transport, was not considered during the planning stage of the constructions (Anfuso et al., 2013).", "In this region, the dominant direction of the littoral transport is from NNE to SSW, and is governed by the monsoon wind waves in the winter season.", "It can be seen in Fig. 1 that sand was accumulating at the northern sides of the structures, whereas on the southern sides erosion has occurred.", "An estimate has shown that, since the construction of the Zeng-Wen Reservoir in 1973, some 256 million m3 of sediments have been retained in the reservoir basin.", "Among these, at least a part of them should be the source of the estuarine sediment.", "In other words, the sediment source for the barrier island has been largely reduced.", "Fig. 1 shows that, approximately 33% of the barrier island was lost in the last 3 decades.", "Alarmed by the severity of the situation of the Bei-men barrier island, the authority has decided to take action.", "Since sand dunes are accepted as coastline protection on a landscape scale world-wide (Borsje et al., 2011), and hard engineering measures are out of the question, it is decided to nourish the sand dunes in environmental-friendly ways.", "The problem now becomes deciding the optimal indigenous materials and species that can be used for sand fences and plant vegetation.", "In the following, we will give some detailed descriptions of the procedures taken, as well as the obtained results.", "Generally speaking, there are two monsoon systems in East Asia, i.e., the East Asian winter monsoon (EAWM) in winter, and East Asian summer monsoon (EASM) in summer.", "The most prominent surface feature of the EAWM is the strong northeasterlies along the east flank of the Siberian high pressure system (SH, also called Mongolian High) and the coasts of East Asia (Chen et al., 2000; Wu and Wang, 2002).", "On the other hand, the EASM is a subtropical monsoon in which the low-level winds reverse primarily from winter northerlies to southerlies in the summer season (Ding and Chan, 2005).", "In Taiwan, the dominant direction of wind comes from southwest in the summer season.", "To increase the chance of a successful sand fencing, it is of vital importance to have some knowledge of the characteristics of the wind on site.", "A year before the actual field observations were carried out, the wind speed, direction and the aeolian sediment transport rates were measured in summer and winter days.", "Measurements were carried out for 20 days both in July and November 2011.", "The results are shown as wind roses in Fig. 2.", "From Fig. 2, it is seen that average wind speeds at a height of 75 cm above the ground are 2.12 m/s in July and 7.26 m/s in November, respectively.", "It is also clear that the dominant wind direction in winter is from the north.", "At the same time, aeolian sediment transport rates were also measured by a sand trapper erected at the measuring site.", "Five heights, 12, 24, 49, 75, and 110 cm, were selected to determine the rates.", "The results are tabulated in Table 1.", "As can be seen in Table 1, the majority of the accumulated aeolian sand is less than 1 m.", "We estimated that the net transport rates in summer and winter are 0.09 and 0.42 g/cm-s, with prevailing directions of 146.0° and 171.7°, respectively.", "The distributions of the grain size of the aeolian transport at each height are summarized in Table 2.", "The ratio of sand to silt decreased rapidly at the height of 49 cm above the ground.", "It is clear that the aeolian sand transport rate in winter is roughly 4.7 times more than that in summer.", "In addition, as mentioned earlier, summer is the typhoon-rich season in Taiwan.", "Taking these into consideration, it was therefore decided that: (1) experiments on dune restoration are to be conducted in the following winter, (2) sand fences should not exceed 1 m in height, and (3) the general direction of the fences is ENE-WSW, to ensure a greater chance of success of the experiment.", "In the Section 2, we will give some details of our experiments.", "The benefits of sand dunes to a coastal region are fourfold (Virginia Marine Resource Commission, 1993).", "They are, flood and erosion protection, sand replenishment, providing habitat for coastal fauna, and increasing the scenic value of the coast (Anfuso et al., 2014).", "To minimize further erosion caused to the barrier beach by waves and surges, the authorities decided to build stable foredunes in long strips.", "It has also been decided to do this in the most economic and ecological possible way.", "Thus, our experiments consist of three parts: sand dunes building, their stabilization, and vegetation transplanting.", "The experimental sites are marked in Fig. 1.", "Different setups with various materials were used to assess their efficiencies in sand dune restoration.", "All the experiments were conducted in the winter monsoon season.", "It should be mentioned that, to shorten the time needed, we have somewhat modified the sequence of our experiment.", "In the usual way, dune stabilization follows sand dune building.", "However, as the time limit of the contract was 1 year, we decided to carry out all the three experiments at the same time and used bull-dozed dunes for the stabilization experiments.", "The primary objective of this experiment was to determine the most suitable materials to be used for sand fencing.", "It is believed that, being both natural and locally available, the impacts on the environment should be kept minimal.", "The experiments were carried out on the northern Bei-men beach, which is relatively flat with a 1/100 slope and is 150–200 m in width.", "3 layouts were used to assess their capability in building up sand dunes.", "These are (1) bundles of dried giant Miscanthus bound to bamboo piles, (2) pile-ups of marine debris such as driftwoods and bamboo racks, and (3) sand fences constructed with bamboos.", "In addition, an empty field with no additional ‘add-ups’ was marked for comparison.", "All test fields were of 12 m × 24 m, with the longer sides facing the dominant wind direction of NNW-SSE.", "It is stressed that, all the materials used in this experiment were natural and/or biodegradable so that there would be no environmental or health concerns.", "Bundles of dried giant Miscanthus on bamboo pole", "Giant Miscanthus is treated as weeds, and can be seen in small groups on water edges all around Taiwan.", "In the experiment, dried giant Miscanthus was tied with hemp rope to a bamboo post at heights of 30, 60, and 90 cm above the ground.", "Three spacing, 2, 3, and 4 m, of the bamboo posts were used for comparison.", "Once a dune is formed, the bamboo posts will be pulled out, and the giant Miscanthus is left to decompose and serve as an organic fertilizer for the transplant vegetation.", "A sketch and the arrangement of this setup can be seen in Fig. 3(a).", "Mounds of marine debris", "At times, marine debris such as driftwoods or broken bamboo racks used for oyster breeding can accumulate on the coastlines of Taiwan after floods linked to typhoons.", "The contaminated zone can be too large that areas are cleaned according to their priority.", "It seems to be a win–win solution if they can be reused for sand dune building.", "The debris was therefore gathered and stacked to have a dimension of, roughly, 12 × 24 × 1 (W × L × H, in meters).", "It is hoped that, with the rugged surface and the abrupt change of the topography, aeolian sands will be trapped more easily.", "With time, sands will fill up the gaps of the debris and, eventually bury them entirely, with the result of a coast having a more or less natural appearance.", "Sand fences are one of the most common man made devices to affect the morphology and vegetation on sandy coasts.", "They are one of the few structures permitted in coastal conservation areas in many countries (Nordstrom, 2000).", "In the meantime, they are inexpensive and easy to emplace.", "In the 2010–2011 period, two attempts of sand fencing in this region have failed.", "Among other possible reasons, we believed that one of them might be that the fences have too large porosity.", "As reported in Lin and Liou (2013), the one with a 75% porosity had trapped only marginal sands, while the other, of 66%, worked better.", "It is also possible that the period of their experiments were too short, as shown by Hotta et al. (1987), fences with any porosity will be buried after a sufficiently long time.", "To prevent the bush at the edge of a shelterbelt to be buried by sands, fences were used by the Forestry Bureau in Taiwan.", "The porosity of the fences used by them is often so small that all the sands were settled in front of the fences, and asymmetrical dunes were formed.", "As a result, the fences collapsed under the weight of the sand dunes.", "Once the fences collapse, the tourists might be injured by the remains.", "Drawing on wisdom from the past, we have decided that our fences should meet two prerequisites.", "In the first place, all the materials should be absorbed easily by the surroundings.", "Secondly, the fences should have adequate porosity so that the sands will be distributed more or less evenly within the fences.", "In this field test, the whole fence was made of bamboo.", "All parts of the bamboo tree were used.", "Lattices of size 1 × 1 m with large openings were made using bamboo splints at first; the openings were then filled with bamboo stems with foliages.", "These lattices were then fixed to bamboo posts to work as bamboo fences.", "Once a dune has reached its designated height the posts are to be removed.", "The remaining bamboo stems and foliages are left to wither, and to be absorbed by the environment.", "The main idea is to use the bamboo foliages to fill up the lattice openings, so that the optimal fence porosity of 50% (Gravens et al., 2001) could be achieved.", "It is estimated that, about 1.5 kg/m of bamboo stems and foliages are to be used to have 50% porosity.", "The setup is shown in Fig. 3(b).", "The distances between each row of the fences are also of importance.", "There is no definite specification in the literature about this distance.", "Usually, a distance that is 4 times the fence height is mentioned (Gravens et al., 2014).", "In this way, a faster sand trapping result at high wind speeds could be reached.", "Since there are many other factors affecting sand accumulation rate (Nordstrom, 2000), we have chosen two fields with spacing of 3 and 4 m for comparison.", "To minimize encroachment of the build-up sand dunes by wind, 3 different mulches were tested.", "The bulldozed dunes for the experiments all have the form of flattop pyramid, with a base of 10 × 10 m and a height of 1 m.", "The slopes of the 4 sides of each pyramid are approximately 1:2.5.", "The experimental site is at the southern side of the Bei-men beach.", "The 3 sand dunes in alignment are designated as sand dune A, B. and C in the following.", "The flat top of each sand dune is further divided into two parts.", "Whereas, the eastern parts without coverage are used as control, the western parts are protected by mulches.", "Materials used for the 3 mulches of sand dunes A, B, and C are, respectively, oyster shells, drift woods, and dried giant Miscanthus.", "Vegetation can enhance the stability of sand dunes, and, in the meantime, increase the surface roughness so that more aeolian sand will be trapped.", "Quite often, however, sand dunes have sparse vegetation due to the harsh environmental conditions they are subjected to.", "Transplantation can help to improve the situation, as many researchers have shown.", "Dahl et al. (1975), for example, have found that the man-built dunes with vegetative transplants have higher rates of success than those without.", "2 indigenous plants, seashore vine morning glory (Ipomoea pes-caprae) and Littoral Spinegrass, were used in our experiment.", "Generally speaking, the success of planting depends on the season.", "Spring and summer are the main growing seasons of the vegetation.", "However, since the experiments were started in fall, the chosen plants were first grown in a nursery garden and then transplanted onto the mulches.", "For comparison, the two plants were arranged in alternative rows of 1 m2.", "There were 50 plants on each of the paved side of the 3 dunes.", "Changes of the nourished sand dunes were surveyed by a GPS model used in real time kinematic (RTK) modalities.", "12 topographical surveys were carried out for this purpose.", "Fig. 4 shows the changes from November 13, 2011 to February 06, 2012.", "The results of each setup are summarized in the following:", "Dried giant Miscanthus bundles on bamboo posts-1", "Sand trapping ability of this setup is rather poor, irrespective of the spacings of the bamboo posts.", "Although there are sands accumulated at the windward sides of the posts, the amount is too small to call them crescentic dunes.", "At the leeward side, strips of sands of about 1 m in length were also formed.", "None of the accumulated sands have heights exceeding 20 cm.", "Since sands in areas with no fencings were clearly seen to be lost through wind action, we consider this setup can work as a “wind erosion prevention” measure.", "Mounds of marine debris-1", "As expected, pile-ups of the marine debris do help in retaining the sands.", "Soon after the pile-ups were completed, they began to trap sands at the windward side and the spacings were filled gradually.", "Finally, the whole pile-up was covered by sands, save some large-sized debris that protrudes from the dune.", "However, at the end of second month soon after the sands have reached the height of the debris, they stopped to accumulate.", "Afterwards, the amount of arrested sands decreased in a continuous way.", "Once the sands have filled the voids of the debris, no more sands can be captured.", "At the same time erosion sets in, leading, eventually, the tips of the larger debris to appear again.", "Sand fences have a long successful history.", "The main objective of our experiments is to determine the specific arrangements required for the climate on site.", "As mentioned before, 2 kinds of arrays of the fences were tested.", "The one is with arrays that are 3 m apart from each other, and the other is 4 m.", "It was found that for the 3 m case, after a period of a month and half, sands have accumulated up to a height of 1 m on the windward sides of the first three rows of the fences.", "For the rest rows the heights are between 50–60 cm.", "3 months after fencing, build-up sand dune began to migrate downwind, so that the tips of fences in the first two rows gradually appeared again.", "Meanwhile, the sand accumulations have the heights of 80–100 cm for the rest rows.", "On the other hand, nourished sand dunes by fences with 4 m intervals have a much flatter top.", "The height is also much lower, of round 40–50 cm.", "It is estimated that the fences with 3 m intervals can arrest about 1.89 times more sands than those with 4 m intervals within the experimental period.", "As mentioned earlier, in majority of the literature the distance between the fence rows is 4 times the fence height.", "However, our experiments here show that a 3 m distance yields much better results.", "This demonstrates once again that the efficiency of sand fencing is locality dependent.", "Experiments on stabilization of man-made sand dune were carried out from October 23, 2012 to February 8, 2013.", "Fig. 5(a) shows the 3 setups at the beginning, and Fig. 5(b) their status at the end of the experiments.", "Changes of the sand dunes were measured with rulers once a week.", "Each sand dune has 13 monitoring points, including 4 of the corners and 9 on the surface of the mounds.", "This is shown schematically in Fig. 6(a).", "Changes of sand dune heights at each monitoring point with time are shown in Fig. 6(b).", "The most severe erosions are at the eastern upper corners (AC3, BC3, and CC3), where wind actions are the strongest.", "It can be seen in Fig. 6(b) that, sand dunes have decreased 80 cm in heights.", "It should be noted that, although the general trend is erosion, the sand carried by wind from adjacent dunes may land here, so that the net loss due to wind erosion is unknown.", "We believe that, this is the reason why the curves in Fig. 6(b) are fluctuating, showing alternating accretion and erosion has occurred.", "At the end of our experiment, sand of the 3 control areas have avalanches down the slop, and all the uncovered parts of the dunes were destroyed.", "Pavement of oyster shells on sand dune A was intended to reduce erosion.", "However, on the western upper corner, which was on the windward side, the height of the dune has reduced 30 cm.", "This is because that, the voids left by the oyster shells are too large so that there is still ample room for winds to blow the sand away.", "The middle and lower parts of the dune have also lost some small amounts of sand.", "A reduction of original thickness of 10 cm was found.", "Sand dune B has a mulch made of driftwoods, and the spacing between these driftwoods are too large so that the dune changes its form continuously.", "It was found that, within a month the windward sides (BC1 and B1) have decreased 40 cm on thickness, while the middle part of the dune has gained 30 cm.", "This means that, part of the eroded sands were transported and settled downwind, making the dune to migrate slowly southwards.", "Dried giant Miscanthus not only keeps sand dune C intact, but has also trapped and added more sands to it.", "This is true even after the strongest winter monsoon winds experienced during the experiment.", "Comparing the results, it is concluded that the setup C performs the best in stabilizing and nourishing sand dune, followed by setup B, and setup A is the least effective one.", "In this paper, we use the term ‘survival rate’ to evaluate the success/failure of the vegetation.", "Here, the survival rate is defined as the number of living plants to the total number of transplanted plants.", "Although watering and/or fertilizing can increase its chance of survival, no such action was taken for the vegetation.", "First of all, fresh water is nonexistent here and must be shipped from land, it is therefore considered as too cost-intensive.", "Secondly, fertilizer may easily leach through porous sand, pollute groundwater, and affect the quality of our water.", "Although the 3 man-made sand dunes were subjected to the same climate condition, it can be seen in Fig. 7 that the same species planted on them have different survival rates.", "For each dune, the Littoral Spinegrass always has a higher survival rate than the seashore vine morning glory.", "The Littoral Spinegrass planted on dried giant Miscanthus (sand dune C) has the highest survival rate of 42%, whereas that of the seashore vine glory is only 18%.", "In whole, plants on sand dune C have the highest survival rate, followed by sand dune B, and sand dune A has the lowest.", "It was also found that vegetation on the windward sides did not survive the test.", "All of them withered eventually.", "Those on the lee sides have a better chance to survive.", "We conclude that, using dried giant Miscanthus as mulch has the advantage of becoming saturated in the humid sea air overnight, providing ample water needed by the plants during the day.", "The 3 basic ways to measure vegetation cover are points, lines, and plots.", "In this experiment, we used the line method to measure vegetation covers of the dunes.", "Coverage rate is defined here as the length covered with plants to the distance from the first to the last plant on the surface of dunes.", "Changes of coverage rates of the planned plants with time on each sand dune are shown in Fig. 8.", "With interchanging accretion and erosion, sand dune A was itself unstable and most of the plants on it withered at the end.", "The coverage rates of sand dune B and C decreased in the first 3 weeks.", "Afterward, the trend reversed and transplant vegetation started to grow.", "On all the dunes, Littoral Spinegrass always have faster growing rates than those of seashore vine morning glory.", "Sand dune C has the highest vegetation cover, followed by sand dune B, and then A, in accordance with the stabilities of the dunes.", "Table 3 summarizes the results of the experiments.", "2 years after the project was completed, we revisited the site.", "Fig. 9 shows the present status of the 3 stabilized dunes.", "Sand dune A with all its transplant vegetations disappeared.", "On the paved side of sand dune B, only one-third of the planted Littoral Spinegrass has survived, and no seashore vine morning glory can be found.", "Sand dune C has merged into the surrounding nature scene, but its original form can still be vaguely recognized.", "The majority of the plants on sand dune C is Littoral Spinegrass, with little seashore vine morning glory scattered around.", "Convinced by the success of the experiments, the Forestry Bureau has used our method in this region since 2013.", "Fig. 9(d) shows one of the restored sand dunes and the restoration is still ongoing.", "In the past few decades, the Bei-men barrier island is found to retreat rapidly.", "The authorities decided to find the most ecologically sound, cost efficient way to prevent its further lost.", "Since only soft-engineering measures are allowed, we focus our attention on the restoration of sand dunes.", "To determine which locally available materials can be used, a series of experiments were carried out.", "Based upon these results, it is therefore concluded that, to restore and nourish sand dunes on the barrier island of Bei-men coast, the best way would be:", "Accumulate aeolian sands using fences made of bamboos, with a spacing of 3 m and 1 m in height.", "It would help in retaining the sand deposits and formed sand dunes.", "However, our results showed that the efficiency of the sand dune nourishments is determined by the local factors.", "It is therefore suggested that field experiments should be conducted first before the emplacement of fences along large areas.", "Once the sand dune reaches the target height, pull out the bamboo stems and leave the rest materials to degenerate and absorb by the environment.", "Use dried giant Miscanthus as mulch for the new-grown sand dunes, and plant Littoral Spinegrass on the dunes.", "The paved and transplanted plants can help to stabilize the dunes, making them more resistant to marine and wind forces.", "In the meantime, it helps to prevent the barrier island from moving landwards.", "It is also worth mentioning that the use of dried giant Miscanthus as a mulch on sand dune has the advantages of (1) preventing the sand dune from wind erosion, (2) keeping it stable, and (3) providing better surroundings for vegetation growth.", "We believe that this measure can be a valuable solution for some coast regions where irrigation facility for the transplant vegetation does not exist." ]	[ "Accumulate aeolian sands using fences made of bamboos.", "Once the sand dune reaches the target height, pull out the bamboo stems and leave the rest of the materials to degenerate and absorb by the environment.", "Use dried giant Miscanthus as mulch for the new-grown sand dunes, and plant Littoral Spinegrass on the dunes.", "The paved and transplanted plants can help to stabilize the dunes." ]
The self–other knowledge asymmetry in cognitive intelligence, emotional intelligence, and creativity	The self–other knowledge asymmetry model (SOKA) assumes that some personality traits might be open to oneself and other persons (‘open area’), while other traits are more accurately perceived by others (‘blind spot’); a third group of traits might be visible only to oneself and not to others (‘hidden area’), and finally a trait might neither be visible to oneself nor to one's peers (‘unknown area’). So far, this model has been tested only for personality traits and general intelligence, not for more specific abilities; to do so was the novel intention of our study. We tested which of six abilities (verbal, numerical, and spatial intelligence; interpersonal and intrapersonal competence; and creative potential/divergent thinking ability) are in which SOKA area. We administered performance tests for the six abilities in two samples – 233 14-year-olds and 215 18-year-olds – and collected self- and peer-ratings for each domain. Numerical intelligence and creativity were judged validly both from self- and peer-perspectives (‘open area’). In the younger sample verbal intelligence was validly estimated only by peers (‘blind spot’), whereas the older group showed some insight into their own abilities as well (‘blind spot’ to ‘open area’). While in the younger group only the pupils themselves could validly estimate their intra- and interpersonal competence (‘hidden area’), in the older group peers were also successful in estimating other's interpersonal competence, albeit only with low accuracy (‘hidden area’ to ‘open area’). For 18-year-olds, spatial ability was in the hidden area too, but in 14-year-olds this could neither be validly estimated by pupils themselves nor by peers (‘unknown area’). These results implicate the possibility of non-optimal career choices of young people, and could, therefore, be helpful in guiding professional career counselling.	[ "MULT" ]	[ "Psychology" ]	[ "1000" ]	[ "“Why do others sometimes know things about us that we don't know about ourselves?”", "(Vazire, 2010, p. 281).", "This tricky question was asked by Simine Vazire, the author of the so-called self–other knowledge asymmetry model (SOKA), in which she described the phenomenon that many people have surprisingly insufficient insight into their own personality.", "This violates the common-sense conviction postulated by Augustin and Descartes that nobody knows yourself better than you do.", "Although self-ratings of personality can be a valid source when predicting professional and other life outcomes, and in spite of the fact that self-perceptions play a very important role in current conceptions of personality (Roberts et al., 2007), it has also repeatedly been shown that people have limited insight into their own traits (Beer and Vazire, 2017; Vazire and Mehl, 2008).", "Much research has shown compelling evidence for the validity of informant reports, such as peers or even unknown persons, called zero-acquaintance judgements (Borkenau and Liebler, 1992).", "That there does not have to be an overlap between self-judgements of personality and informant judgements has been demonstrated in studies (Carlson, 2013; Helzer and Dunning, 2012; Vazire and Carlson, 2011) inspired by Vazire's (2010) proposition of the SOKA model.", "She started her research on the basis of Luft and Ingham's Johari window (1955), which contains four quadrants (see Fig. 1): (a) aspects of personality known to both the self and others (open area); (b) aspects not known to the self but known to others (blind area); (c) aspects known to the self but not to others (hidden area); and (d) aspects unknown to both self and others (unknown).", "As pointed out by Vazire (2010), the problem of people's limited insight into their personality traits not only has important theoretical implications, but also practical ones in several fields, including clinical psychology as well as in work and organizational psychology.", "So far, the SOKA model has been studied mostly in the field of classical personality traits like the Big Five, as well as in two very general abilities (general intelligence and creativity).", "In several studies (e.g., Vazire, 2010; see also Beer and Vazire, 2017; Bollich et al., 2011) these authors had participants providing self-ratings and informant ratings from friends and from strangers in a round-robin design.", "Through this approach, Vazire (2010) showed that in the open area (a) there are the observable, nonevaluative traits like extraversion; in the blind spot (b) there are evaluative traits like intelligence, and in the hidden area (c) there are internal (unobservable) non-evaluative traits like neuroticism.", "Since then, surprisingly little research has taken up the general idea; the few studies on the SOKA model deal solely with clinical fields (Carlson et al., 2013) or social relations (Uziel, 2012).", "What is completely unknown is: Where in the SOKA model are specific cognitive abilities like verbal, numerical and spatial intelligence, or other abilities like creativity and emotional competencies located?", "So far, no study has simultaneously assessed a broad variety of well-known abilities/performance traits and at the same time obtained self- as well as informant-ratings of the very same abilities.", "Therefore, we want to analyze the location of six defined abilities in the SOKA model: verbal, numerical and spatial intelligence (which are part of most current intelligence models; Hunt, 2010), creativity (or more exactly the creative potential as measured by divergent thinking tasks), as well as two central factors of emotional intelligence, namely intra- and interpersonal intelligence/competence (Freudenthaler and Neubauer, 2005).", "This research question is not only relevant from a theoretical point of view but also has important practical implications regarding career counselling, especially for young people: Are they (or their peers, parents, etc.) aware of their abilities and talents, so that they can base decisions regarding their vocational choices not only on their interests but also on their abilities?", "This is particularly important, considering that abilities and interests mostly display surprisingly low relationships, as shown in the meta-analysis by Pässler et al. (2015).", "The large importance of abilities for later professional success has been documented in several meta-analyses (Schmidt and Hunter, 2004, 1998; Strenze, 2007), which mostly showed correlations around .5 for cognitive intelligence tests.", "For the big five, however, meta-analytically smaller correlations with job success have been reported, mostly between .1 and .2, maximally up to .3 (Barrick et al., 2001).", "For emotional abilities, however, evidence on the importance for job success is much less clear.", "Two recent meta-analyses (Andrei et al., 2016; Joseph, Jin, Newman, & O'boyle, 2015) demonstrated that also ‘emotional intelligence’ (EI) is relevant for job success, and although the relationships are small, EI shows an incremental contribution over and above cognitive intelligence.", "For creativity, however, no meta-analysis on relationship with job success is available.", "What do we know about the validity of self- versus informant ratings of abilities?", "Except for Vazire's (2010; Vazire and Mehl, 2008) studies, so far no one has obtained performance measures, self- and informant ratings in the same study.", "To derive hypotheses, we thus needed to look into studies of (a) self-estimates of abilities and (b) informant estimates of abilities.", "A recent metasynthesis by Zell and Krizan (2014) has shown that self-estimates of quite diverse abilities are of low to medium validity; the mean correlation being only .29, with a considerably higher average correlation only for verbal ability (r = .63), whereas for some abilities it was considerably lower than .29 – in particular, for nonverbal skills it was only .09.", "Another meta-analysis by Freund and Kasten (2012), which focused on abilities covered in intelligence tests, however, reported valid self-estimates only for numerical but not verbal and spatial abilities.", "This discrepancy might have arisen due to different conceptualizations of verbal abilities: In their meta-analysis, Freund and Kasten referred to verbal intelligence (as measured in intelligence tests), whereas the meta-synthesis by Zell and Krizan referred to foreign language ability.", "With regard to informant estimates of abilities, two kinds of studies can be found: First, some studies analyzed the validity of zero-acquaintance reports for general intelligence, which is mostly around .3 (Borkenau and Liebler, 1993; Borkenau et al., 2004).", "Studies on peer reports of intelligence have been summarized by Denissen et al. (2011), who concluded that they “were reliable, stable and weakly correlated with objective intelligence” (p.108).", "The found intelligencepeer correlations that varied from .22 to .27.", "A further restriction is that pertinent research has been conducted only in adults, not in children or adolescents.", "However, from the perspective of career counseling we argue that the validity of self- and informant estimates of abilities is particularly important for young people facing important career decisions.", "In choosing a career path, young people might either rely on the self-perception of their abilities, or seek advice from friends, parents, teachers, and so on.", "But do self-estimates of abilities and/or informant estimates of abilities give a sufficiently valid picture of the real (psychometrically determined) ability?", "And for which abilities should (young) people rely on their self-perception or on the advice of informants?", "In order to answer these questions, we want to analyze validity of self- versus informant ratings of select abilities at two age points, where important career decisions are made.", "These are at 13/14 and at 18 years of age.", "In many Western countries adolescents around 13–14 years of age decide whether to choose a further career at (higher secondary) school leading eventually to academic studies at university, or whether to go directly into professional education (such as apprenticeships in Germany, Austria, other countries; see Dumfart et al., 2016).", "The second timepoint for career decisions is at 17–18 years, when (senior) high school students decide about a career path at college or university.", "Therefore, we want to analyze the validity of self- and informant reports of select abilities at 14 compared to 18 years of age.", "The following hypotheses are being tested:", "Verbal and interpersonal ability should be located in the open area (I, see Fig. 1), that is, self- and informant reports should both be valid sources (Furnham et al., 2005; Vazire, 2010).", "We based our prediction regarding verbal ability on the meta-synthesis by Zell and Krizan (2014), instead of the meta-analysis by Freund and Kasten (2012), given the larger scale of the former.", "Our prediction for interpersonal abilities arose from the finding that they usually correlate at medium-effect size with extraversion (Freudenthaler et al., 2008), which was in the open area in Vazire (2010).", "For numerical ability, Freund and Kasten (2012) found valid self-estimates, whereas Zell and Krizan (2014) did not analyze this ability separately.", "With regard to informant ratings, no study was found that analyzed the validity of numerical ability.", "However, because of the high visibility of numerical ability in school contexts, we predict numerical ability to be also in the open area (I).", "Spatial abilities have been shown not to be correlated with self-estimates (Freund and Kasten, 2012), therefore it could be in the blind area (II) where informant ratings of performance might be more valid.", "The same prediction is made for creativity (which was in the blind area in Vazire, 2010).", "Finally, we expect intrapersonal competence to be located in the hidden area (III) of the SOKA window.", "Vazire (2010) placed neuroticism in the hidden area and in a former study we showed that intrapersonal competence is substantially correlated (negatively) with neuroticism (Freudenthaler and Neubauer, 2005).", "Finally, we ran a set of more exploratory multiple regressions to see how good the six abilities can be predicted from self- and peer-perspective in combination.", "The aim was on one hand to see whether the self- and other perspective can complement each other and for which abilities they can do so.", "On the other hand, in case both perspectives could contribute, we wanted to see the total prediction (multiple R2) and compare that between the six abilities.", "We recruited two convenience samples of adolescents in lower secondary school (LSS; grade 8, around 13/14 years, n = 237) and higher secondary school (HSS; grade 12, around 17/18 years, n = 225).", "However, since we wanted to compare correlations between measures, we excluded all participants with missing values on any of our key variables (performance in ability tests, self-estimates, and peer estimates).", "This left 448 participants (55.8 % female), with 233 of them in LSS (Mage = 14.07, SDage = 0.60, 53.2 % female) and 215 of them in HSS (Mage = 18.00, SDage = 0.70, n = 215, 58.6 % female).", "The studies were approved by the Ethic Commission of the University of Graz and the School council of the Austrian province of Styria.", "According to Austrian law, this fulfills the requirements for consent as long as tested pupils have the right to refuse – which they had in the studies conducted.", "We aimed at recruiting a diverse and representative sample, therefore inviting pupils from both public and from private schools that were located in rural areas and cities across two Austrian provinces (Styria and Upper Austria).", "A total number of thirteen schools participated.", "Descriptive statistics and reliabilities of all measures can be found in Table 1.", "Verbal, numerical and spatial abilities were measured by means of three subtests from Intelligenz-Struktur-Analyse (ISA; Fay et al., 2001).", "The subtests were for verbal ability ‘Gemeinsamkeiten finden’ (commonalities), numerical ability ‘Zahlenreihen’ (number series) and spatial ability ‘Figurenauswahl’ (figure completion), and these are well-established, reliable, and valid measures of these abilities for use in German language.", "Creativity cannot be assessed directly, particularly not in these young people.", "Therefore, we measured the creative potential through divergent thinking tasks.", "We used three alternative uses tasks, namely umbrella, plastic bottle, and shoe, each given with a time limit of 2 min.; these tests have proven reliable and valid in a former study (Jauk et al., 2013).", "In these tasks, the students had to name as many original uses as they can for the given object within the time limit.", "Each task was analyzed for fluency (number of answers) and originality, the latter being judged independently by three raters (authors A.P. and A.W. and a student; see Table 1 for interrater-agreement measure κ).", "Since the results for both measures showed similar tendencies but those for fluency were less clear-cut, we only report the originality measure here (see also Diedrich et al., 2018).", "Fluency and originality were correlated at .53 in LSS and at .60 (both p <.", "001) in HSS.", "Emotional abilities were measured by means of a previously developed test for emotional management ability (EMA, inter- and intrapersonal) for which reliability (αs > .7) and validity has been demonstrated (Freudenthaler et al., 2006; see also Freudenthaler and Neubauer, 2005; Freudenthaler et al., 2008).", "This is a 28-item situational judgement test (SJT) with 14 items each for intrapersonal and interpersonal emotional management ability.", "Items are of the following type: A short description of an emotionally laden situation is given and the testee has to choose from four response alternatives, which are scored between one and four where ‘4’ = maximally efficient handling of the situation and ‘1’ = least efficient handling of the respective situation.", "In the present study alphas were rather low (see Table 1), which is surprising considering that it was acceptable in prior studies.", "However, this is a common phenomenon with tests for social and emotional abilities (see Archer and Akert, 1977; Costanzo and Archer, 1989).", "For each of the six performance domains a self- and a corresponding peer-estimate scale was developed by describing the respective ability in 9–10 items, reflecting its various aspects as good as possible.", "Peer estimates came from classmates: Each target person was rated by two peers/classmates, who were selected randomly.", "For each ability, the two peer-estimates were averaged, in order to obtain a measure that is less susceptible to distortions by extreme values.", "Items for the self-estimate questionnaires were statements like “I can easily rephrase a text using different wording.”", "(verbal intelligence) or “Estimating the size of a room is easy for me.”", "(spatial intelligence).", "The peer estimate questionnaires were based on the same items but slightly rephrased so that they referred to others instead of oneself (e.g., “_____ can easily rephrase a text using different wording.”)", "Participants were instructed to mentally fill the blanks with the names of the peers they were to judge.", "The appendix shows translations of all items used.", "As can be seen in Table 1, all alphas were either high (α > .80) or at least satisfactory (α > .70), with the exception of the self-estimate for intrapersonal ability (αLSS = .64, 95% CI [.56, .71], αHSS = .63, 95% CI [.55, .70]).", "Tests were given in regular class hours and took two hours and 50 minutes each.", "The order of tests was: 1.", "verbal, numerical and figural tests with time limits of 8, 11 and 7 minutes, respectively; 2.", "the three alternative uses tasks with a time limit of 2 minutes each; 3.", "the test for emotional management ability without a strict time limit (this took about 20 minutes).", "In order to obtain a maximum estimate of selfability correlation, each self-estimate was given directly after the corresponding performance test.", "Peer estimates were taken at the end of each testing session, that is, after all other tests had been administered.", "At this point, individuals also had to complete the German version of the Inclusion of the Other in the Self Scale (IOS scale; Aron et al., 1992) for each peer they rated.", "This is a measure of relationship closeness and resulted in a score between one (no relationship at all) and seven (very close relationship/best friends).1", "The validity of self- and peer estimates of abilities was investigated in two ways: First, we computed correlations between self-estimates and performance, as well as, between peer estimates and performance for each of the six ability domains.", "Bonferroni-correction was applied to all tests of significance involving multiple subscales of the same test, that is the ISA with three subscales (p = .05/3; resulting in a significance threshold of p < .0167), and the EMA with two subscales (p = .05/2; resulting in a significance threshold of p < .025).", "In addition to this significance-testing approach, we also developed a more descriptive approach when considering the minimal effect necessary to define a perspective as reasonably accurate.", "The aim of this effect-size-based approach is to obtain results that are comparable across studies and less dependent on sample size than those obtained by looking at significance levels alone.", "Similar to Vazire and Mehl (2008), we consider all estimateperformance correlations starting from r = .2 to indicate a relevant level of accuracy.", "This lies exactly between what would be considered a small and a medium sized effect according to common conventions and appears to be a reasonable threshold, considering the associations between self- or peer estimates and abilities found in past studies (Denissen et al., 2011; Freund and Kasten, 2012; Zell and Krizan, 2014).", "We then compared the resulting self-estimateperformance and peer estimateperformance correlations with each other.", "In line with Vazire (2010), we defined a difference between two correlation coefficients of more than .15 as substantial.", "Second and with respect to the initially proposed practical research question of whether self- and informant views of one's own abilities could provide independent contributions to predicting actual (psychometric) performance, we performed a series of multiple regression analyses with performance in each of the ability tests as outcome.", "Gender was entered in the first step as control variable.", "In the second step, self- and peer estimates of the respective ability were added simultaneously to assess how much unique information each perspective can provide.", "All common assumptions of multiple regressions (i.e., homoscedasticity, absence of multicollinearity, independent errors, and normality of residuals) were assessed and met.", "Intercorrelations of all performance measures for both samples are displayed in Table 2.", "As can be seen, all classical intelligence facets (i.e., verbal, numerical, and spatial intelligence) correlated positively with each other in both samples, as did both facets of emotional management ability.", "While creativity correlated with all classical intelligence facets in the older sample, it correlated with verbal and numerical but not with spatial intelligence in the younger sample.", "There was no significant difference between the closeness of the friendship between the first peer and the target (LSS: M = 3.31, SD = 2.14; HSS: M = 3.13, SD = 1.72) and the one between the second peer and the target (LSS: M = 3.36, SD = 2.11, t(224) = −0.22, p = .826, d = 0.02; HSS: M = 3.37, SD = 1.83, t(212) = −1.44, p = .153, d = 0.10).", "Table 3 shows the correlations of the six performance domains with self- and peer estimates for the respective domain in lower versus higher secondary school, as well as the selfpeer correlations.", "Relationships of abilities with self- and peer estimates are quite diverse.", "Surprisingly, verbal intelligence does not correlate with self-estimates in lower secondary school.", "It also shows only a small to medium correlation with self-estimates in higher secondary school and, albeit significant, this correlation is slightly below the above defined accuracy threshold of r ≥ .2.", "Even peer estimates of verbal intelligence are only of rather low validity (rs between .26 and .28, albeit significant).", "Considering Vazire's (2010) suggestions that a difference in r values of more than .15 indicates a relevant difference between the perspectives, we conclude that in the younger sample the peer-perspective is somewhat more valid than the self-perspective, while in the older sample the difference falls short of that criterion.", "In contrast, numerical abilities show mostly medium to high validities, both for self- and peer estimates.", "Nevertheless, in higher secondary school the self-perspective is substantially more valid than the peer-perspective (difference in rs of .18).", "Spatial ability is quite badly self- and peer estimated in both samples, with the exception of self-estimates in the older age group; only in this group the difference between both perspectives is .15.", "Creativity (originality in divergent thinking tasks) gave low to medium validities (rs between.22 and .34); and according to the ‘Vazire criterion’ the differences in perspectives are irrelevant.", "Intrapersonal ability can be self-estimated well, with medium to high r values (rs between .37 and .39), whereas peers show no insight into this ability; the difference in r values exceeds .15 in both samples.", "For interpersonal abilities self-estimates show good validity (rs around .46), whereas peer-estimates show low validity in the older sample (r = .19, significant but below our proposed threshold) and no correlation with performance in the younger sample (r = −.01).", "Also here the difference between perspectives is substantial and conforms to the ‘Vazire criterion.’", "Table 3 also shows selfpeer correlations: In both samples self- and peer perspectives agree at medium to high size for verbal and numerical abilities, and (lower) for creativity.", "In higher secondary school there is a medium selfpeer correlation for interpersonal ability.", "Spatial and intrapersonal abilities show (almost) no correspondence between self and peers.", "The amount of variance explained in each step of the hierarchical multiple regression analyses as well as beta-values of each predictor can be seen in Table 4.", "Our control variable gender was a significant and positive predictor of numerical intelligences in both samples (albeit in LSS only in the first step), of spatial intelligence in lower secondary school and intrapersonal abilities in higher secondary school, indicating that boys performed better than girls in the respective performance measures.", "Girls outperformed boys in creativity in lower secondary school and interpersonal abilities in both age groups.", "In both samples, verbal intelligence can only be predicted from peer estimates and the overall amount of variance explained is comparatively low (LSS: F(3, 228) = 5.80, p = .001, R2 = .071, Cohen's f2 = .076; HSS: F(3, 211) = 6.42, p < .001, R2 = .084, Cohen's f2 = .092).", "Numerical intelligence can be predicted with high effect sizes and both self- and peer estimates contribute significantly to its prediction (LSS: F(3, 228) = 28.67, p < .001, R2 = .274, Cohen's f2 = .377; HSS: F(3, 211) = 38.69, p < .001, R2 = .355, Cohen's f2 = .550).", "Spatial intelligence shows low predictability in both samples and can only be predicted from self-estimates in higher secondary school, while neither self- nor peer estimates can contribute to its prediction in lower secondary school (LSS: F(3, 228) = 4.66, p = .003, R2 = .058, Cohen's f2 = .062; HSS: F(3, 211) = 8.73, p < .001, R2 = .110, Cohen's f2 = .124).", "For creativity, a medium-sized amount of variance can be explained and both perspectives contribute to its prediction (LSS: F(3, 228) = 13.62, p < .001, R2 = .152, Cohen's f2 = .179; HSS: F(3, 211) = 11.32, p < .001, R2 = .139, Cohen's f2 = .161).", "When it comes to intrapersonal emotional management abilities, only self-estimates contribute to its prediction and the overall amount of explained variance is also medium-sized (LSS: F(3, 228) = 12.53, p < .001, R2 = .142, Cohen's f2 = .166; HSS: F(3, 211) = 15.81, p < .001, R2 = .184, Cohen's f2 = .225).", "Like numerical intelligence, interpersonal emotional management abilities can be predicted with high effect sizes in both samples.", "However, here only self-estimates can significantly contribute to the prediction (LSS: F(3, 228) = 26.03, p < .001, R2 = .255, Cohen's f2 = .342; HSS: F(3, 211) = 26.87, p < .001, R2 = .276, Cohen's f2 = .381).", "The questions and hypotheses of this study were derived from three sources: 1.", "Vazire's (2010) SOKA model; 2.", "the literature on self-estimates of abilities; 3.", "the validity of informant ratings of abilities.", "We made the following predictions regarding the four quadrants of the SOKA window (see Fig. 1): Verbal and numerical, as well as interpersonal ability should be in the open quadrant (I); spatial intelligence and creativity in the blind spot (II); intrapersonal ability in the hidden area (III).", "Most predictions were not confirmed.", "Since the pattern of results was similar for both age groups, we discuss most findings without differentiating between age groups (unless otherwise noted):", "In lower secondary school verbal intelligence was not located in the open area but in the blind spot, meaning that these adolescents have no accurate self-views here, while peers judge them more accurately, but also with rather low validity (r of .26).", "Also in line with this result, only peer estimates could significantly predict verbal intelligence in the respective multiple regression analysis.", "Pupils in higher secondary school seem to have at least some sense of their verbal abilities, at least according to classical significance-testing.", "However, the correlation is slightly below our threshold of .2, speaking for low validity and putting verbal intelligence at the verge between the open and blind spot.", "Again, peers have a bit more insight, albeit the difference between both perspectives is lower than what Vazire (2010) suggested to be relevant.", "The accuracy of both perspectives was on the one hand unexpectedly low, considering the supposedly high visibility of this ability and the findings on language competence from Zell and Krizan's metasynthesis (2014).", "On the other hand, it conforms well with results from Freund and Kasten's meta-analysis (2012), which operationalized verbal abilities as performance in verbal intelligence subtests and could therefore be considered more alike to the present study.", "Moreover, multiple regression showed that verbal ability had (among) the lowest total ‘estimability’ (R2s of .07 and .08), putting it almost in the unknown area.", "This might be the most puzzling finding of the current study.", "There are several possible explanations for the low associations between psychometrically measured and estimated verbal intelligence: The verbal intelligence test might not be a good measure of verbal ability, but the ISA used here is a well-established verbal intelligence test suited for people from 14 years and older, which has satisfactory reliability and also showed the expected intercorrelations with the other intelligence factors (see Table 2).", "In addition, the substantial correlations with divergent thinking tasks clearly speak in favor of a valid measurement of verbal ability in our samples (verbal fluency, which is also measured in divergent thinking tasks, is an important aspect of verbal ability, see Thurstone and Thurstone, 1943).", "The self- and the peer-estimate scales for verbal ability were of sufficient reliability (see as in Table 1) and the self-peer correlations were rather high (see Table 3).", "Therefore, it seems rather that the implicit concept of verbal ability in adolescents might not correspond to our (the psychologists) concept of verbal intelligence.", "A closer look at the items in the self- and peer estimate scales shows that some items might reflect oral expression abilities, which might not be correlated substantially with the abilities tapped by verbal intelligence tests.", "There is another – maybe most plausible – explanation though: These days, the supposedly weak verbal proficiency of young people is often complained about, with the low rate of (focused) reading and other factors being held responsible (e.g., De Naeghel, Van Keer, Vansteenkiste and Rosseel, 2012).", "This might be a well-founded reason, with the following argumentation: Individuals of lower verbal ability might be particularly weak at self- and peer estimating because this might require exactly what they are missing: a good verbal ability (see also the Dunning-Kruger-effect; Kruger and Dunning, 1999; Dunning et al., 2003).", "The finding that numerical ability is in the open area is in agreement with Freund and Kasten's meta-analysis (2012) and can presumably also be explained by the high visibility of this ability in the context of school classes (i.e., adolescents' mathematical performance) and the amount of feedback pupils get in this context.", "Spatial intelligence we had hypothesized to be located in the blind spot.", "However, it turned out to be in the unknown area (14 years) or in the hidden area (18 years).", "This is the only ability that changes its position within the Johari window between both age groups.", "This seems less surprising than some other findings, as spatial abilities might not play a major role in school performance and therefore do not receive as much feedback from school performance in the younger sample compared to the higher secondary school, where in math classes aspects of spatial ability are also required (e.g., geometry).", "The third unexpected finding is that creativity is not in the blind spot (Vazire, 2010) but in the open area.", "We also found that self-ratings of creativity were of medium validity, being even higher than those from the informant perspective.", "This deviation from Vazire's (2010) finding might be explained by the age group studied here.", "Children/adolescents might get more feedback in school about their creativity than adults usually get, but this is only a conjecture.", "Apart from verbal intelligence, maybe the second most surprising result is that, especially in the younger age group, interpersonal competence was not located in the open area but in the hidden area.", "This means, young adolescents have accurate self-views but no insight into each other's interpersonal abilities.", "Here it might be that – after filling out the SJT for interpersonal ability – the testees have a conceptually valid view of their own interpersonal competence.", "However, when judging peers (which was done at the end of class) that conception might have vanished already.", "It is also possible that the peer estimate reflects some general aspects related to liking, although this is purely speculative at the moment.", "Nevertheless, it is interesting to note that this ability is the only one where some change in peers' accuracy with age can be seen: While in 14-year-olds peer estimates showed zero validity, in the 18-year-olds it was around .19 and even though this is still slightly below our threshold for accuracy, the change between both age groups is considerable.", "Self-peer-agreement also rose from .04 to .28.", "This means that interpersonal competence might need some ontogenetic development – and possibly also feedback from experiences with the other gender – so that people can properly estimate other peoples' emotional competence.", "In explaining the weak validity of peer estimates, one could also be tempted to blame the performance test itself.", "However, upon closer examination the test mostly showed the expected correlations with other abilities, namely medium-size correlations of .34 in the younger sample and .26 in the older sample with intrapersonal ability (see Table 2), which corresponds well with earlier findings (Freudenthaler and Neubauer, 2005; Freudenthaler et al., 2008).", "Finally, the fact that intrapersonal competence is in the hidden area conforms well with the initial prediction.", "Turning to the practical issue of career counseling in the field of work and organizational psychology, using multiple regression analyses we asked whether complementing self- and peer perspectives of six central ability domains gives the adolescents a good perspective on their real performance level in the respective domains.", "Numerical ability as well as interpersonal ability can be judged quite well in terms of the amount of variance explained (≥25%).", "In the case of numerical ability both the self- and the peer-perspective make important contributions to its prediction, while for interpersonal ability only the self-perspective can contribute (Table 4).", "On the contrary, spatial and verbal ability have quite low levels of explained variance (≤11%), implicating that young people who face the situation of selecting a career have no accurate account of either their verbal or their spatial abilities.", "Intrapersonal abilities and creativity lie in between with around 15% variance explained.", "These findings have important practical implications (see also Zell and Krizan, 2014): Spatial ability in particular appears to be a construct, for which neither oneself nor others can make an accurate judgement of the real performance level.", "This is not surprising – we usually do not get much feedback on our spatial ability, particularly not at a very young age.", "Somehow more surprising is the low validity of self- and peer perspectives on verbal ability, which might pose a serious problem for young people.", "Even though a replication of these findings is needed, it seems that in the present sample it is, if anything, the peer perspective that gave a satisfying although not high validity of verbal ability.", "It seems most puzzling how poor the self-estimates of verbal ability are, which could not be expected on the basis of Zell and Krizan's (2014) metasynthesis, but it is in line with Freund and Kasten (2012), as explained above.", "Thus, it seems that the weak self-estimation of verbal ability is a ubiquitous phenomenon and that the young people of our sample indeed have a weak meta-perception of their verbal proficiency.", "Finally, between the well estimable numerical intelligence and interpersonal competence on one hand and the poorly estimable verbal and spatial intelligence on the other hand, we find intrapersonal ability and creativity.", "Between around 15 and 20 % of variance of these abilities can be estimated, which means (vocational) counselling is important here too, as people themselves as well as peers do not give the adolescents a particularly adequate estimate of how good they are in these domains.", "The study does have some limitations: While our samples are rather large and we have put effort into making them representative for the specific age groups, it has yet to be tested whether the findings would generalize to (older) adults.", "We restricted our study to 13/14-year- and 18-year-olds here, because in these age ranges important decisions regarding vocational choices are being made (at least in countries with an apprentice system).", "A second restriction concerns the rather low reliability of both scales of the test for emotional management abilities and the self-estimates of intrapersonal abilities, which could have potentially impacted the correlations in these domains.", "As already stated, this is not uncommon with tests for social and emotional abilities (Archer and Akert, 1977; Costanzo and Archer, 1989).", "Bonett and Wright (2015) argued that Cronbach's alpha often underestimates internal consistency and suggested always reporting the confidence interval around it.", "As can be seen in Table 1, upper limits for Cronbach's alpha are around what is considered acceptable for self-estimated intrapersonal skills but still potentially problematic for the EMA performance measures.", "Nevertheless, self*performance correlations for inter- and intrapersonal ability were around .40 for both samples, which is why we do not expect a strong restriction of generalizability here.", "Moreover, as we gave self-estimates directly after each corresponding ability test, we think that we obtained rather ‘upper-limit’ estimates of the validities of the self-estimates.", "If the self- and also the peer-estimate scale were given out of the context of the ability testing, validities might have been lower.", "Another restriction might be that we did not take self-efficacy into account; this should be properly addressed in future studies.", "Bearing in mind that Vazire (2010) proposed that acquaintance or relationship closeness between target and rater might be important for a trait's position within the SOKA model, future research should investigate their impact in a more systematic manner than the present study.", "For example, one could look into different groups of peer-raters with different kinds of relationships to the target.", "We have found no moderating influence of self-reported closeness of the friendship between peer-rater and target on the association between peer-ratings and performance.", "However, this result might have been affected by specifics of our research: We assigned peer-raters randomly, thereby reducing the possibility that raters were the participants' best friends or classmates who do not like them at all.", "Since relationship closeness was not our main interest in this study, we even further minimized its potential implications by using the average of both peer-raters in our analyses.", "Similarly, it might be interesting to investigate gender differences when it comes to the accuracy self- and peer estimates of intelligence.", "Some research indicates that men give higher self-estimates of intelligence than women do (see the meta-analysis by Syzmanowicz and Furnham, 2011) and that others also give higher intelligence-estimates for men than for women (Borkenau et al., 2004; Steinmayr and Spinath, 2009), both being particularly apparent for spatial and numerical intelligence.", "There is also some indication that these higher self- and other-estimates do not reflect existing gender differences in actual intelligence alone, but also a tendency for overestimation (Steinmayr and Spinath, 2009; Syzmanowicz and Furnham, 2011).", "We found gender differences in intelligence and emotional competence that are largely consistent with existing literature (Freudenthaler et al., 2008; Halpern & LaMay, 2000).", "Therefore, it would be interesting to see whether gender also affects the position of an ability within the SOKA model.", "However, the aim of the present study was to provide first insights the allocation of abilities into the model, which is why we controlled for gender in our regression analyses instead of investigating it as potential moderator.", "Summarizing, and viewed from a practical career counseling perspective, the weak self- and peer validities corroborate quests for professional career counseling on the basis of psychometrically sound ability tests.", "As young people might have biased views of their own but also of their peers' abilities, scientifically based career counseling should include not only tests for interests but also performance tests to avoid erroneous vocational decisions.", "Aljoscha Neubauer: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.", "Anna Pribil, Alexandra Wallner: Performed the experiments; Analyzed and interpreted the data.", "Gabriela Hofer: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.", "This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.", "The authors declare no conflict of interest.", "Data associated with this study has been deposited at https://doi.org/10.17605/OSF.IO/V8E5X.", "Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2018.e01061." ]	[]
Planar Differential Growth Rates Initiate Precise Fold Positions in Complex Epithelia	Tissue folding is a fundamental process that shapes epithelia into complex 3D organs. The initial positioning of folds is the foundation for the emergence of correct tissue morphology. Mechanisms forming individual folds have been studied, but the precise positioning of folds in complex, multi-folded epithelia is less well-understood. We present a computational model of morphogenesis, encompassing local differential growth and tissue mechanics, to investigate tissue fold positioning. We use the Drosophila wing disc as our model system and show that there is spatial-temporal heterogeneity in its planar growth rates. This differential growth, especially at the early stages of development, is the main driver for fold positioning. Increased apical layer stiffness and confinement by the basement membrane drive fold formation but influence positioning to a lesser degree. The model successfully predicts the in vivo morphology of overgrowth clones and wingless mutants via perturbations solely on planar differential growth in silico.	[ "BIOC" ]	[ "computational modeling", "finite element", "folding", "morphogenesis", "tissue mechanics" ]	[ "1300", "1307", "1309", "1312" ]	[ "Epithelial folding is a fundamental morphological process that is encountered abundantly during the development of multiple organisms.", "It is used to sculpt organs from flat epithelial sheets into complex structures such as tubular, undulated, and branched tissues (Nelson, 2016).", "Folds may function as a means of compartmentalization, surface area increase to facilitate material exchange, or may emerge as a side effect of pathology, such as overgrowth in cancer (Gutzman et al., 2008; Hruban et al., 2000; Nelson, 2016).", "Possibly the most extensively studied driver of folding is apical constriction via accumulation of non-muscle myosin II (Dawes-Hoang, et al., 2005; Granholm and Baker, 1970; Lecuit and Lenne, 2007; Lewis, 1947; Polyakov et al., 2014).", "Basal relaxation, lateral constriction (Štorgel et al., 2016; Sui et al., 2012, 2018; Wang et al., 2016; Wen et al., 2017) and cell shortening (Conte et al., 2012; Gutzman et al., 2008; Sherrard et al., 2010), constriction of the regions surrounding the prospective fold (Kondo and Hayashi, 2013; Monier et al., 2015; Röper, 2012), and constriction of supporting structures by other cells (Hughes et al., 2018), have all been demonstrated as potential folding mechanisms.", "Other force generation mechanisms, such as cell rounding in mitosis, adhesion shifts, or basal extrusions can also induce folds (Kondo and Hayashi, 2013, 2015; Monier et al., 2015; Wang et al., 2012).", "In all these scenarios, what defines the position of the prospective fold is a biochemical signaling mechanism responsible for selecting the cell population to actively generate the forces.", "Beyond cellular forces, differential growth between tissue layers can induce patterns of buckling such as those of the gut (Savin et al., 2011), dental epithelium (Marin-Riera et al., 2018), brain (Tallinen et al., 2014), lungs (Kim et al., 2015), and the rippled edges of plant leaves (Dervaux and Ben Amar, 2011; Liang and Mahadevan, 2009; Marder et al., 2003).", "External structures, such as the extracellular matrix (ECM), can provide sufficient confinement to growing tissue to induce folding (Diaz-de-la-Loza et al., 2018).", "Uniform growth and constriction will induce folds in predictable patterns following the physical rules of buckling (Karzbrun et al., 2018; Pocivavsek et al., 2008; Shyer et al., 2013; Wang and Zhao, 2015).", "The patterns can then be refined by the overall shape of the tissue (Tallinen et al., 2016) or further local perturbations such as constriction by smooth muscles (Kim et al., 2015), adhesive forces, and ECM alterations (Sui et al., 2012).", "While shaping a tissue, various mechanisms are likely to occur in parallel, such that once a fold is initiated in a selected position, a combination of modifications of the confinement and active force generation can help its progression.", "A key question is, how are the initial positions of the folds defined to achieve the precise tissue morphology (Nelson, 2016)?", "As an emergent mechanical phenomenon, fold position selection is likely to depend on a combination of the forces accumulating in the growing tissue, the dynamics of surrounding structures, and the inherent properties of the tissue such as stiffness or shape prior to folding.", "None of these factors are trivial to investigate independently in an experimental system—how would one eliminate the influence of the shape of a tissue on its form?", "Therefore, the topology of folding morphogenesis is a problem particularly suitable for computational exploration.", "Drosophila melanogaster is an established model system for studying morphogenesis.", "The wing imaginal disc of Drosophila forms three distinct folds, perpendicular to the dorsal-ventral axis.", "These major folds are highly reproducible in their number and positions, marking the boundaries between the notum, hinge, and pouch regions of the wing disc (Figure 1).", "There is evidence that basal relaxation, lateral constriction, and stiffness changes within the cell compartments play roles in the generation of the folds (Sui et al., 2012, 2018; Wang et al., 2016).", "However, what determines their positions and drives the initiation of these folds is an open question.", "This makes the wing disc an ideal experimental system to investigate general mechanisms that control the position of folds in complex epithelia, a problem that has been under-investigated but critical in determining the final functional architecture of the tissue.", "Here, we investigate the minimum set of requirements for initiating the correct topology of the complex multi-folded wing disc epithelia.", "Our search allows us to postulate planar differential growth as a mechanism for fold initiation.", "We measure the differential planar growth rates of wing imaginal disc development with high spatial resolution.", "Utilizing a computational approach, and from experimental measurements, we demonstrate that the differential growth in the plane of the tissue, especially in the early stages, under the compression of the ECM, drives the initiation of three folds from the apical surface.", "When challenged with mutants, our model can successfully recapitulate the morphology of overgrowth clones.", "We also predict that a reduction of early growth in the hinge region, prior to any folds being visible, can affect the number and position of folds that form later.", "We experimentally validate this prediction against a wingless mutant, which has reduced proliferation specifically in the hinge region (Neumann and Cohen, 1996).", "Our simulations show that the alterations of planar growth rates are sufficient to explain the observed fold perturbations in overgrowth and undergrowth mutants.", "The wing imaginal disc is a monolayered epithelial sac.", "The peripodial layer, positioned as the top layer throughout this paper, is formed of squamous cells.", "The bottom layer is the columnar layer, which forms the folds by the end of third instar (Figures 1Aiv, 1Av, and 1B).", "The apical surfaces of both layers face each other toward the lumen, and the basal surfaces face outwards.", "Both apical and basal surfaces harbor ECM of different compositions (Pastor-Pareja and Xu, 2011; Ray et al., 2015).", "We focus our attention on the columnar layer and characterize the two-day period from 48 h AEL, when the tissue is flat (Figure 1Ai), to 96 h AEL, when the columnar layer has formed three folds at the hinge region (Figures 1Av and 1B).", "From dorsal to ventral tips, these folds are termed notum-hinge (NH), hinge-hinge (HH), and hinge-pouch (HP) folds (Figure 1Bii).", "Between the HH and HP folds, the tissue forms additional lateral folds (LF) that do not reach the midline (Figures 1Avi, 1Avii, and 1Bii).", "There are multiple smaller folds at the ventral tip of the wing disc, where the tissue loops connect the columnar layer to the peripodial layer.", "These smaller folds are beyond the scope of the current work, as the largely unknown dynamics of the peripodial layer are not included in the current model.", "In our analysis, we segment the development into three morphological stages (Figures 1A and S3A): (1) The early stage before initiation of folds, ending prior to approximately 80 h AEL.", "The tissue is relatively flat, and folds do not start forming (Figures 1Aii and 1Ci).", "(2) The intermediate stage where the folds are starting to initiate on the apical surface, with the possibility that the HH fold has fully formed, ending by 88 h AEL (Figures 1Aiii, 1Aiv, and 1Ci).", "(3) The stage where all folds are established, ending approximately 96 h AEL (Figures 1Av and 1Avi).", "The folds are formed at reproducible, precise positions as normalized to the tissue DV length (Figure 1Cii).", "We match the initial state of our simulations to the tissue size and shape at 48 h AEL and start simulations using simple growth rates derived from the changes in dimensions of the wing disc as described above (Figure 1Ci).", "Step by step, we add in external confinement with apical ECM and BM, physical property heterogeneities, and fine growth patterns to characterize the requirements of fold initiation in the wing disc.", "For the purposes of identifying the mechanisms driving wing disc folding, we develop a finite element (FE) model (Bonet and Wood, 2008) of tissue morphogenesis.", "The goal of this model is to realistically capture the mechanical behavior of the tissue, then simulate its shape dynamics for different tissue physical properties and growth scenarios.", "The dynamics of the tissue emerge by balancing the elastic forces upon deformation and viscous resistances against the shape changes at each time step.", "In our model, the tissue is defined as a non-homogeneous continuous material.", "It is composed of a set of elastic FEs in the shape of triangular prisms.", "These prism elements are independent of the cells in the tissue and can have sizes varying from subcellular to multicellular scales (Figures 2A–2C and S1A).", "These elements have fixed “reference shapes,” which represent the initial shape of the element (Figure 2B, red wireframe).", "The growth is defined in the form of a deformation relative to the element’s reference shape.", "The shape resulting from the combination of growth and the reference shape becomes the element’s current grown or “preferred” shape (Figure 2B, blue wireframe).", "The total deformation of each element is calculated with respect to its reference shape at each time step.", "This total deformation is then decomposed into two components, the expected deformation due to growth of the element and the remaining elastic deformation (Rodriguez et al., 1994; Taber, 1995) (Figure 2B).", "Elements resist the elastic deformation with reactive forces (Figure 2C).", "The dynamics of morphogenesis then is governed by the forces depending on the viscoelastic properties and growth of the tissue.", "The relationship between the elastic deformations and resistance forces is modeled as a Neo-Hookean material, and a viscous resistance from the environment is applied on the exposed surfaces.", "In our approach, we extend the existing decomposition methodology to include oriented growth that follows the plane of the tissue (Figures 2D and S1B).", "In addition to active growth, the model allows for remodeling of tissue sub-compartments, such as the BM.", "In remodeling, the growth of an element is slowly updated such that the preferred shape slowly approaches to its current (deformed) shape, relaxing the tissue strains in the process.", "The model starts with the preferred and current shapes of each element being equal to the element’s reference shape.", "As the tissue grows in time, a mismatch between the preferred and current shape emerges, creating elastic deformations and generating consequent reactive forces.", "Balancing these elastic forces against deformation with the viscous resistance to movement at each time step, the new current shape of each element, therefore the whole tissue is obtained.", "Further details of the modeling procedures, meshing (Shewchuk, 2005), boundary conditions (Muñoz and Jelenić, 2004), linearization (Hughes, 2000), a pseudo code of the simulations and a table summarizing all utilized model parameters can be found in Methods S1, Methodology for the Computational Model.", "We start our simulations with uniform, constant growth rates that would bring the tissue AP and DV contour lengths from their sizes at 48 to 96 h AEL, in a 48-h time window (0.028 h−1 in AP and 0.033 h−1 in DV).", "Simulations with uniform growth on a tissue with minimal external resistance and homogenous physical properties do not form any folds (Figure 3A, top-left corner).", "This result reinforces that the tissue must have external factors driving compression.", "During morphogenesis, both apical ECM and BM can exert resistances to tissue growth and movement and contribute to correct tissue architecture (Diaz-de-la-Loza et al., 2018; Hannezo et al., 2015).", "Initially, we represent this as a viscous external resistance on the apical and basal surfaces.", "As the viscous resistance is increased, the tissue starts forming ripples with compact, regular folds, initiating from the center and distributing toward the dorsal and ventral edges (Figure 3Ai, first column).", "To assess the fold initiation, we generate apical indentation maps (Figure 3Aii), then calculate the total deviation in fold positions as a percentage of the DV length, each missing fold deviation contributing 100% to the sum (Figure 3Aiii).", "Within the tested range with uniform tissue physical properties (Figures 3Ai–3Aiii, first columns), the tissue can form only two folds.", "The peaks of these folds are also higher than the apical surface of the pouch and notum regions, which is not the case for the wing disc.", "Next, we consider physical property heterogeneities as a source of breaking symmetry.", "It is likely that the imaginal disc epithelium has heterogeneities, especially along its apical-basal axis.", "The dense actomyosin mesh on the apical surface, the actin of the basal surface, or the accumulation of the cell nuclei in the middle zone could all result in higher stiffness than the rest of the cell (Farhadifar et al., 2007; Meyer et al., 2011; Sui et al., 2018).", "To account for all these possibilities, we simulate wing disc growth with an increased stiffness on the apical surface (Figure 3A), on apical and basal surfaces (Figure S2A), or on the midline (Figure S2B).", "Of the tested cases, only stiffness increase on the apical surface can generate three folds; with 15%–19% deviation from the correct fold positions at tissue midline (Figures 3Ai–3Aiii bottom rows, B; Video S1).", "However, none of the cases generate folds similar to the experimental morphology (Figure 1Av).", "Tissue growing from 48 to 96-h AEL, with uniform in-plane growth rates.", "The growth rate is 0.033 h-1 in DV, and 0.028 h-1 in AP, as calculated from Figure 1C.", "Apical stiffness is 200 Pa (green), and stiffness for the rest of the cell body is 25 Pa (blue).", "Apical ECM or BM effects modeled as viscous resistances and external viscous resistance coefficient applied to both surfaces at 8,000 Pa s μm−1.", "Scale bar is 20 μm.", "Simulation time depicted on the frames.", "With this analysis, we conclude that external resistance to growth is essential for buckling the tissue and that increased apical stiffness can induce correct number of folds.", "However, defining growth rates as uniform and the BM as a simple viscous resistance is not sufficient to induce folds in the correct positions and shapes.", "Therefore, we construct detailed maps of tissue growth at fine spatial and temporal resolutions to improve the implemented growth rates.", "We experimentally measure the local growth rates via clonal analysis.", "By inducing sufficiently sparse single cell clones at different ages AEL, we quantify the local growth rates and generate spatial growth maps (Figure 4A).", "The extent of growth is defined by the number of nuclei in each clone; the orientation and aspect ratio of growth are defined by the ellipse fitted to the clone shape (Figure 4Aii).", "This analysis results in coupled growth rate heatmaps and orientation maps, in three-time windows as identified from the morphological quantifications (Figures 4B, S3A, and S3E; STAR Methods, Growth Rate Analysis).", "The maps reveal that the wing disc harbor high spatial and temporal variability in its planar growth patterns.", "Specifically, notum and pouch regions have significantly higher growth than the hinge region at early stages (Figures 4B and S3E).", "Similar to the previous observations for the pouch region (Mao et al., 2013), the overall growth rates of the tissue are reduced as the tissue ages.", "Next, we implement these measured growth rates in our simulations and model the 48 hours of development (48–96 h AEL).", "Here, the measured planar growth rates of three morphological age groups are applied sequentially in three equal (16 h) time windows (Figure S3Aiii), and growth is assumed to be constant through each window.", "With apical and basal confinement from viscous resistances, the experimental growth patterns result in folding patterns distinct from those of the uniform growth (Figures 4C and 4D; Video S2).", "Some indications of apical indentations emerge around the hinge region (Figure 4Dii, red arrows), and two lateral apical indentations that merge at tissue midline in later stages form (Figure 4Diii).", "None of the tested cases can initiate three distinct folds.", "This suggests that additional to the spatial-temporal variability in growth patterns, the BM should also be modeled in finer detail.", "Tissue growing from 48 to 84-h AEL, with experimental planar growth rates (Figure 4B).", "Apical stiffness is 200 Pa (green), and stiffness for the rest of the cell body is 25 Pa (blue).", "Apical ECM/BM effects modeled as viscous resistances and external viscous resistance coefficient applied to both surfaces at 8,000 Pa s μm−1.", "Scale bar is 20 μm.", "Simulation time depicted on the frames.", "To characterize the morphology of the BM structure, we acquire electron microscopy (EM) images of wing discs at pre-folding stages (72 h AEL) and at the end of third instar (120 h AEL) (Figures 5Ai and 5Aii) and quantify the thickness of the BM (Figure 5Aiii).", "The images reveal the BM is an approximately 0.1-μm thick uniform sheet in young discs.", "For older discs, the BM is a more complex structure with multiple layers and thickness in the range 0.4–0.6μm.", "Assuming a constant thickening rate of BM at the pouch center, the average thickness between 72 and 96 h AEL is then 0.2 μm.", "We thus define the BM in our model as a 0.2-μm thick elastic layer encapsulating the tissue (Figure 5B).", "In the simulations, the BM grows with remodeling upon deformation.", "Each BM element grows in the orientation of its current deformation, at a rate set by the local remodeling half-life (Methods S1).", "This leads to the gradual relaxation of BM deformations, and an emergent, non-homogenous growth of the BM influenced by the growth of the cellular layer.", "For clarity, the remodeling is represented in a 2D schematic in Figure 5C.", "We then simulate the development of the wing disc with a series of BM stiffness, remodeling half-life, and apical viscous resistance coefficient parameters.", "Upon definition of the elastic BM, we initially investigate apical-basal tissue stiffness heterogeneity ranges (Figures S4A–S4C), using the experimentally measured growth rates (Figure 4B).", "As we increase the apical viscous resistance, the tissue starts forming buckles (Figures S4A–S4C).", "For the cases with increased apical stiffness and increased stiffness on both surfaces, three apical indentations emerge (Figures S4A–S4Ci).", "Of the two scenarios, increased apical stiffness initiates a more dome-like pouch, proportional hinge fold indentations, and lower percentage deviations from the experimental fold positions (Figures S4A–S4Cii), better mimicking the in vivo fold pattern.", "Simulation snapshots for a setup with 100 Pa apical and 25-Pa cell body stiffness demonstrate the emerging indentations and their depth as the development progresses (Figures 5Di–5Dvi; Video S3).", "In the apical indentation maps, we can see the emergence of all three folds, the curved pouch border marked by the HP fold (gray, then cyan) and the emergence of the LF (cyan) (Figure 5Dvii).", "The folds are concentrated to the central region of the tissue, close to the experimental fold positions (Figure 5Dviii), with a total deviation of 4%.", "We predict the initiation of the folds requires the BM to be an order of magnitude stiffer than the cellular layer, which indeed has been inferred to be the case (Keller et al., 2018).", "As long as the BM is dramatically stiffer than the cellular layer, this phenotype can be produced with a large range of stiffness and remodeling half-life parameter sets for the BM, relative changes in one compensating for the other (Figure S5A).", "Simulating the same parameter set as Figure 5D with uniform growth reveals numerous symmetric ripples on the apical surface (Figure 5E).", "This signifies the importance of planar differential growth in the precise selection of the number and position of the folds.", "Tissue growing from 48 to 84-h AEL, with experimental planar growth rates (Figure 4B) and an explicitly defined BM (yellow).", "Apical stiffness is 100 Pa (green), and stiffness for the rest of the cell body is 25 Pa (blue).", "Apical ECM effect modeled as a viscous resistance with external viscous resistance coefficient of 16,000 Pa s μm−1.", "BM stiffness is 1,600 Pa, and BM renewal half-life is 8 h. Scale bar is 20 μm.", "Simulation time depicted on the frames.", "Our simulation also initiates an ectopic buckle on the pouch, which is not observed in live tissue (Figure 5Dvii, red line, Fi).", "To decipher what may be driving the resistance of the pouch region to such buckling, we turn our attention to tissue thickness.", "Our analysis reveals, through the 48 h of our interest, the wing disc increases its thickness in a non-uniform manner (Figure 5Fii), with the pouch region becoming relatively thicker than the rest of the tissue.", "The emergent tissue thickness increase during the simulations brings the pouch height to 17.3μm, and notum height to 13.3μm at 84 h AEL, from the initial uniform height of 12.5μm at 48 h AEL.", "This is well below the experimental observations (Figure 5Fii); therefore, the thickness increase should be an active growth input.", "Implementing this growth in the tissue thickness prevents pouch surface buckling (Figures 5G and S4D, and Video S4), with a total fold position deviation of 10% (Figure S5A).", "Our simulations predict that within the mechanical context where the hinge folds are initiated, the relative thickness of the pouch region protects it from further buckling that would be otherwise induced by the compression.", "Tissue growing from 48 to 84-h AEL, with experimental planar growth rates (Figure 4B), differential tissue thickness increase (Figure 5Fii, see STAR Methods), and an explicitly defined BM (yellow).", "Apical stiffness is 100 Pa (green), and stiffness for the rest of the cell body is 25 Pa (blue).", "Apical ECM effect modeled as a viscous resistance with external viscous resistance coefficient of 16,000 Pa s μm−1.", "BM stiffness is 1,600 Pa, and BM renewal half-life is 8 h. Scale bar is 20 μm.", "Simulation time depicted on the frames.", "Throughout the analysis, number of nuclei is used as a surrogate for tissue growth.", "To identify the potential impact of cell volume variations, we generate volume variability maps from the clone analysis and obtain volume scaled growth maps (Figures S5Fi–S5Fiii).", "Simulations with the volume scaled growth maps can capture the main pattern of the three folds, but additional ectopic buckling along the anterior-posterior axis between HH and HP folds is seen (Figure S5Fiv).", "The core pattern of fold initiation with differential growth is robust against observed variations in cell volume, but approximating the tissue growth as a combination of the number of nuclei and observed volume does not improve model predictions.", "The observed volume of a clone is a combination of the cell’s preferred volume and its deformations, as opposed to the nuclei count and the definition of growth in the model.", "This disparity, combined with the challenges of accurate volume measurements in the complex pseudostratified wing disc epithelia, negates potential additional information a true volume measurement could provide.", "As such, in line with our parsimonious approach throughout the paper, we continue to approximate growth with number of nuclei and assume constant cell volume throughout development in further model tests.", "Our results so far demonstrate the importance of planar differential growth in defining fold positions.", "The simulations reveal the fold initiation starts by the end of the early growth phase (Figure S4E).", "It has been shown that wing discs dissected immediately prior to fold initiation are still able to initiate folds ex vivo upon blocking cell division (Sui et al., 2018).", "With these, we investigate if the early growth rates are sufficient to initiate the folds upon significant perturbation to growth thereafter.", "We run simulations where the early growth rates in Figure 4Bi are applied for the first 16 h (48 to 64 h AEL) as in the control case, and then the growth is continued in the simplest form, with the uniform rates.", "The early growth rates followed by uniform growth are sufficient for the emergence of in vivo mimicking fold morphology (Figure S6Ai).", "Similarly, WT growth up to 64 h AEL, followed by non-oriented growth at the experimental growth magnitudes, does not alter the emerging fold morphology (Figure S6Aii).", "Additionally reducing the growth rate to 50% of the experimentally measured rates results in a minimal discontinuity in the fold pattern, but the structure is preserved (Figure S6Aiii).", "When the simulations are run with control growth rates, but the accumulated forces are relaxed prior to the emergence of the folds, the emerging morphology is perturbed.", "The final topology has loose fold formation, and the pouch curve shrinks compared to the control simulations (Figure S6B).", "These results suggest that the early growth rates measured prior to initiation of the folds, and the corresponding force accumulation, are critical for correct tissue morphology.", "Next, we challenge our simulations with perturbations in tissue growth, both overgrowth and undergrowth.", "Given the sufficiency of early growth rates for patterning the tissue, we must select perturbations that are effective prior to the formation of any folds.", "First, we experimentally induce overgrowth clones via increasing insulin signaling at 48 h AEL, by expressing a constitutively active form of the insulin receptor.", "The clones induce apical and basal bulging of the tissue, with ectopic fold initiation at the clone boundaries (Figure 6A).", "We simulate overgrowth clones with a range of growth rates, and we can capture the same deformations (Figure 6B).", "The apical bulging is visible from 200% growth increase, ectopic folding can be observed at 300% and above (Figure S6C), which is in line with overgrowth magnitudes observed for other perturbations in the wing disc (Genevet et al., 2009).", "Next, we test undergrowth.", "Again, this mutant must have an effect before the folds form.", "The expression of wingless (wg) localizes in two concentric rings encapsulating the pouch region from early third instar, before the folds start initiating.", "spadeflag (spdfg) mutations in wg lead to loss of wg expression at the inner ring, coinciding with the hinge.", "Consequently, the mutant has reduced growth in the hinge, and reduced size in the corresponding region of the adult wing (Neumann and Cohen, 1996; Rodríguez Dd et al., 2002).", "First, we carry out a clone growth rate analysis to demonstrate growth is reduced in spdfg mutant during 2nd instar stage (Figure 6C).", "We quantify the clone size in the region corresponding to the dorsal side of the inner ring that is lost in spdfg.", "This quantification reveals a 35% reduction in clone size for the mutant with respect to the wild type (Figure 6Ciii).", "Wg expression in the pouch DV boundary is preserved in the mutant.", "As an internal control, we quantify the clone size in this wg expression band and show that there is no reduction in growth at the DV boundary (Figure 6Ciii).", "These clearly demonstrate that the spdfg mutant reduces growth specifically in the wing disc hinge during early wing disc development (48–72 h AEL), prior to fold initiation.", "Next, to predict the wing disc phenotype of this mutation, we run simulations where hinge growth is reduced in the range 75% to 25% of WT.", "At 50% growth reduction (Figure 6D), the simulated mutant loses the three-fold morphology and displays only two folds at the midline of the disc (Figure 6E).", "On the lateral side, a third fold starts emerging and collapses on the pouch side fold before reaching the midline (Figure 6Eiv).", "It follows that the spdfg mutant should be able to form the NH fold, while HH and PH folds and the LF will collapse into a single fold at the tissue midline (Video S5) (Figure 6F).", "To test our prediction, we examine spdfg mutant wing discs (Figure 6G).", "The morphology of mutant wing discs at sequential developmental stages clearly demonstrates the emergence of a two-folded morphology (Figures 6Gi–iii) instead of three.", "Further, investigating the lateral cross-sections of the tissue at 96 h AEL shows that a third fold initiates at the lateral regions, yet collapses with the pouch side fold before reaching the tissue midline (Figures 6Giv and 6Gv), matching with the model predictions (Figure 6Div; Video S5).", "Simulations also reveal a dose dependent perturbation of the fold structure.", "At growth rates as low as 25% of WT, the third fold does not emerge from the lateral sides (Figures 6Hi and 6Hii), whereas at 75%, three folds form at the midline, albeit being more compact than the WT (Figures 6Hiii and 6Hiv).", "Then going back to the experiments, we could identify cases where small peaks emerged at the groove of the pouch side fold at tissue midline (Figure 6I).", "This further supports the hypothesis that the HH fold collapses with the HP fold and the LF in the spdfg mutant.", "Depending on the level of growth perturbation, the loss of the HH fold can be observed gradually, again matching with the predictions of the simulations.", "Tissue growing from 48 to 84-h AEL, with planar differential growth rates mimicking the spdfg mutation of the wingless.", "The growth of the hinge is reduced by 50% for this particular simulation (Figure 6A).", "There is differential tissue thickness increase (Figure 5Fii, see STAR Methods).", "Explicitly defined BM (yellow) has stiffness of 1,600 Pa, and renewal half-life of 8 h. Apical stiffness is 100 Pa (green), and stiffness for the rest of the cell body is 25 Pa (blue).", "Apical ECM effect modeled as a viscous resistance with external viscous resistance coefficient of 16,000 Pa s μm−1.", "Scale bar is 20 μm.", "Simulation time depicted on the frames.", "Here, we present a computational model of tissue growth and morphogenesis, incorporating spatial and temporal heterogeneity in growth rates and orientations, BM mechanics and remodeling, and physical property heterogeneities within different layers of the tissue.", "Coupled with the stiff apical surface and BM mechanics, we demonstrate that the planar differential growth rates are key in defining the positions of epithelial folds of the wing disc.", "Upon identifying early growth phases as sufficient for the initiation of folds, we make predictions on the emergent morphology upon perturbation of early growth rates.", "By changing only the planar differential growth rates in silico, we successfully predict overgrowth clone and wingless mutant morphologies in vivo.", "With our computational analysis, we propose a mechanism whereby planar differential growth rates define epithelial fold initiation positions.", "In the BM mechanics, we identify, a large range of parameters will suffice as long as the BM is elastic and sufficiently stiff (8 times the apical stiffness and above).", "This observation could explain the robust folding of the wing disc against the high spatial and temporal variability in BM structure (Figure 5A).", "Our simulations also show that some form of viscous resistance on the apical surface is necessary.", "The apical ECM has a different composition (Ray et al., 2015) and likely independent dynamics from the BM (Keller et al., 2018).", "The composition of the lumen, and any possible tethering interactions with the peripodial layer (Gibson and Schubiger, 2000), would resist apical surface movement with fluid-like viscous mechanics.", "Currently, we do not have any means of acquiring this viscous resistance of the apical ECM, yet our modeling approach signifies its importance.", "While our model is able to successfully generate the three-fold pattern, the timing of fold initiation in our simulations deviate somewhat from the experimental observations (65 h AEL in simulations compared to 76–80 h AEL (Sui et al., 2018) in vivo, Figure S4E).", "Temporal dynamics of tissue and BM physical properties could lead to the offset in our fold initiation timing.", "With its complex structural variability (Figure 5A), it is not possible to assume monotonically increasing or decreasing BM stiffness, without extensive further studies.", "The stiffness of the tissue itself is most likely altered through the same period, given that the residual tension of the tissue has been shown to reduce over time (Rauskolb et al., 2014).", "The fact that we can observe fold initiation in the simulations prior to the emergence of the folds in the experiments indicates the accumulation of cell mass due to planar differential growth is sufficient for fold initiation, yet the tissue may not have reached the enabling physical state before 76 h AEL.", "At later stages of the simulations, the successfully initiated hinge folds do not progress into fully established folds (Figure S5B).", "Our simulations suggest that once the folds are initiated with planar differential growth rates, additional mechanisms should be activated to progress these indentations into folds.", "Indeed, cell shortening, alteration of the microtubule and actin networks, modification of both interaction with the BM through integrins and the BM structure itself through MMPs, have all been reported as necessary requirements for progression of wing disc folding, each fold requiring particular subsets of modifications (Shen et al., 2008; Sui et al., 2012, 2018).", "These mechanisms and the mechanics of the accumulated cell mass due to differential growth can act together in a sequence of events that are temporally difficult to separate, acting concurrently or consecutively.", "Our findings set the scene for further theoretical and experimental investigation on the feedback mechanisms between the morphology of the apical surface and the signaling pathways regulating local growth, cell shape, and BM interactions.", "In our model, we impose the growth patterns from experimental observation.", "Further analysis is needed in order to determine the coupling between this growth distribution and other genetic or chemical factors.", "In conclusion, our results suggest planar differential growth as a mechanism for determining tissue fold positions, independent of active force generation.", "With wider implications, we suggest that the growth patterns giving tissues their final size can also regulate their architecture.", "We show that forces may not always result in instantaneous morphology changes, but can result in delayed morphogenesis.", "Stresses may accumulate early during development, even without any obvious changes in tissue morphology, but these may be critical for the precise sculpting of the tissue later in development.", "Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Yanlan Mao ([email protected]).", "All unique and/or stable reagents and the custom code generated for the manuscript are available from the Lead Contact upon request.", "Fly stocks were raised in non-crowded conditions on standard cornmeal molasses fly food medium at 25°C, unless otherwise indicated.", "Briefly, the fly food consisted of, per 1L, 10g agar, 15g sucrose, 33g glucose, 35g years, 15g maize meal, 10g wheat germ, 30g treacle, 7.22g soya flour, 1g nipagin, 5ml propionic acid.", "Male and female larvae were dissected at a range of developmental stages from 48hr AEL to 120hr AEL for experiments.", "Strains used are listed in Key Resources Table, and include: For clone generation, tissue shape, and morphology measurements: hsFLP;; (BDSC8862) and w;; Act<CD2<GAL4, UAS-GFP (Neufeld et al., 1998).", "For wildtype height measurements: yellow white (yw;;) (BDSC).", "For wingless (wg) mutant analysis: ; wg spd-fg; (BDSC1005), wg clones ywhsFLP; wgspd-fg; S-T.", "For overgrowth clones with perturbation of the insulin pathway: hsFLP; UAS-InRA1325D/CyO; MKRS/TM6B and w; Act<y<Gal4, UAS-CD8-mCherry/CyO; arm-GFP/TM6.", "To generate heat shock flip-out GFP clones of the correct density for growth rate analysis, the following regimes were used: for growth rates at 48–72h, 56-80h and 64–88h, heat shock was performed at 48h, 54hr or 64hr AEL respectively, for 12-20 min and dissected 24h later.", "For growth rates at 72–96 h, heat shock was performed at 72h for 10 min and wing discs dissected 24h later.", "All heat shocks were carried out at 37°C.", "To investigate spontaneous expression of GFP, we carried out non-heat shock controls (n=22 wing discs).", "Spontaneous GFP expression was indeed observed in larval epidermis, within the trachea and myoepithelial cells in the proximity of the wing disc.", "No such spontaneous expression is observed upon inspection of the wing disc columnar epithelium.", "For induction of insulin overgrowth clones marked by CD8-mCherry, heat shock was performed at 48hr AEL for 10 min at 37°C, followed by a 48hr growth before dissection.", "For the induction of flip-out GFP clones for growth rate analysis of the wingless mutant, the heat shock was performed at 48hr AEL for 10 min at 37°C and grown for 24 hrs before dissection.", "Larval wing imaginal discs were dissected and stained as per the procedure described in (Gaul et al., 1992).", "In brief, wing discs were dissected at the appropriate age in ice cold PBS for up to 15 min and fixed in 4% formaldehyde in PBS, at room temperature, for 30 min.", "For wingless mutants, fixed discs were repeatedly washed within a 40 min period in 0.3% PBT, followed by repeated washes with 0.5% BSA, 0.3% PBT for a further 40 min.", "Primary antibody, mouse anti-Wingless was prepared in 0.5% BSA, 0.3% PBT at 1:100 concentration and incubated overnight at 4oC.", "Washes were repeated as prior to primary antibody incubation.", "Secondary antibody, goat anti-mouse RRX (Jackson ImmunoResearch) (1:500), Alexa fluor 647-Phalloidin (Cell Signalling and Life Technologies) (1:20) and Hoechst (Sigma-Aldrich) (1:500) were prepared in 0.5% BSA, 0.3% PBT and incubated for 1 h at room temperature.", "Wing discs were washed repeatedly for 1 h in 0.3% PBT, prior to rinsing in PBS.", "For yw larva used for height measurements and flip-out clone larva used for growth rate analysis, dissected and fixed wing discs were washed in 0.3% PBT repetitively for 20 min, then immediately incubated with Alexa fluor 647-Phalloidin (Cell Signalling and Life Technologies) (1:20) and Hoechst (Sigma-Aldrich) (1:500) in 0.3% PBT for 15 min at room temperature.", "Wing discs were washed repetitively for 30-40 min, and then rinsed with PBS.", "Fixed and stained wing discs were mounted in fluoromount G Slide mounting medium (Southern Biotech) for imaging.", "Wing discs were imaged on a Leica SP5 and SP8 inverted confocal microscope with a 40X oil objective at 1-2X zoom, 0.341 μm depth resolution and 512 by 512 or 1024 by 1024 pixel resolution.", "Wing discs were fixed in 2% formaldehyde/ 1.5% glutaraldehyde in PBS for 30 min prior to being flat, sandwich-embedded in 2.8% low melting point agarose dissolved in PBS.", "Once set, asymmetric cubes of agarose were cut out containing the wing discs, and they were secondarily fixed for 1 h in 1% osmium tetroxide/1.5% potassium ferricyanide at 4°C.", "Further fixation and contrast enhancement was achieved with, 1% tannic acid for 45 min.", "Samples were then dehydrated in increasing concentrations of ethanol solutions and embedded in Epon resin (TAAB 812).", "The 70nm ultrathin resin sections were cut with a diamond knife (Diatome) using an ultramicrotome (UC7; Leica) and sections were collected on formvar-coated slot grids and stained with lead citrate.", "Discs were imaged using a 120kV transmission electron microscope (Tecnai T12; FEI) equipped with a ccd camera (Morada; Olympus SIS).", "A detailed definition of the computational model, simulation results analysis methods and a table providing the parameter definitions together with the values used in the simulations are provided in the Methods S1, Methodology of the Computational Model.", "The tissue size is measured from maximum projection images.", "DV length is defined as the longest axis from ventral tip of the pouch to the dorsal tip of the notum.", "AP length is measured to be the longest axis of the tissue perpendicular to the measured DV axis.", "The number of discs measured for each stage are: 4 discs at 48hr AEL; 32 discs for early stages with no fold initiation; 38 discs for DV and 32 discs for AP for middle stages with some fold initiation; 22 discs for DV contour length, 31 discs for DV length and 17 discs for AP length for 96 h AEL discs.", "Fold positions are measured at the longest axis in tissue midline, corresponding to the axis of DV length measurement.", "Each fold position is normalized to DV length, dorsal tip being 0 and ventral tip 1.", "The NH fold position is averaged from 19 wing discs, HH fold from 26 and HP fold from 16 discs.", "The clone position, aspect ratio, and orientation were calculated with automated segmentation and ellipse fitting, number of nuclei in each clone was counted manually, all using ImageJ (Schneider et al., 2012).", "To convert the growth information of each clone to spatio-temporal maps of tissue growth, we go through age classification of the wing disc, alignment of wing morphology to average, binning the clone positions on a 2D projection of the tissue, and averaging data points, followed by overlaying the pouch growth rates from the literature (Mao et al., 2013), to generate the growth maps of Figure 4B. Next, the data points on the growth maps are intra- or extra-polated to cover the empty spaces of the map grid, to allow for the maps to be smoothly read during the simulations (Figures S3D and S3E).", "For each clone, the manually counted number of nuclei determine the growth rate, the fitted ellipse determines the growth orientation and aspect ratio (Figure 4Aii).", "The centre of the fitted ellipse defines the position of the measured growth as normalised to the tissue bounding box.", "Assuming symmetry in the anterior-posterior axis, all single clone measurements are binned on one half of the bounding box (Figure 4Aiii).", "The wing discs are divided into three age groups depending on their morphology.", "The first indication of folds on wing discs occurs between 72-80 h AEL (Sui et al., 2018).", "Therefore the wing discs with no visible fold initiation, except for minor actin accumulation on the fold region, corresponding to up to 80hr AEL age (Figure S3Ai) are classified into the “early phase”.", "The wing discs with at least one, mostly two to three initiated folds, but not reached to fully folded morphology, corresponding to the range 80-88hr AEL age (Figures S3Aii–S3Aiv) are classified into mid-phase and finally, wing discs with all three folds formed, corresponding to 96 h AEL age are classified into “late phase”.", "The age definitions are not clear-cut at all times, therefore we will refer to disc growth periods as early, mid, and late phase throughout the manuscript, referring to the above morphological characterisation (Figure S3A).", "Upon division of the data points into age groups, where the wing disc has any markers for initiation of the first (HH) fold, the HH fold position of the individual disc is aligned to the average HH fold position, and the position of the clones updated (Figure S3B).", "This alignment step is not applicable to wing-discs of the earliest growth phase, where no folds are visible.", "Next, all the clones for a selected time point are binned on a 20 by 10 grid, according to their normalised planar positions within the bounding box of the tissue (Figure 4Aiii).", "Any grid bin with less than two data points is treated as empty.", "The growth data from the clones in each bin are averaged, Gaussian average is applied to number of nuclei, and the orientation angles, the orientation of the long axis of the fitted ellipse, are defined to be within π/2 degrees of each other before taking a Gaussian average.", "The aspect ratios are averaged with geometric average.", "The growth rates of the pouch region of the wing disc have previously been characterised in high detail by Mao et al. (2013).", "We overlay these measurements onto the generated growth and orientation maps by defining the position of the HP fold as the dorsal tip of the pouch and using the pouch sizes measured in this study (Figure S3C).", "Finally, with the assumed anterior-posterior symmetry, the resulting growth map is reflected to the remaining half, and our final spatial-temporal growth profiles to be utilised in our simulations are obtained (Figures 4B, S3Aiii, and S3E).", "The simulation requires a complete map, without gaps, such that each element can read its growth rate at each time step.", "Therefore, we fill the empty points of the grid by interpolating the existing measurements.", "Starting from the centre of the grid and moving out radially (Figure S3Di), once an empty grid point is detected, all the populated points within its eight immediate neighbors are averaged to fill the grid point (Figure S3Dii).", "The order of filling is of significance, as once a point is filled with averaging the neighbors, it will be counted as a populated point in following iterations.", "This allows us to fill the grid points at all regions, and we obtain the maps in Figure S3E. Of note, the corner points are not necessarily sampled in the simulation, as the emergent simulation tissue shape is similar to that of the experiments, nevertheless, the map should cover a slightly larger area then the immediate experimental boundary to ensure continuity.", "For an example of the region sampled throughout the simulations, see Figure 6D.", "While reading the growth rates from these maps, each element of the simulation takes its centre point normalised to tissue bounding box, reads the closest four corner values from the growth and orientation maps, and interpolates the actual growth rate/orientation to apply depending on its distance from each of the four corners.", "Growth in apical-basal axis is calculated from measurement of tissue thickness (Figures 5Fii–5Fvii and S5D).", "The growth rate is directly calculated form the height increase, yet one complexity here became the pseudostratification of the tissue.", "As the tissue grows, the nuclei become pseudostratified, first in the pouch, followed by the notum (Figures 5Fiii–5Fvii and S5D).", "Coinciding with the relative pouch thickness increase, the pseudostratification is visible in the pouch region as early as the initiation of the HH fold as an apical indentation (Figure 5Fv), while notum nuclei are still organized in a single layer (Figure 5Fiv).", "As the development progresses, the pseudostratification can be seen everywhere, the extent being significantly higher in the pouch (Figures 5Fvi and 5vii).", "The measurements demonstrate tissue thickness can increase without pseudostratification (Figure 5Fiv), indicating addition of material to tissue height independent of cell division, i.e., the nuclei count from which we derive our planar growth rates.", "On the other hand, the pouch region of the tissue increases in height faster, to a greater extent, and pseudostratification is more predominant in this region (Figures 5Fiii and 5Fvi), which should influence our definition of planar growth.", "To account for this difference, we allowed for tissue height increase to the level of the notum thickening, without altering the planar growth rates, reflecting the cell height increase independent of nuclei stratification.", "The additional height increase observed for the pouch region, the difference between the notum and pouch height increases, is reduced from the planar growth rates, “using up” the increase in nuclei numbers.", "We quantify the volume variability of the cells through an analysis analogous to growth rate analysis.", "The total volume of each clone is approximated by the maximum projected clone area and the height the clone spans in the z-stack.", "Then combining this data with the number of nuclei in the clone, we obtain the average cell volume in each clone.", "Normalising the measured cell volume to the average of the sample set, and following the same spatial and temporal classification of clones as in the growth rate analysis, we generate the volume variability maps (Figure S5F, top panels).", "Then we scale our growth maps with these volume maps, and obtained the volume scaled growth maps (Figure S5F, bottom panels).", "Prism 7 was used for statistical analysis (Figure 6C).", "Two-tailed t test was used with the exact n values used for each of the experiments.", "The following statistical significance cut off was applied:", "∗ p < 0.05,", "∗∗ p < 0.01,", "The authors declare that the data supporting the findings of this study and custom code generated for the manuscript are available from the corresponding author, Yanlan Mao ([email protected]), upon reasonable request." ]	[ "Drosophila wing discs grow with spatial and temporal heterogeneity", "This differential growth determines the positions of epithelial folds", "Constriction from the basement membrane is necessary for correct fold initiation", "Our computational model correctly predicts the shape of growth mutants" ]
Longitudinal serum S100β and brain aging in the Lothian Birth Cohort 1936	Elevated serum and cerebrospinal fluid concentrations of S100β, a protein predominantly found in glia, are associated with intracranial injury and neurodegeneration, although concentrations are also influenced by several other factors. The longitudinal association between serum S100β concentrations and brain health in nonpathological aging is unknown. In a large group (baseline N = 593; longitudinal N = 414) of community-dwelling older adults at ages 73 and 76 years, we examined cross-sectional and parallel longitudinal changes between serum S100β and brain MRI parameters: white matter hyperintensities, perivascular space visibility, white matter fractional anisotropy and mean diffusivity (MD), global atrophy, and gray matter volume. Using bivariate change score structural equation models, correcting for age, sex, diabetes, and hypertension, higher S100β was cross-sectionally associated with poorer general fractional anisotropy (r = −0.150, p = 0.001), which was strongest in the anterior thalamic (r = −0.155, p < 0.001) and cingulum bundles (r = −0.111, p = 0.005), and survived false discovery rate correction. Longitudinally, there were no significant associations between changes in brain imaging parameters and S100β after false discovery rate correction. These data provide some weak evidence that S100β may be an informative biomarker of brain white matter aging.	[ "BIOC", "MEDI", "NEUR" ]	[ "Aging", "Longitudinal", "S100β", "Small vessel disease", "White matter" ]	[ "1302", "1309", "2717", "2728", "2800" ]	[ "The calcium-binding protein S100β has clinical value as a proteomic biomarker of central nervous system damage.", "It is primarily found in glial cells, but also in some neuronal populations and in melanocytes, among other cell types (Donato, 2006; Donato et al., 2013).", "At nanomolar concentrations, S100β exerts neuroprotective and neurotrophic influences, but elevated S100β may contribute to further negative effects, as its presence at micromolar concentrations increases expression of proinflammatory cytokines, leading to apoptosis (Kleindienst et al., 2010; Rothermundt et al., 2003; Steiner et al., 1999).", "S100β is elevated after traumatic brain injury in both cerebrospinal fluid (CSF) and serum (Ingebrigtsen and Romner, 2002; Petzold et al., 2003; Vos et al., 2010), with greater S100β concentrations prognostic of poorer outcomes and recovery (Goyal et al., 2013; Thelin et al., 2017).", "Serum S100β levels are also influenced by blood-brain barrier (BBB) leakage (Kapural et al., 2002; Kleindienst et al., 2010; Ucar et al., 2004), as well as from other sources such as bone fractures, exercise, muscle injury, burns and melanoma (Anderson et al., 2001; Harpio and Einarsson, 2004; Koh and Lee, 2014; Mocellin et al., 2008; Mohammed et al., 2001; Pelinka et al., 2003).", "Although S100β has been investigated as a biomarker (in serum and CSF) in studies of head injury, depression, and neurodegenerative diseases such as Alzheimer's disease (Chaves et al., 2010; Peskind et al., 2001; Polyakova et al., 2015), the neurostructural correlates of S100β and its longitudinal trajectories in nonpathological aging are underinvestigated.", "S100β concentrations are positively associated with age (Nygaard et al., 1997; Schroeter et al., 2011; van Engelen et al., 1992), although some (Portela et al., 2002; Wiesmann et al., 1998) found no age effect in adulthood.", "Identifying possible biomarkers of brain aging is a key challenge (Academy of Medical Sciences, 2016; Jylhävä et al., 2017), and serum S100β is one of the logical candidates, yet data on S100β and multimodal brain analyses in older participants are lacking.", "Two prior cross-sectional studies indicate that serum S100β is specifically associated with poorer white matter microstructure (assessed with diffusion tensor MRI) in a small sample of healthy participants (N = 41, effect found in females only; Streitbürger et al., 2012), and in a small study of schizophrenia patients versus controls (total N = 39; Milleit et al., 2016).", "Neither study found a significant association between S100β and gray matter (GM)—however, it should be noted that both adopted a voxel-based–morphometry approach which results in reduced power in the large areas of the cortex that show highly individualized patterns of gyrification, and insensitivity to discrete lesions; Tisserand et al., 2004).", "Another study (N = 102; van der Leeuw et al., 2017) found no association between S100β and either white matter fractional anisotropy (FA) or cortical thickness in a mixed sample of patients with psychosis, relatives, and controls.", "Thus, well-powered, longitudinal, multimodal imaging studies—in participants at an age that confers relatively high risk of brain structural decline—are required to examine the possible differential sensitivity of S100β to cross-sectional levels of, and longitudinal declines in, various imaging parameters and brain tissues.", "Other candidate MRI parameters that may relate to S100β are markers of cerebral small vessel disease (SVD) burden.", "There is increasing evidence that BBB leakage occurs as an underlying pathology in SVD (Wardlaw et al., 2017; Zhang et al., 2017).", "The presence of white matter hyperintensities (WMHs) and perivascular spaces (PVS) are important markers of SVD pathophysiology that increase with age, in cerebrovascular disease, are linked to increased risk of stroke, and are associated with both cognitive impairment and dementia (Doubal et al., 2010; Wardlaw et al., 2015).", "PVS are also associated with elevated plasma markers of inflammation in older participants (Aribisala et al., 2014).", "They are also more frequent in patients with lacunar stroke and WMH, and are more visible with increasing evidence of BBB leakage in patients with SVD-related stroke (Wardlaw et al., 2009).", "PVS are also more visible with inflammation and BBB leakage in active multiple sclerosis plaques (Wuerfel et al., 2008).", "Although plasma S100β is influenced by BBB leakage in head injury as well as general cerebral pathology, it remains unknown whether S100β is associated with these important SVD markers on brain MRI in healthy community-dwelling older adults.", "In the present study, we investigated the level and change in S100β and indices of structural and diffusion MRI, in a large cohort of older individuals measured at ages 73 and 76 years.", "Given that S100β concentration in blood may rise due to age, central nervous system (CNS) damage, and BBB disruption, we hypothesized that relatively higher and increasing concentrations of plasma S100β would be coupled with lower and decreasing measures of brain structural and microstructural health.", "Prior evidence indicates that S100β is particularly strongly expressed in the human brain's white matter tracts (histological data showed co-localization of S100β with oligodendrocyte markers in the human brain; Streitbürger et al., 2012), and that serum S100β is cross-sectionally associated with poorer white matter microstructure (assessed with diffusion tensor MRI) in small mixed samples (N ≤ 102) with wide age ranges (Milleit et al., 2016; Streitbürger et al., 2012).", "Thus, we hypothesize that elevated and increasing serum S100β would be particularly pertinent to poorer and decreasing white matter structure, beyond measures of global atrophy and GM volume.", "Data are drawn from waves 2 and 3 of a longitudinal study of aging: the Lothian Birth Cohort 1936 study (LBC1936; Deary et al., 2007; 2012), when participants were a mean age of about 73 and 76 years, respectively.", "In 1947, Scotland tested the intelligence of almost all schoolchildren born in 1936, and the LBC1936 follows up some of those individuals—now in older age—who mostly live in the Edinburgh and Lothians area.", "The initial wave of LBC1936 (wave 1) took place between 2004 and 2007.", "It assessed 1091 individuals on aspects of their health, and physical and cognitive function, at around 70 years old (M = 69.53, SD = 0.832).", "At wave 2 (2007–2011) and wave 3 (2011–2013), 866 and 697, respectively, returned at mean ages of about 73 and 76 years, a detailed MRI brain scan was added to the protocol at both waves (Wardlaw et al., 2011).", "During a medical interview at each wave, participants reported their medical history (including a self-reported diagnosis of hypertension, diabetes, melanoma, and dementia).", "The Multi-Centre Research Ethics Committee for Scotland (MREC/01/0/56), the Scotland A Research Ethics Committee (07/MRE00/58), and the Lothian Research Ethics Committee (LREC/2003/2/29) approved the use of the human participants in this study; all participants provided written informed consent and these have been kept on file.", "Serum samples were obtained from participants during the main physical and cognitive testing appointment at waves 2 and 3.", "The mean lag between waves was 3.77 years (SD = 0.28).", "After collection, samples were stored at −80 °C at the Wellcome Trust Clinical Research Facility, Western General Hospital, Edinburgh, until the conclusion of the wave.", "They were then transferred to the Department of Clinical Biochemistry, King's College London using cold-chain logistics, where they were stored at −20 °C until assays were conducted using a chemiluminescence immunoassay S100β kit (distributed by DiaSorin, Berks, UK) on a LIAISON chemiluminescence analyzer.", "The lag between sample dispatch at the end of sampling and assay completion (i.e., time stored at −20 °C rather than −80 °C) was an average of 44 days (SD = 26) for 4 batches at wave 2, and 8 days (single batch) at wave 3, respectively.", "The minimal detectable concentration of the assay was 0.02 μg/L.", "Intra- and inter-assay precision for both waves is reported in Supplementary Table A.1.", "Participants underwent whole-brain structural and diffusion MRI using the same 1.5 T GE Signa Horizon scanner (General Electric, Milwaukee, WI, USA) at wave 2 and 3.", "The scanner is maintained with a careful quality control programme.", "Scans took place at the Brain Research Imaging Centre, Edinburgh, shortly after serum collection (mean lag for the present study sample: wave 2 M = 65.39 days, SD = 34.69; wave 3 M = 38.69 days, SD = 28.37).", "Full details of acquisition and processing are available in an open access protocol article (Wardlaw et al., 2011).", "Briefly, T1-, T2-, T2*-, and FLAIR-weighted sequences were co-registered (voxel size = 1 × 1 × 2 mm).", "Total brain (TB), GM, and white matter hyperintensity volumes were quantified using a semiautomated multispectral fusion method (Valdés Hernández et al., 2010).", "WMHs were explicitly defined as punctate, focal, or diffuse lesions in subcortical regions and distinguished from lacunes and PVS by signal characteristics (Wardlaw et al., 2013).", "Cortical or discrete subcortical infarcts were excluded by careful manual editing blind to other features.", "PVS were defined as fluid-containing small spaces running parallel with the expected direction of perforating vessels, appearing punctate in cross section and linear in longitudinal section, with <3 mm diameter.", "PVS were differentiated from lacunes or WMH on morphology, signal, and size criteria as previously defined (Wardlaw et al., 2013; Potter et al., 2015a).", "From the T2-weighted volumes, PVS ratings were performed by a trained neuroradiologist (JMW, ZM; Potter et al., 2015a; Potter et al., 2015b).", "Change in PVS between waves was scored by comparing scans at wave 2 and wave 3 side by side, blind to any other participant characteristics, and scored on a 5-point scale from −2 (reduction, i.e., improvement) to +2 (increase, i.e., worsening), where 0 denotes no visible change.", "The diffusion tensor MRI (DT-MRI) acquisition comprised a single-shot spin-echo echo-planar diffusion weighted volumes (b = 1000 s mm−2) acquired in 64 noncollinear directions, alongside 7 T2-weighted images (b = 0 s mm−2).", "This yielded 72 contiguous axial slices (FoV = 256 × 256 mm, matrix 128 × 128, 2 mm isotropic voxels).", "Repetition and echo times were 16.5 s and 95.5 ms, respectively.", "After preprocessing (brain extraction, removal of bulk participant motion and eddy current–induced distortions), water diffusion tensor parameters were estimated using FSL tools (FMRIB; Oxford, UK; http://www.fmrib.ox.ac.uk).", "A 2-fiber model with 5000 streamlines was then used to create brain connectivity data using the BEDPOSTX/ProbTrackX algorithm in 12 tracts of interest: the genu and splenium of the corpus callosum, bilateral anterior thalamic radiation, cingulum, uncinate, arcuate, and inferior longitudinal fasciculi.", "Probabilistic neighborhood tractography as implemented in the TractoR package (http://www.tractor-mri.org.uk; Clayden et al., 2011) identified the tracts of interest from the connectivity data (Bastin et al., 2010; Muñoz Maniega et al., 2017).", "White matter tract-averaged FA and mean diffusivity (MD) were then derived as the average of all voxels contained within the resultant tract maps.", "All segmented images were visually inspected for accuracy, blind to participant characteristics, to identify and correct errors.", "We excluded from the analyses those with self-reported history of dementia, or Mini-Mental State Examination score of <24 (n = 35).", "This was based on prior reports of elevated S100β in dementia (Chaves et al., 2010), the likelihood that these individuals were undergoing pathological CNS degeneration, and their low numbers in the current cohort (yielding low statistical power with which to reliably detect associations).", "Given that elevated serum S100β is associated with melanoma (Harpio and Einarsson, 2004; Mocellin et al., 2008; Mohammed et al., 2001), those who reported melanoma at either wave (n = 26) were also excluded.", "S100β concentrations for excluded participants are shown in Supplementary Table A.2.", "Both WMH measures were log transformed to correct skewness.", "Extreme outlying points (>4 SDs above the mean) for S100β at wave 2 (n = 6) and wave 3 (n = 4) were removed, along with 7 points at wave 3 that were below the sensitivity threshold of the assay (<0.02 μg/L).", "After exclusions, a total of 776 and 619 participants provided S100β data at ages 73 and 76 years, respectively, 593 and 414 of whom also provided brain MRI data.", "We used the maximum available sample size in all analyses.", "The main questions we addressed were (1) are there associations between serum S100β and brain imaging variables cross-sectionally at age 73 years?", "and (2) are the changes in S100β from age 73 years to age 76 years correlated with changes in brain imaging variables across the same ages?", "We used bivariate change score models (McArdle, 2009) in a structural equation modeling (SEM) framework to test these cross-sectional and longitudinal associations between S100β and brain MRI variables (Fig. 1), specifying a separate model for each brain MRI outcome.", "In the case of PVS analysis, the visual rating of PVS change was used in place of a latent change score, and correlated with the latent S100β change score.", "The volumetric brain indices were expressed as a proportion of intracranial volume in our main SEMs, and we also provided a supplementary analysis for uncorrected measures.", "Using the FA and MD measures across multiple white matter tracts, we derived a latent variable (hereafter referred to as gFA and gMD) for waves 2 and 3, respectively, using the following: genu and splenium of the corpus callosum, and left-right averages of the anterior thalamic radiation, inferior longitudinal fasciculus, uncinate, arcuate, and cingulum.", "We imposed strong factorial invariance (as was previously shown to be possible for these data; Ritchie et al., 2015), constraining the intercepts of each tract measure and their loadings on the latent variable to equality across waves.", "We also included correlated residuals between corresponding tracts across waves, alongside 5 other significant tract-tract residual paths for gFA and 6 for gMD.", "This builds on our and others' prior work, which found that there is substantial shared variance in white matter microstructural properties across tracts of the brain in early life, middle, and older age (Cox et al., 2016; Penke et al., 2010; Ritchie et al., 2015; Telford et al., 2017).", "Thus, these general, latent, factors reflect common microstructural properties (FA and MD) across white matter pathways.", "Finally, based on evidence of local white matter variation in S100β expression (most strongly expressed in the corpus callosum and cingulum bundle) and cross-sectional associations between FA and S100β (Streitbürger et al., 2012; Milleit et al., 2016; van der Leeuw et al., 2017; Allen Institute for Brain Science, 2010), we used the same framework as above to examine associations between S100β and tract-specific microstructure in each white matter tract of interest for FA and MD.", "Given that there was a short delay between serum collection and MRI scanning at both waves, we corrected MRI and S100β for their respective age in days at data collection within each model, along with sex, diabetes, and hypertension.", "To account for missing data bias due to attrition between waves, we took account of all available data, using full information maximum likelihood estimation.", "We assessed model fit according to the χ2 minimum function test statistic, the root mean square error of approximation (RMSEA), comparative fit index (CFI), Tucker-Lewis index (TLI), and the standardized root mean square residual (SRMR).", "All statistical analyses were conducted in R version 3.2.2 “Fire Safety” (R Core Team, 2015).", "SEM was conducted with the “lavaan” package (Rosseel, 2012) and the resultant p-values for the associations of interest (see asterisks in Fig. 1) were corrected for multiple comparisons with false discovery rate (FDR; Benjamini and Hochberg, 1995) using the “p.adjust” function in R.", "Participant characteristics are shown in Table 1, and bivariate associations among study variables are reported in Supplementary Table A.3.", "Descriptive plots of S100β (density, and age 73–76 years correlation, boxplot, and trajectory) are in Fig. 2.", "S100β concentrations showed substantial stability of individual differences from age 73 years to age 76 years (Pearson's r = 0.585, p < 0.001).", "S100β concentrations were significantly higher at age 76 years than at age 73 years (when considering returners only: wave 2 S100β M = 0.085, SD = 0.035; wave 3 S100β M = 0.092, SD = 0.040; t (1190.70) = 3.244, p = 0.001, Cohen's d = 0.118).", "Males showed lower S100β than females at both waves (wave 2: t (773.28) = 3.655, p < 0.001; wave 3: t (614.69), p = 0.002), but did not exhibit differences in their rate of change with age (p = 0.546; Fig. 2).", "When comparing baseline values of those who returned to provide an S100β sample at age 76 years from those who did not, there were no significant differences for S100β (t (270.19) = 0.115, p = 0.908), TB volume (t (167.74) = 1.577, p = 0.117), WMH volume (t (152.70) = 1.277, p = 0.204).", "However, individuals who returned at age 76 years had significantly more GM at age 73 years than nonreturners (t (174.00) = 2.800, p = 0.006).", "To satisfy the assumption of missing at random (MAR; Rubin, 1976), under which FIML operates, baseline GM volume was included as an auxiliary variable (Schafer and Graham, 2002) when modeling associations between S100β and all other imaging variables.", "Significant increases in WMH volume and white matter tract MD, and significant decreases in TB volume, GM volume, and white matter FA exhibited by this cohort between wave 2 and 3 have been previously reported elsewhere (Dickie et al., 2016; Ritchie et al., 2015).", "In the context of the current sample, all brain measures showed statistically significant mean changes over time, considering only those who provided scans at both waves.", "There were significant reductions in raw TB (t (851.73) = 2.815, p = 0.005, Cohen's d = 0.191) and GM volume (t (844.37) = 2.861, p = 0.004, Cohen's d = 0.197), and increases in WMH volumes (t (809.50) = −4.267, p < 0.001, Cohen's d = 0.293).", "The visual ratings of PVS change across waves showed that PVS load either stayed consistent (N = 426 received a score of 0) or became worse over time (N = 42 received a score of +1).", "Those who provided S100β and did not undergo an MRI scan were not significantly different from those who provided both—at either wave 2 or wave 3— in terms of age, S100β concentrations, and Mini-Mental State Examination score (all t values <0.779, p > 0.437).", "However, those who undertook both elements of the study comprised a significantly larger proportion of males at wave 2 (χ2 = 5.708, p = 0.017), although not at wave 3 (χ2 = 2.303, p = 0.129).", "Individuals showed substantial variation in the degree to which S100β and the continuous MRI indices changed over time, as indicated by significant slope variances (all p < 0.001); slope means and variances from age- and sex-corrected univariate change score models are reported in Supplementary Table A.4.", "Results of the SEM analyses are shown in Table 2, with bias-corrected 95% confidence intervals from 1000 bootstraps.", "Model fit statistics are shown in Supplementary Table A.5.", "Models examining associations between the level and change of S100β and volumetric MRI indices showed adequate fit to the data (WMH: χ2(28) = 42.629, RMSEA = 0.023, CFI = 0.993, TLI = 0.988, SRMR = 0.026; GM: χ2(24) = 42.337, RMSEA = 0.027, CFI = 0.987, TLI = 0.977, SRMR = 0.022; TB volume: χ2(28) = 131.237, RMSEA = 0.060, CFI = 0.931, TLI = 0.887, SRMR = 0.039).", "None of these measures showed significant cross-sectional associations with S100β at age 73 years (all absolute r-values ≤0.061, all p-values ≥0.113) or longitudinally (all absolute r-values ≤0.082, all p-values ≥0.095).", "Running these volumetric analyses without correction for intracranial volume did not substantially alter the results (Supplementary Table A.6).", "The model of visually rated PVS change showed an adequate fit to the data (χ2(14) = 39.439, RMSEA = 0.042, CFI = 0.963, TLI = 0.936, SRMR = 0.032).", "There was no association between S100β at age 73 years and visually rated PVS change (r = −0.034, p = 0.475), and the nominally significant association with longitudinal S100β concentrations (r = −0.096, p = 0.041) did not survive FDR correction.", "The models examining associations of S100β with white matter diffusion parameters both fitted the data well (gFA: χ2(205) = 284.004, RMSEA = 0.019, CFI = 0.977, TLI = 0.969, SRMR = 0.035 and gMD: χ2(205) = 322.817, RMSEA = 0.024, CFI = 0.970, TLI = 0.959, SRMR = 0.048); tract loadings are reported in Supplementary Table A.7.", "At wave 2, higher S100β was significantly associated with “less healthy” white matter gFA (i.e., poorer directional coherence of water molecular diffusion; r = −0.150, p = 0.001), which survived correction for multiple comparisons.", "The 3-year association between declining gFA and increasing S100β was nonsignificant (r = −0.083, p = 0.154).", "Associations between gMD and S100β were nonsignificant for both level (r = 0.003, p = 0.941) and change (r = 0.019, p = 0.717).", "Next, we examined the level and change associations between S100β and average white matter microstructure (FA and MD) within each of the tracts of interest.", "Fit statistics indicated that all models fitted the data well (Supplementary Tables A.8 and A.9).", "Results of the models are shown in Fig. 3, and Supplementary Tables A.10 and A.11.", "A higher concentration of S100β at age 73 years was significantly associated with “poorer” FA at the same age in the anterior thalamic radiation (r = −0.155 p < 0.001) and cingulum bundle (r = −0.111 p = 0.005).", "Both survived FDR correction.", "There were also nominally significant associations with the level of the splenium (r = −0.087 p = 0.030) and arcuate (r = −0.087 p = 0.032) in the same direction, but these did not survive multiple comparison correction.", "The corresponding associations for tract MD were all nonsignificant cross-sectionally (all absolute r values ≤0.059 p-values ≥0.138) and longitudinally (all absolute r values ≤0.069 p-values ≥0.158).", "These data represent the first large-scale study of longitudinal S100β concentrations and their association with longitudinal multimodal brain vascular and neurodegeneration MRI markers in community-dwelling older adults.", "We focused on multiple MRI indices of brain white matter because S100β is predominantly found in glial cells.", "We also considered measures of GM and global atrophy as comparators.", "Notably, our results suggest that individual differences in serum S100β concentrations may be potentially informative for specific aspects of brain white matter aging.", "We found that higher S100β was, in cross-sectional analysis at age 73 years, significantly associated with generally poorer white matter microstructure (as indexed by gFA), with a small effect size (Cohen, 1992).", "Further investigation of tract-specific effects indicated that this association is predominantly driven by lower FA in the anterior thalamic, arcuate, cingulum, and callosal fibers.", "The significant gFA-S100β association at age 73 years reported here contradicts some (van der Leeuw et al., 2017), but corroborates other (Milleit et al., 2016; Streitbürger et al., 2012) previous cross-sectional associations in smaller (N ≤ 102) samples.", "Our well-powered longitudinal design provides important new data on the coevolution of this serum biomarker with brain MRI, including several measures that had not previously been examined, such as white matter MD and markers of SVD.", "Given the prevalent expression of S100β in the corpus callosum (Streitbürger et al., 2012), it is notable that associations between tract-specific change and S100β change for both FA and MD in the genu of the corpus callosum were not significant.", "This merits further investigation in longitudinal samples over a longer period with more sampling occasions (which could also take account of nonlinear age-related trajectories).", "Although there has been relatively little research on the association between S100β and age-related brain and cognitive decline, our findings that higher concentrations are related to poorer white matter FA could partly be related to deleterious effects due to systemic inflammation.", "Systemic inflammatory challenge reportedly elicits increased BBB permeability in humans and rodent models (Elwood et al., 2017), and there are relationships between higher inflammatory markers and lower brain metrics, including white matter markers of SVD (Aribisala et al., 2014; Corlier et al., 2018, reviewed in; Persson et al., 2014 and in; Wardlaw et al., 2013).", "Consequently, it will be of interest to quantify the degree to which the relationship between inflammation and cognitive decline is mediated by S100β and brain structural outcomes, as well as to identify the potential genetic and lifestyle determinants of inflammation (e.g., Corlier et al., 2018) in well-powered longitudinal designs.", "Taken together, our results provide some limited support for the hypothesis that both (serum markers and brain MRI) provide meaningful and overlapping biomarkers of age-related white matter degradation.", "This study also provides novel information about the concentrations and stability of individual differences in serum S100β (i.e., the correlation between samples taken 3 years apart), in generally healthy older adults.", "These may suggest that serum S100β concentrations in the same individual may represent a relatively stable trait, although establishing this more robustly would require many more sampling occasions.", "We also provide information on sex differences in the context of important confounds of age, melanoma, and dementia.", "Significant associations between greater S100β at older ages have been reported in some studies (Nygaard et al., 1997; Schroeter et al., 2011; van Engelen et al., 1992), but were nominally negative in others (Wiesmann et al., 1998) or null (Portela et al., 2002).", "With respect to sex differences, our finding that females exhibited higher S100β corroborates the findings from some studies (Gazzolo et al., 2003), whereas others report the converse pattern (Nygaard et al., 1997) or no significant difference (Portela et al., 2002; Streitbürger et al., 2012; Wiesmann et al., 1998; van der Leeuw et al., 2017).", "Unlike the present study, those cited previously were all cross-sectional and represent a mix of single studies across very wide age ranges (neonate to 70 years old), across serum and CSF sampling (with varying sensitivity; Wiesmann et al., 1998), and comprising participants with various characteristics (healthy controls, mood disorders, those undergoing diagnostic lumbar puncture, or surgery with spinal anesthesia).", "Moreover, as far as the authors are aware, there is no large-scale data on the stability of individual differences in serum S100β concentrations across time in nonpathological older adults.", "The results reported here therefore address a substantial gap in our understanding of stability and longitudinal S100β trajectories in older community-dwelling adults.", "Although it has been hypothesized that observed increases of S100β with age could reflect (1) age-related increases in myelin loss, it could also be that (2) CNS cell “turnover” remains stable, but that cellular S100β concentrations are simply higher (Nygaard et al., 1997), or that (3) S100β does not change, but that serum concentrations are driven by greater age-related BBB leakage.", "Our findings lend some support to the first or third interpretations.", "Nevertheless, it should be noted that white matter FA can be affected by multiple microstructural properties, including myelination, but also extending to axonal bore, cell membranes, microtubules, and other structures (Beaulieu, 2002; Jones et al., 2013).", "As such, inferences about the weak associations of S100β with any specific microstructural property of the brain's white matter should be undertaken with caution.", "There are several study limitations.", "We note that our measure of change is based on a relatively brief (3 years) period.", "Although older individuals are at higher risk of brain structural changes than their younger counterparts, the brief sampling window limits the opportunity for large brain structural changes to take effect, especially because this group was broadly healthy, but fairly typical of similarly aged community-dwelling adults in Europe.", "Further study with a longer sampling period or a larger sample is merited to increase our ability to reliably assess these potentially subtle coupled changes, and to account for the likelihood that observed changes over time are nonlinear.", "On a related note, our models of latent change derived from single-indicator latent measures did not allow for the independent estimation of measurement residuals, meaning that our measures of change here should be considered as essentially difference scores (not accounting for the covariates).", "These analyses at only 2 time points also preclude tests of nonlinear change and of lead-lag relationships of change in brain and serum markers.", "We also reiterate that S100β concentrations may be influenced by a number of factors, such as exercise, melanoma, dementia, sleep apnea, depression, time of year/season, bone fractures, muscle injury, and burns (Anderson et al., 2001; Chaves et al., 2010; Harpio and Einarsson, 2004; Koh and Lee, 2014; Mohammed et al., 2001; Morera-Fumero et al., 2013; Pelinka et al., 2003; Peskind et al., 2001; Polyakova et al., 2015; Traxdorf et al., 2016), only some of which (dementia and melanoma) were accounted for in the present analyses.", "Our measure of PVS and its change is likely to be relatively insensitive; the rater could not be blinded to time, and the binary and disproportionate nature of visually rated PVS change mean that the estimates reported here should be interpreted accordingly.", "Computational methods for PVS quantification that are currently in development (Ballerini et al., 2016) may improve sensitivity to detect important aging-related changes.", "Finally, the narrow age range, ethnic homogeneity (all participants were White British), and relative good health of study participants limits the degree to which our findings can be generalized to groups of different ages, ethnicities, and patients.", "Nevertheless, the fact that these characteristics obviate such strong potential confounds in the current analysis can be viewed as an important strength.", "Combined with the large sample size, longitudinal data, rich multimodal imaging parameters, same-scanner MRI acquisition, advanced and appropriate statistical modeling, and inclusion of important covariates, the present study is well-situated to test hypotheses about cross-sectional and short-term longitudinal associations between serum S100β and brain structural aging.", "High and increasing concentrations of serum S100β at this age is identified here as a potentially meaningful marker of poorer brain white matter health and, with further testing, risk of future dementia.", "These findings require replication in other well-powered healthy and pathological aging samples, and across a longer time period.", "The authors have no actual or potential conflicts of interest." ]	[]
Dataset of leisure time among students at Kermanshah University of Medical Sciences and its relationship with health-related quality of life (HRQOL)	The present data article aimed to investigate the leisure time among students at Kermanshah University of Medical Sciences and its relationship with health-related quality of life. In this descriptive and correlational data article, the statistical population consisted of 420 students at faculties of Health, Paramedics, Nursing and Midwifery in Kermanshah University of Medical Sciences who were selected through multi-stage cluster sampling. For data collection, the demographic questions, 36-item short form survey (SF-36) and a researcher-made leisure time questionnaire were utilized. for data analysis, the descriptive and inferential statistics (Pearson correlation coefficient) were employed in the SPSS0.23 Statistics Software. The obtained data of the present data article demonstrated that the means and SDs of students’ leisure time and health-related quality of life measured 3.25 ± 0.61 and 2.50 ± 0.41, respectively. Also, the obtained data indicated that the quality of life significantly and positively correlated with students’ leisure time. In addition, the obtained data showed that in-person communication had the highest relationship with the total quality of life. In contrast, no relationship was found between the artistic activities and the total quality of life.	[ "MULT" ]	[ "Health-related quality of life (HRQOL)", "Leisure time", "University students" ]	[ "1000" ]	[ "Of the whole 420 subjects, 220 subjects (52.4%) were female and 200 subjects (47.6%) were male, and the average age of participants was 21.96 ± 3.70.", "In addition, the under-20 age range was in the majority (221 subjects or 52.6%).", "In terms of marital status, the majority of participants were single (390 subjects or 92.9%).", "Besides, in terms of education, 339 subjects (80.7%) were doing a bachelor׳s degree, and the majority of samples were majoring in nursing and midwifery (198 subjects or 47.1%).", "Moreover, in terms of the Residence, 201 subjects (47.9%) were living in dormitories, and the majority of students were born in urban areas (316 subjects or 75.2%) (Table 1).", "The mean and standard deviation of the total score of students’ leisure time measured 3.25 ± 0.61.", "The obtained data also demonstrated that in-person communication and training classes had the highest and lowest relationships with the total quality of life (3.40 ± 0.72 and 3.14 ± 0.80, respectively).", "It was found that the mean and standard deviation of the total score of students’ quality of life measured 2.50 ± 0.41.", "As for the health-related quality of life of samples, the obtained data indicated that vitality and limitations related to physical problems had the highest and lowest means and standard deviations (3.53 ± 1.18 and 1.18 ± 0.19, respectively) (Table 2).", "The obtained data demonstrated that the relationship between the total leisure time and total quality of life of students was positive and significant.", "It was found that in-person communication had the highest relationship with the total quality of life.", "In contrast, no relationship was found between the artistic activities and the total quality of life (Table 3).", "Here, 420 students at Kermanshah University of Medical Sciences were studied in 2017.", "Moreover, research samples were selected in two stages.", "In the first stage, out of seven colleges affiliated to Kermanshah University of Medical Sciences, three colleges (health, nursing and midwifery) were selected, and in the second stage, the samples were selected through multistage cluster sampling and Cochran sample size formula.", "Further, the ethical principles employed included obtaining the necessary permits, retaining the rights for the schools being researched to either accept or reject to participate, and ensuring the confidentiality and non disclosure of the personal information of samples.", "Then questionnaires were distributed among the target sample.", "To this end, the objectives were explained to the target subjects Informed consent was obtained from all participants before data was collected.", "Not to mention, the exclusion criteria were the samples disinterested in participation in the study and handing over incomplete questionnaires.", "As for data collection, three questionnaires were used: a demographic questionnaire, a researcher-made leisure time questionnaire, and the health-related quality of life questionnaire by Ware and Sherbourne.", "Demographic Questionnaire: It consisted of seven items: gender, age, place of birth, marital status, residence, education, and faculty.", "Leisure Time Questionnaire:This 45-item instrument was developed by the researcher to assess the status of spending leisure by the samples under data article.", "In the formulation of this questionnaire, the studies conducted by the National Youth Organizations about the young׳s leisure time and lifestyles were reviewed [2–9,1,10–14].", "The questions were carefully designed and tailored to the students’ conditions.", "The subscales of this questionnaire were in-person communications (3 items), training classes (6 items), recreational activities (14 items), data article (3 items), artistic activities (3 items), unplanned activities (5 items), sports activities (5 items), and religious activities (6 items).", "Furthermore, the questions were answered on a five-point Likert scale: everyday (100), several days a week (75), several days a month (50), rarely (25), never (0).", "Each question had a score in the range of zero to 100.", "A score of zero means that the person does not do the activity under data article, whereas a score of 100 indicates that the person is highly engaged in doing the activity.The face and content validities of the instrument were confirmed by the health and sociology experts.", "In addition, the reliability of the instrument was confirmed through calculating the Cronbach׳s alpha (0.84).", "36-item Short Form Survey (SF-36):This 36-item scale, developed in America by Ware and Sherbourne [1,15–18] was designed for assessing health and quality of life.The subscales of this questionnaire were physical functioning (10 items), limitations related to physical problems (4 items), emotional role functioning (3 items), vitality (3 items), mental health (6 items), social role functioning (2 items), bodily pain (2 items), general health perceptions (6 items) [16,19,20].", "A score ranging from 0 (indicating the worse health status) to 100 (indicating the best health status) was assigned for each domain [18].", "Furthermore, the Cronbach׳s alpha was used to determine the reliability of this instrument (0.85) [21].", "Additionally, this instrument was standardized by Montazeri et al. [22].", "in Iran, too (0.70).", "Furthermore, for data analysis, the descriptive (frequency distribution, mean, and standard deviation) and inferential statistics (Pearson correlation coefficient) were employed in the SPSS Statistics Software Version 23.0, and it should be noted that the related presumptions were examined prior to the above-mentioned tests." ]	[]
Ten essentials for action-oriented and second order energy transitions, transformations and climate change research	The most critical question for climate research is no longer about the problem, but about how to facilitate the transformative changes necessary to avoid catastrophic climate-induced change. Addressing this question, however, will require massive upscaling of research that can rapidly enhance learning about transformations. Ten essentials for guiding action-oriented transformation and energy research are therefore presented, framed in relation to second-order science. They include: (1) Focus on transformations to low-carbon, resilient living; (2) Focus on solution processes; (3) Focus on ‘how to’ practical knowledge; (4) Approach research as occurring from within the system being intervened; (5) Work with normative aspects; (6) Seek to transcend current thinking; (7) Take a multi-faceted approach to understand and shape change; (8) Acknowledge the value of alternative roles of researchers; (9) Encourage second-order experimentation; and (10) Be reflexive. Joint application of the essentials would create highly adaptive, reflexive, collaborative and impact-oriented research able to enhance capacity to respond to the climate challenge. At present, however, the practice of such approaches is limited and constrained by dominance of other approaches. For wider transformations to low carbon living and energy systems to occur, transformations will therefore also be needed in the way in which knowledge is produced and used.	[ "ENER", "SOCI" ]	[ "Action research", "Sustainability", "Sustainability transitions", "Transdisciplinarity" ]	[ "2102", "2103", "2104", "2105", "3301" ]	[ "In a world with a changing climate significant societal change is inevitable.", "Keeping the world well below 2 °C rise in temperature relative to pre-industrial levels will require extensive and rapid social and technological transformations, including in the systems, structures, worldviews and beliefs underpinning climate change and other contemporary challenges [1,2].", "This raises a critical question for humanity: how can rapid and transformational societal change be achieved to prevent dangerous levels of global warming?", "While science has so far excelled at understanding the climate problem and identifying techno-centric solutions, it has so far largely failed to seriously engage with the critical question of how to make transformational change happen.", "Addressing this and other related questions requires a diversity of approaches to knowledge production [3].", "Importantly, many contemporary challenges have emerged through the success of science over the last 300 years, such as through technologies to extract and use fossil fuels that have led to human induced climate change.", "Thus, while science has clearly brought many benefits, it has also resulted in new challenges that require new ways of thinking to address them [3,4].", "These approaches need to be able to take into account normative aspects, inequalities, politics and power, and work more directly across the interface of science and practice [4–6].", "Many alternative forms of research that are more democratic, inclusive, action-oriented and integrate different forms of knowledge have emerged over the last three decades.", "This includes mode 2, transdisciplinarity, post-normal, participatory, sustainability science and action research [7–12].", "As yet, however, there has been no integration of these insights specifically for researchers aiming to inform and facilitate the transformational changes necessary to address climate change and help achieve more sustainable societies.", "Further, while all forms of research have value, effective responses to climate change require a much more direct and concerted effort towards learning from and through action [13].", "This paper therefore presents 10 essentials we believe are important for researchers to achieve greater impact from their work in relation to energy transformation and climate change.", "The paper does not suggest that research that does not apply all of the essentials is not useful, and working towards applying any of these will add value.", "However, when applied as a collective, the essentials represent a considerable shift in the way research is conducted that will generate more significant impacts for addressing the climate challenge and legitimise the inclusion of a greater diversity of kinds of knowledge, perspectives, values, imaginations and approaches needed to facilitate transformations to a low-carbon, resilient world.", "Overall, while the emphasis is on climate change and transformation, the paper will be of wide relevance to any field of study that seeks to enhance societal outcomes.", "The paper first explains the need for more action-oriented research and the concept of first and second-order science, which frames the rest of the paper.", "We then explain the 10 essentials, followed by a discussion about the challenge of encouraging greater attention to the kinds of research that will more effectively accelerate the learning needed to stimulate transformations in the context of climate change.", "There is a growing emphasis on research agendas and programmes relating to understanding how to achieve deliberate societal transformations to avert the threat of climate change [1,14,15].", "While there are many definitions [16], transformation is broadly a process leading to marked and qualitative change [17] and processes that lead to fundamentally different forms of thinking, actions, systems and structures (Table 1) [18].", "Clearly, such long-term changes come about as a collection of short-term, and often emergent actions [18] and that research processes are critical in shaping these.", "Thus, while there is extensive debate about whether transformative change can be achieved sufficiently quickly to mitigate and adapt to climate change [19,20], the challenge of finding ways to create the necessary shifts in the systems, structures, assumptions and worldviews underpinning climate change remain [21].", "A considerable volume of untapped knowledge about social change from the arts, humanities and social sciences already exists that can inform transformations towards low-carbon, resilient living [22,23].", "This includes, for example, a large and growing body of knowledge on large-scale systems change [18], historical dependencies, social and technical innovations, practices and processes for change [24–26], individual, cognitive, systemic, cultural, corporate, legislative, power and political dimensions that inhibit or enable change [2,27–31], climate policies and strategies [32–34], climate insurance [35], normative aspects (values, ethics, aesthetics) and how to work with uncertain futures [36–38].", "Yet despite the vast amount of knowledge already accumulated, there is still limited emphasis on understanding how to implement change.", "This ‘how to’ question is now arguably the most important question for climate research.", "One of the key reasons for limited engagement with the ‘how to’ question is because implementation has traditionally been confined to the domain of practice, in part due to a dominant culture in science where implementation is viewed as political, normative and future oriented and hence not amenable to scientific analysis [39].", "This is highlighted by the work of influential organisations, such as the Intergovernmental Panel on Climate Change (IPCC), which has focused on providing evidence of the problem and identifying broad pathways.", "As a matter of principle, and through influences from UNFCCC, Conference of the Parties (COP) and national focal points, the IPCC aims to stay away from being policy-prescriptive.", "That is, it avoids normative statements about how assessment findings should be acted upon, under the assumption that the latter is the role of politicians.", "While there are good reasons for the approach (e.g. to appear impartial in a highly politicised context and avoid the problem that implementation often requires locally specific approaches), it has meant that most of the focus has been on understanding the causes and impacts of climate change.", "It also produces largely descriptive mitigative and adaptive solutions, including of their costs and barriers to implementation, but providing very little critical assessment of how solutions are being implemented and to what effect.", "This not only means that critical knowledge about implementation gets omitted in reports, but also contributes to wider perceptions about the usefulness of the different kinds of research and knowledge needed to address ‘how to’ questions, ultimately slowing progress towards identifying and learning about implementing solutions.", "There are many examples of work at the interface of academia and practice and a growing trend towards more impact driven knowledge, co-creation of outcomes from research and practice, and greater engagement of researchers in interventions seeking to enact change [40].", "Yet such work still remains at the fringes of more dominant approaches.", "If the goal is to enhance understanding about how to make transformative change happen, a massive upscaling of research that works more directly with practical domains will be needed.", "Nothing less than a radical shift towards large-scale expansion of more action-oriented knowledge production will be required that: takes into account the real world of politics, values, and ethics that characterise societal change [3]; works with academic and practical forms of knowledge; embraces creativity, imagination and innovation as a form of knowledge production [6,41]; and is more explicit about its relationship to society [4].", "Thus, while the major advances in knowledge production over the last 300 years are extremely important, a major shift towards acceptance and incorporation of new forms of science and research is also needed [4].", "In short, transformations towards more viable systems of knowledge production and use are required for wider societal transformations in response to climate change to occur.", "One of the reasons why there has been limited attention in research on solutions and their implementation is the primary assumption underpinning much of the sciences and social sciences that an observer is, and can be, independent to that which is observed [42].", "Invoking this assumption has had a powerful effect on the ability of humanity to produce certain kinds of knowledge, even though this assumption is largely flawed.", "It is widely recognised in the social sciences and humanities, for example, that it is impossible for a researcher to be independent: cognition, prior experience, understanding, scientific paradigms, and societal influence such as cultures, politics and the ‘hot topics’ that receive funding all affect how research is conceived, conducted, interpreted or used [13,43–46].", "Through theories, concepts, and findings researchers also influence society, which in turn reinforces how researchers or the public perceive and approach the world in which they are embedded [45].", "Thus, researchers are inevitably embedded within, and not separate from, the systems they seek to observe.", "Researchers are also arguably always interveners.", "Intervention is the “purposeful action by a human agent to create change”, where action is influenced by knowledge, including perceptions, implicit understandings, conscious and unconscious motivations, as well as values, morals, ethics and norms and behavioural habits ([47], p. 113).", "Science can thus be understood as an active process of intervention, either directly in practice or more indirectly through the generation of knowledge.", "This includes both applied science (e.g. climate science to develop knowledge to inform policy or agricultural science to directly improve farming practices) as well as curiosity-driven research (e.g. the production of a research paper which ‘intervenes’ in the thinking of other scholars).", "Because observation is just one type of intervention, scientific techniques are part of a more pluralistic set of intervention methods, including methods for exploring values, reflecting on subjective understandings and planning future activities [48].", "Viewing science as intervention places greater responsibility on researchers to be more explicit about the reasoning behind the decisions they make throughout the process of scientific enquiry.", "Importantly, choosing to focus on one form of intervention means avoiding doing another.", "Researchers therefore need to be more explicit about what kind of intervention they choose to engage in [47].", "For an unprecedented issue like climate change where urgent action is required, what is researched and where resources are allocated matters [49].", "Focusing on gaining a better understanding of the climate problem on the assumption that this will lead to formation of policy and change may be laudable, but in the context of constrained research budgets and value-driven budget allocations, a focus on problems may be at the expense of arguably more urgent ‘how to’ questions that can no longer be ignored.", "Acknowledging that science is essentially a choice about focusing on a particular kind of intervention thus frees up possibilities for new questions, domains of application and different ways of learning about, and influencing change.", "These issues are well recognised in the field of cybernetics [50] where distinctions are made between first- and second-order forms of science.", "Second-order science rejects the assumption that an observer can or should be independent to what is observed [42].", "This then leads to the opening up of many possibilities (Table 2).", "For example, researchers making choices about how they intervene highlights the normative nature of science and that it occurs in conditions in which truth is not absolute [13].", "Acknowledgment of this can then lead to greater acceptance of multiple ways of knowing and recognition of the need for transdisciplinary approaches to science that actively include diverse stakeholders, which then provide new opportunities to learn more directly from and shape action [13,42,51].", "It also demands critical reflexive practice by individuals or collectives involved in research about their role as interveners and how they carefully balance the challenges of achieving methodological rigour while also being part of the system they are studying [51,52].", "In second-order science this may include researchers ‘dipping in and out’ of action to enable them to enhance learning about practical elements of change while also providing opportunities for more critical thought and analysis [47].", "Viewing research through the framework of first- and second-order science helps clarify the nature of different kinds of transformation and climate change research (Table 3).", "First-order transformation research involves describing and analysing processes of change [53,54], where results are then disseminated to beneficiaries through some kind of knowledge transfer [55].", "Examples include research on technological innovations, systems science research on global change and climate change [56], and historical analyses of large-scale socio-technical transitions [26], as well as many of the research questions proposed as core for climate and sustainability science [8].", "In comparison, second-order transformation research is more likely to view action, learning and the generation of new knowledge as being more closely intertwined (Table 3).", "It places greater emphasis on the research as a reflective practice [51], and focuses on creating change from within the system being studied rather than viewing it as an external problem [57].", "Importantly, while both first- and second-order science are inherently normative, a key distinction is that second-order science directly accepts this normativity and actively seeks ways to work with it in a meaningful way.", "First-order science, however, tends to assume that it is possible and desirable to work in a normative vacuum.", "This has enabled first-order science to make major advances through developing certain kinds of knowledge, but at the exclusion of others.", "This, in turn, has limited development of new kinds of strategies for addressing contemporary challenges like climate change.", "Overall, second-order science is more likely to privilege approaches like purposive transdisciplinary and action research which may explicitly focus on practical resolution of real-world issues and which challenge traditional notions of the validity of different kinds of knowledge [36,58,59].", "Some of the many examples of the application of key assumptions from second-order climate research are outlined in Table 4.", "Viewing research as first and second-order is helpful because it provides a meta-framing that makes explicit the underlying assumptions and features of many research approaches like transdisciplinarity, mode 1 and 2 science, or action research.", "The concepts of second-order science are also just as applicable to bio-physical as well as social research, with many aspects of first-order science still having a legitimate place within second-order science when observation is the intention [47].", "Importantly, while second-order transformation research itself has many challenges and is not a panacea [42,59–62], it does open up possibilities for more direct development of practical forms of knowledge, which are essential for informing how to implement change.", "At present, however, effort is still massively skewed towards first-order approaches [41,63].", "If the intention is to help bring about transformative change for societal benefit, then much greater focus on second-order science will be required [13].", "As yet there has been no synthesis of the diverse insights from second-order science and other traditions specifically used to guide those attempting to engage in more action oriented transformation, energy transition and climate change research (Fig. 1).", "In the following sections we therefore present ten essentials for second-order action-oriented transformation research.", "The essentials emerged over a two-year period from individual and collective reflection of the authors during three consecutive workshops at the Transformation conference in Sweden in 2015, a two-day professionally facilitated intensive International Futures Forum workshop in 2016 in Scotland on second-order science, further conversations at the International Sustainability Transitions conference in Germany in 2016, and many iterations and different discussions among the authors involved in this work.", "The process began with a collective frustration about the limited acceptance of second-order science in a world dominated by first order approaches, despite a real and urgent need for a greater diversity of scientific work.", "The iterative process initially led to the identification of key essentials, but without a conceptual foundation and sufficient clarity about their underlying assumptions.", "The concept of second-order science was then chosen as the most appropriate foundation and the essentials were further modified and developed under this framing.", "The process led to the essentials outlined below.", "The primary intention is to highlight critical assumptions and outline what is needed for a comprehensive approach to second-order transformation research rather than prescribe how to apply the essentials in practice.", "Nevertheless, many examples and references are also provided about their application.", "The first three essentials generally relate to the focus of the research: transformations; solutions; and practical forms of knowledge.", "These are then followed by seven essentials for practicing second-order transformation research that begin with wider aspects relating to how research is framed and approached and then moving towards more specific methodological aspects.", "The first essential is the need for research to explicitly focus on transformational change and how this is brought about.", "This can be through, for example, examining drivers and mechanisms of transformational change, the role of different kinds of disruptive changes and/or different transformative pathways, and the range of possibilities and uncertainties involved [64].", "It also requires being explicit about what is meant by transformation to avoid all types of change being labelled as transformative [65] and clarifying what is to be transformed and for whom transformation is intended.", "Importantly, the criteria for deciding what is or is not transformation is normatively defined by what is desired or considered legitimate.", "For climate change, consideration is needed as to whether change is of sufficient depth (e.g. intensity, quality, or whether it results in systemic forms of change), breadth (widely distributed), and speed (rapidly resulting in intended outcomes) [6].", "These dimensions may apply to different domains, such as cognitive, structural, relational, and functional aspects of the social, ecological, and technological (Table 5).", "Importantly, sustainable human-environment relationships are also critical aspect of considerations of what counts as transformation, and ecological and environmental aspects cannot be ignored in the attempts to shape societal change [66].", "This highlights the need for clarity about the normative goals of any transformative research (essential 5), which in the case of this paper is broadly towards social and environmental sustainability, such as that outlined in the UN 2030 Agenda for Sustainable Development [67].", "Overall, transformation research is inherently subjective, requiring researchers to be explicit about how their understanding of transformation and values and motivations shape their work and how they can more effectively contribute to facilitating transformative change.", "Second-order transformation research also needs to focus less on understanding problems and more on how to effectively and efficiently steer and facilitate transformations towards mitigation and adaptation [68].", "Problem-oriented research typically involves identifying the dynamics, impacts and causes of climate change and assessing who or what is affected [69,70].", "Understanding causes and effects, however, does not explain how to mitigate and/or adapt to such changes [71].", "Research instead needs to generate actionable knowledge contributing to processes giving rise to solutions to climate change problems.", "In research on solution processes, problem analysis is typically conducted less comprehensively and is viewed as a means to an end, and not an end in itself.", "Research on solution processes also then addresses two additional aspects: First, it addresses the aspired outcomes (e.g. desired states and dynamics) such as the future state of climate conditions suitable for human and ecological wellbeing; what the (transformed) human activities and emission pathways might look like in such a future; and aspects that need to change for such conditions to materialize, such as the values, norms, and legal structures [31,72].", "Second, it focuses on the processes that lead to the outcomes [73], such as understanding the pathways needed to achieve transformation; who would need to take what kinds of action and when; the resources needed to deliver the actions; and the kinds of barriers likely to be encountered on the pathway to change and how to overcome them [74–77].", "Examples of these kinds of projects include work on disaster recovery, renewable energy provision, alternative administrative purchasing practices, as well as substitution of chemicals and materials that have adverse impacts [63,68,78].", "These examples are a form of experiment, which is itself a key component of second-order transformation research (essential 9).", "Overall, this indicates that such action-oriented research requires combining problem analysis, visioning, assessment, and intervention methodologies [79].", "Learning about change requires more than identifying solutions: it also requires knowing how to implement change in practice.", "It is often not acknowledged, however, that this involves engaging with different kinds of knowledge to that traditionally found in much of academia.", "Academia is dominated by ‘episteme’, which is teachable and abstract [80].", "Practical knowledge, however, includes both ‘know how’ knowledge (techne), such as that used to install solar panels or to facilitate complex climate negotiations, and ethical and political-practical knowledge (phronesis), which relates to the ability to know what makes a good end and a viable, morally defensible path toward that end [80].", "Practical knowledge is typically embodied, difficult to articulate and often built experientially over many years for particular circumstances or contexts [81,82].", "Failing to acknowledge distinctions between academic and practical forms of knowledge has led to a largely misconceived gap between research and practice and attempts to make academic knowledge or theory more practical, rather than focusing on development of practical forms of knowledge [83].", "That is, reducing carbon emissions cannot be taught only through abstract knowledge like a powerpoint presentation: a learner also has to learn from doing it in practice [84,85].", "Failure to focus on these practical forms of knowledge can result in knowledge being produced that is distantly related to the actual and action-oriented needs of practitioners.", "There are three broad ways in which research around practice can be conceptualised [86].", "First, there is research into practice, where researchers observe that practice.", "Second there is research for/as practice, where practices are the intended outcome and researchers work with practice or as practitioners to develop knowledge, such as a when developing a new process of change or new technologies through experimentation and iteration [86].", "Here the outcome is usually a written report on the practice or the lessons learned and the impacts emerging from action.", "Research methodologies and theories are also likely to be developed during the process of doing practice rather than prior to engaging in it.", "Finally, there is research through practice, where the act of practice itself becomes the research [86].", "Here the emphasis is more towards developing the practice (techne and phronesis) rather than the epistemic knowledge about that practice.", "Thinking is then embodied in the artefact that emerges, such as a process or tool for change.", "Research through practice is generally lacking in the humanities, social sciences and sciences, although it may sometimes emerge in transdisciplinary and action research.", "It is, however, much more common in the arts.", "Artists are increasingly finding ways to demonstrate their work has rigor and quality that does not rely on the written epistemic word [86].", "Shifting towards research through practice that more directly recognises techne and phronesis has major potential for encouraging a more engaged and rapid approach to transformation research.", "For example, it is not inconceivable to have a research project that creates an imaginative and transformative policy.", "Here the policy development is the act of knowledge creation itself, with the outcome being the final policy (artefact) that embodies extensive creative, discursive and collective know-how and phronesis forms of knowledge.", "Thus, in addition to developing more actionable epistemic forms of knowledge (essential 2), approaches from the arts can provide more radical and direct approaches to learning about social and environmental change.", "Examples of such work already exist from action, transdisciplinary and activist research traditions [87,88].", "Ultimately, a focus on practical knowledge for stimulating transformations and moves towards low-carbon, resilient living will only emerge when different forms of knowledge and different ways of assessing them are more widely accepted.", "This is challenging given that existing disciplinary structures and cultures tend to be very resistant to more pluralistic forms of producing and using knowledge.", "The fourth essential involves conceptualising research as being conducted from within the system being studied.", "Developing practical knowledge requires a shift from researchers viewing themselves as being ‘apart from the universe’, such as looking as if through a peephole upon the unfolding universe, to viewing themselves as ‘a part of the universe’, implying that when they act, they are also changing themselves and the world around them ([89], p. 293].", "These different ways of conceptualising science have significant implications (Table 6).", "Importantly, moving towards conceptualising science as being from within allows for a powerful widening of the scope of research processes and for a shift towards explicitly acknowledging research as being an actor that is part of the process of promoting change [90].", "While there are a large and growing number of examples of approaching research from within (e.g. [91,92]), the vast majority is still conceptualised as being from outside that which is being studied.", "This leads to an emphasis on working with data, statistical methods, models, and development of theory rather than practical problem solving, how research findings can be implemented, and limiting reflection back to question how the research is being framed [39].", "This is particularly prevalent in many high-profile climate change research syntheses.", "Conceptualising research as being from within enables the goal to focus on social improvement as opposed to primarily knowledge production which dominates research that is viewed as being conducted from the outside [39].", "It encourages greater acceptance that applied work lies within the realm of science and a greater focus on knowledge about implementation and action [39].", "Finally, it places considerable emphasis on the need for researchers to be more cognisant of the role of their own underlying assumptions that shape the nature of the questions posed (e.g. essential 6) and to continually reflect on their role and influence in the processes of research and change (essentials 8 and 10).", "Viewing science as being conducted from the outside has led to separate institutions (e.g. universities) as the legitimate producers of knowledge which perpetuate distinctions between knowledge production and practice.", "There are now a growing number of alternative institutions that have a stronger focus on climate change solutions rather than on generating knowledge directly.", "Examples include the Coastal Resources Centre (http://www.crc.uri.edu/), the Dutch Institute for Research Transitions (https://www.drift.eur.nl/), the Edinburgh Centre for Carbon Innovation (http://edinburghcentre.org/), and the MIT Climate CoLab (http://climatecolab.org/).", "Such institutions are situated at the interface between academia and practice and involve participatory projects with measures of success relating to implementation and change as well as generating knowledge.", "They represent examples of the kinds of transformative institutional innovations that are needed that can challenge structures and norms developed to support first-order patterns of knowledge production and use [93].", "While there has been extensive research on identifying values [94] and identifying the normative challenges in transformational change processes [31], researchers across the social and natural sciences rarely acknowledge the values and ethics that shape their own research [95].", "Values and norms always shape what and how research is conducted [96].", "Failure to acknowledge this can, at best, lead to omission of important perspectives and opportunities for learning that affect what and how something is understood.", "At worst, it can also lead to deliberate production of ignorance, where science may be used to exploit uncertainty, such as has occurred for tobacco, asbestos and climate change [97].", "Engaging with climate research, for example, raises important ethical responsibilities of researchers in supporting others to adapt and find ways to reduce carbon emissions [49].", "Given that all researchers are essentially interveners [47] climate researchers need to be more than just informed by past science: they also need to be transparent and accountable about the choices made about what science is undertaken, and how it is funded and communicated [49].", "This requires acknowledging the normative role of the researcher, critically questioning and reflecting on the values underlying choices early on in research [98], being transparent about these, and taking responsibility to consider how their research will contribute to addressing the unprecedented challenge of climate change.", "One way to approach this is to actively acknowledge and build normative aspects into research programmes.", "Normative aspects have, for example, been explicitly integrated into the British Permaculture Association’s research strategy, where researchers trained in formal research working with and within the permaculture movement conform to three permaculture ethics (earth care, people care, fair shares).", "This has significantly changed the aims, processes and outcomes of the research [85] and has encouraged framing of research in societal terms rather than research framing society [99].", "Greater receptivity to ethical concerns and the need to negotiate normative aspects in science will grow as demand for practical outcomes from research increases [39].", "More specifically, key issues emerge when focusing on transformation and climate research which implies being involved in a process of changing the status quo.", "Second-order transformation research is thus inherently political, as it involves exploring how incumbent systems and power might break down allowing for a broader societal shift towards transformative alternatives.", "This poses key challenges for operationalizing second-order transformation research in a world dominated by assumptions that knowledge creation is separate from politics and where knowledge creation occurs first, followed by dissemination and then decision-making about how to act.", "One solution to help address such challenges is to co-develop ethical codes with participants [100] and facilitate dialogue over important concepts such as value, resilience, agency, ideology, knowledge, and power as part of the research process[101].", "Without doing so, there is a danger of transformation research becoming a powerful depoliticizing practice [102], unintentionally reproducing unfavourable market settings, social inequalities and exploitive institutional relations inherent in the systems and structures of society that continue to contribute to climate change.", "Transformation is a change process that involves creation of previously unimagined possibilities including new ways of thinking and action (Table 1) [18].", "As Moran highlights, being intelligent about complexity involves exploring possibilities without being restricted to what is formally probable [103].", "Yet science and research are naturally conservative, with a tendency to progress incrementally rather than through more fundamental change.", "The seventh essential therefore involves seeking new ways to open up space for new questions, insights and solutions that can transcend current paradigms and disciplines.", "An example of such an approach is re-entry, where first order concepts are applied back on themselves resulting in new meta-concepts and emergence of new questions (see examples in Table 7).", "Through re-entry, old concepts “appear to close around upon themselves” while also leading outward to transcend existing boundaries in ways that seem to “have turned inside out, [where] the inside is the outside” ([104], p. 131).", "An example of applying re-entry is asking questions about the sustainability of sustainability research and initiatives, such as the extent to which sustainability research and action projects contribute to production of carbon emissions [105].", "By asking how sustainable sustainability projects are, attention is drawn to wider systemic issues associated with the delivery of sustainability initiatives that may conveniently be ignored because they are usually seen as too difficult to address.", "For example, asking how sustainable approaches to sustainability research might be implemented raises questions about the way in which research is part of and reinforces internationalisation and economic growth agendas or why funding regimes for research construct ever more intricate understanding of problems without proper investment in solutions.", "Applying the process of re-entry to many other questions would stimulate new thinking about transformations, such as when asking about how transitions of transition research occur; how systems of systems inhibit or enable change or how knowledge about knowledge influences transformation.", "Re-entry thus provides new ways to frame problems, identify questions, generate new research fields, and to enhance innovation [106,107].", "By applying re-entry, attention is opened outwards as well as forced back on the assumptions underpinning the methods, approaches, and the paradigms underpinning research and climate change projects.", "Re-entry is an example of highlights the need for new ways to generate questions that will lead to more transformative findings.", "Such approaches require higher degrees of integration, recognition of the systemic nature of issues like climate change, reflexively thinking back on the systems being studied and the need and possibilities for more post-disciplinary forms of science [13,42,103,104,106,107].", "Thus, while many important first-order questions need to be answered to inform sustainability [8,108], transformation research needs to find new and diverse ways to help researchers transcend and critique the very systems in which they are themselves embedded.", "The sixth essential to support second-order transformation research in the context of climate change involves taking a multi-faceted approach.", "It is well known that different paradigms, methodologies and methods affect the interpretation of phenomena and the way in which subsequent actions are prescribed [45,109–114].", "Climate research has been heavily criticised for being dominated by particular ontological perspectives about what is considered real and epistemological perspectives about what constitutes knowledge and knowing.", "These lenses have led to the promotion and acceptability of certain kinds of questions, approaches and knowledge at the expense of others [46,112,115–117] and to the framing of climate change as an environmental rather than a social or political problem [46,115].", "Ontological and epistemological lenses also have a major influence on the nature of the solutions that emerge from analytical insights [115,118].", "A well-documented example includes analysis of decision-making for fisheries management in Sweden, where positivist lenses led to a focus on calculations and modelling of the fisheries at the expense of social complexity, such as human agency, power, and influence [118].", "This resulted in the predominance of market-based resource management as the primary guide for solutions, such as emphasis on privatization and on values related to entrepreneurship or investment power, as opposed to solutions focusing on participation and dialogue that acknowledge dynamics of conflict [118].", "This example shows how ontological perspectives implicitly shape both social reality and the nature of outcomes at the expense of others and illustrates how closely research is intertwined with politics, norms, values and culture [118].", "Pluralistic approaches can help to avoid narrowly prescribed solutions [109,111].", "In pluralistic approaches the objective is not to unify multiple lenses, which can reduce diversity, but to mesh or link different ways of understanding the world.", "This may involve diverse societal values and perspectives.", "One way to approach this is through applying different ontological perspectives relating to four domains of complexity [109]: (1) Natural-world complexity through focusing on ‘what is’ through the search for truth; (2) Social-world complexity through focusing on ‘what ought to be’ in relation to actual or potential action through the search for what is right; (3) Subjective-world complexity through focusing on understanding what individuals are thinking, intending, or feeling; and (4) Interaction complexity, by focusing on the interactions between the other three domains.", "These domains can be used to help guide the development of pluralistic methodologies (e.g. quantitative approaches for natural world complexity; quantitative and qualitative approaches for social world complexity; and qualitative approaches to subjective complexity) that then lead to the application of mixed methods [109].", "Importantly, this approach does not claim that it is possible to work across paradigms.", "Instead, it seeks to promote the setting of a new position to encourage learning from different paradigms by incorporating different ontological positions [109].", "In order to have significant (transformative) shifts in thinking through these processes it is critical to find a common language to facilitate exchange and ensure that potentially transformative transdisciplinary work does not get subsumed back into multi-disciplinary ways of operating.", "Overall, to generate more relevant, salient, legitimate and transformative insights, second-order transformation research will need to apply such multi-faceted approaches and find ways to facilitate communication across paradigms.", "Enhancing learning about how to foster change often requires greater flexibility in the roles undertaken by its participants to assist collaboration, co-creation of ‘know how’ knowledge and to stimulate action [84,119,120].", "Sometimes multiple roles are taken, with those involved regularly switching between them and which evolve over time [121,122].", "There are diverse roles that may be undertaken by researchers (Table 8), such as facilitating interactions [123], mediating between different frames [123], acting as knowledge brokers [73,124], assisting putting sustainability into action [125] or guiding action [84,87,88,126] (Table 8).", "Practitioners may also take a much more active role in research as they are often in better positions to learn about practice than an external researcher [120].", "They can play important roles in framing questions, developing methodologies or even conducting research [101], such as through Science Shop arrangements, (http://www.livingknowledge.org/livingknowledge/science-shops), Public Laboratory Networks (http://publiclaboratory.org/) or in participatory action research and evaluation [127,128].", "Applying alternative roles is common in design [129], transdisciplinary [59], participant observation [43,130] and action research [84,120].", "A good example where both researchers and practitioners took on different roles was the UK’s Soil Association programme on research into organic farming, where farmers conducted their own on-farm experiments while academics provided advice about how they could improve the research (http://www.soilassociation.org/innovativefarming/duchyfuturefarmingprogramme/fieldlabs).", "Here the farmers were the researchers while the academics helped to improve the learning.", "Together this generated more practically focused work but also capitalised on the expertise researchers have in enhancing the production of knowledge.", "While such approaches can enhance learning, they also present challenges.", "This includes potential conflicts between roles, such as when a researcher simultaneously needs to be an independent facilitator while also being dependent on activities that enable data collection [128], or when trying to shape action whilst attempting to retain rigor in their analysis of change [131].", "Attention therefore needs to be given to cultivating skills and dispositions for towards self-reflexivity (essentials 4, 5, 7, 10) and developing awareness of the limits to skills and knowledge regarding the different roles undertaken.", "An important task for second-order transformation research is to produce evidence and enhance learning about the actions and solutions that lead to desirable social, cultural, ecological and economic futures.", "Experimenting with change processes, such as through local and context-specific actions, projects and initiatives is therefore a critical part of transformation research [132–136].", "In first-order science, external control is a critical hallmark of experiments.", "In second-order science experimenting, however, the intervention happens from the inside where researchers have differing degrees of control on the intervention, system and context (essential 4).", "This is different to the concept of natural experiments, which still generally assume that research is conducted externally [137].", "Second-order transformation experiments are more akin to learning by doing.", "This can include four processes: (i) Integration, where external knowledge is incorporated and coordinated to inform interventions and actions; (ii) trial and error, where new actions are undertaken until it succeeds; (iii) repetition, whereby activity is improved by undertaking it multiple times; and (iv) extension, where the learning is used for larger and more complex activities [138].", "Participatory methods are often likely to be used to foster learning among actors, encourage systemic thinking, enhance social outcomes and encourage questioning of underlying assumptions [128], as well as simultaneously producing evidence about, and action for, solutions.", "Overall, conceptualising second-order transformation research as experimenting helps ensure focus on learning from the action as much as generating tangible and actionable outcomes.", "It places onus on the actor and/or researcher to be clear about who is supposed to learn (the researcher, immediate stakeholders, or wider practices or academic communities) and on finding the best way to improve rigor of both the methods used to learn (the research) and the intervention itself (the action).", "Opportunities for second-order experimenting emerge from a focus on solution processes (essential 2) with diverse examples from: community development [119,139]; mobility and healthcare; sustainable urbanization [140] and climate change (e.g. urban, transition and social innovation labs) [133,141–143].", "Such experiments deliver innovative practices and require an open and inclusive governance context [140].", "They follow a prescriptive and normative logic and actively seek to be part of the process of fulfilling societal needs in fundamentally new ways [39,136].", "Second-order experimenting is not without challenges.", "Standardized methods to replicate results are not likely to be possible and notions of reliability, scalability and transferability will not always hold [72,144].", "The advantage of second-order experimenting, however, is that it provides real-time opportunities for learning that cannot be achieved through traditional approaches and flexibility in being able to respond to the challenges of attempting transformation in practice.", "The key issue for second-order experimentation is thus to capitalise on the opportunities for learning provided by interventions that are already happening and more quickly feed this back to enhance action.", "Engaging in second-order transformation research requires those involved to be reflexive (Table 1) [13,30,145,146].", "Reflexivity is the critical exploration of how perceptual, cognitive, theoretical, linguistic, political and cultural circumstances influence interpretations [147,148].", "It is a deeper learning process to the relatively superficial act of reflection [149] and enhances capacity for innovation and insight (e.g. [145,146,149]).", "Reflexivity entails scrutinizing the “things usually taken for granted, in such a way that their historically grown self-evidence is challenged” ([150], p. 84), similar to what Latour framed as critique and critical inquiry as core principles of science [151].", "This includes engendering scepticism concerning one’s knowledge and value stances as well as the views of others and making explicit underlying values and assumptions [114].", "Reflexivity is important for ensuring research opens up, rather than closes down, space for active critical contention over the emergence of alternative societal pathways and attention to marginalised interests [152].", "It can also help actors or researchers of change examine how they are part of the system in which change is desired [52,148].", "Transcendent forms of reflexivity [148], for example, may involve the researcher critically reflecting on how they may need to undergo their own transformations in order to be in a better position to understand or shape transformative change in the systems in which they are embedded.", "An example of an effective approach that capitalises on deep reflexivity to learn from and simultaneously encourage change is Reflexive Monitoring in Action.", "This involves continual reflection on long-term goals and supports learning and systemic level changes [30].", "A reflexive monitor (e.g. researcher) stimulates reflection through being a sparring partner of the change agents involved.", "Emphasis is placed on the extent to which new rules, relations and systemic level changes are being achieved while maintaining sufficient distance to take a critical stance if and when needed [30,153].", "The reflexivity keeps ambitions high and helps break through path-dependency in situations where relapses into old patterns are likely to occur [153].", "Such approaches have helped reorient existing socio-technological systems (e.g. animal husbandry, water management, and local food chains) [153–155] and are applicable to the arts, humanities and the social and natural sciences [156].", "These examples highlight how reflexivity is important for helping action-oriented research to: maintain emphasis on transformational change (essential 1); ensure continued focus on practice (essential 2–3); assist in identifying questions, conception, design and implementation (essentials 4–9).", "Reflexivity is also a skill and capacity, which in turn requires practice, training, and being disposed towards applying it on a regular basis [131].", "Overall, reflexivity provides an overarching approach to assist the other essentials outlined in this paper and to enhance the ethical conduct of second-order transformation research.", "This paper has provided ten mutually reinforcing essentials for second-order transformation research in the context of climate change which incorporate many of the rich and diverse traditions of action-oriented ways of producing and working with knowledge.", "The essentials are underpinned by explicit assumptions and together open up space for more engaged and interactive forms of research that can work with normative dimensions, seek new ways of understanding, more directly support action and societal goals and provide new ways to enhance learning about the practice of transformative change for a low-carbon, resilient world.", "While application of individual essentials will enhance action and understanding about the implementation of transformative solutions, the greatest impacts will be achieved when the essentials are applied together.", "Jointly practicing the essentials will create a highly adaptive, reflexive, relational, collaborative and impact-oriented form of research that has a strong impetus to engage with action.", "Their joint application would provide coherence and intellectual depth in ways that expand the explicit and normative aspects of research needed to address the climate challenge while also encouraging recognition of the responsibility of research and researchers in this process.", "Applying appropriate forms of monitoring and evaluation will be important to both guide the joint application of the essentials and to enhance learning about the practice of second-order climate change and transformation research.", "While the essentials highlight much about what needs to be applied, they do not fully prescribe how this should be done.", "A full prescription is beyond the scope of this paper and much has already been written relating to the specifics of method and how to achieve standards of quality [87,88,157].", "Nevertheless, application of the essentials is challenging and requires developing skills not often supported in current research training and learning from what is already known about how to conduct action-oriented research.", "Thus, in addition to working more closely to develop practical knowledge about transformation, researchers and practitioners will also need to develop practical knowledge about the practice of doing new kinds of research [158].", "Greater attention to the practice of second-order science would have many benefits.", "It would encourage expansion of the scientific focus from being predominantly theoretical to being a more mixed form of theoretical and practical problem solving.", "This, in turn, would help facilitate the development of more practical theory while also improving theoretical approaches to practice [39].", "There is extensive demand for a greater mix of the conceptual and practical, as highlighted in the recent publication of the top 100 solutions to climate change, which was instigated by a non-academic frustrated with the lack of emphasis in research on climate solutions and which underwent four re-printings within six weeks of initial publication [159].", "Such work helps form new bridges between basic and applied research and can lead to more rapid diffusion of new research approaches and solutions.", "Unfortunately, wider-scale engagement in second-order science is still heavily constrained by societal and scientific norms, worldviews, values and political and institutional structures.", "While some shifts are beginning to occur, many of the ideas are not widely accepted or are only implemented in tokenistic ways.", "Some of the positive changes include tendencies towards interdisciplinary research, demands for research to be accountable to society which provide greater opportunity for engaging with practice and different perspectives [4,39,40,160] (essentials 2–6, and 8–10), and increasing access to technology for collecting and analysing data which enhances opportunities for second-order level investigations (e.g. through re-entry, essential 7) [161].", "Access to outcomes for research is also increasing (e.g. a strong emphasis on ‘open science’ in EU research funding) as well as trends towards greater participation in science by the public that encourages inclusion of more diverse stakeholders in research (essentials 3, 5 and 8).", "Finally, growing recognition that current ways of doing things are no longer fit for purpose are resulting in new practices associated with transformation and change [162,163] (essential 1).", "While these changes are helpful, they cannot always be taken at face value.", "Very little has changed in climate research over the last 25 years [23] and existing systems and structures that support first-order kinds of thinking have a powerful influence on what research gets funded.", "Climate research lacks certain critical voices [112] and interdisciplinary research can be more about realigning elite power bases in the face of change, such as through co-opting language and discourses, rather than representing deeper transformational change [4,99].", "Further, an integration imperative does not always equate to pluralism, and can exclude more innovative approaches to understanding and responding to climate change [164].", "Importantly, when usefulness is defined by those who seek to maintain the status quo, it constrains acceptance of the kinds of research which promote transformative change or which question dominant paradigms [163].", "Who decides what research is useful and for whom therefore matters.", "Overall, in the experience of many of the authors of this paper, there is a growing frustration by those outside of academia with the inability of traditional knowledge producers (e.g. higher education and research institutes) to contribute more directly to climate solutions.", "Much of the climate innovations come from the world of practice and the authors are aware of many academics who are struggling to understand how their work can become more relevant in a rapidly changing world.", "One of the major barriers to the application of second-order science is that funding for traditional institutions of knowledge production are usually tied to, and revolve around knowledge production rather than social and environmental improvement [39].", "New approaches are often not accepted or may even face fierce resistance because they do not conform to established first-order traditions.", "Incentives for academics are also heavily directed to first-order kinds of knowledge production, which severely limit opportunities for engaging in more action-oriented research.", "New ways of recognising and validating second-order science will therefore be needed, such as those that encourage development of practices of change through research as a practice [86] (essential 3).", "Shifts towards new forms of training of undergraduates and postgraduates that involve students being embedded in the practical context of their studies will also be required [50,131,165].", "The challenges of moving towards more fluid, iterative and action oriented research are not just confined to academia: deeply entrenched views on what research is supposed to look like, how it should be conducted, and how knowledge is used are also held in government agencies, non-government organisations and the public.", "Action-oriented research is inherently political and counter cultural [166,167] and can be heavily constrained by the limited time and resources of non-academics that may need to engage in the co-production of knowledge [101].", "Such research often experiences resistance, such as when organizational and individual norms and beliefs are challenged during a reflexive process [60,168].", "This is particularly the case for transformation research, which is likely to require questioning the social, governance, institutional and power dynamics that reproduce unsustainable or inequitable patterns in society.", "Change can be hard to realize, and unless there is generalized commitment to developing an organisation by learning from practice and to move away from formalized and codified forms of science and policy, outcomes will likely be instrumental rather than transformative [118,169].", "As such, researchers engaging in second-order science will require political acumen and well-honed adaptive expertise to flexibly apply a variety of strategies appropriate to different circumstances [167,170].", "They will also require appropriate support and enabling conditions to help them navigate the complexities of the political and normative issues involved.", "To enable second-order transformation and climate research, extensive and concerted efforts will be needed that challenge current systems and structures of the way in which knowledge is produced and used.", "This is a major challenge given the dominance of first-order approaches and powerful disciplines which are backed up by well-established assumptions, structures, institutions and finance mechanisms.", "Given the need for system transformation to enable greater engagement with second-order science, joint action at four levels will be required.", "First, support is needed for innovative researchers to actively practice and further develop second-order science as a viable and mainstreamed complement to the practices of first-order research.", "This would include initiatives such as building knowledge networks of champions of innovation; directly funding action-oriented research; strategically funding research on research (e.g. how to more effectively combine normative dimensions and rigour) and providing safe spaces at the boundary of science and society to test and apply innovations.", "Second, at a political and structural level bold and strategic action of politicians and funders is needed to pioneer and provide the supportive conditions necessary for second-order transformation research to flourish.", "Third, intermediary actors, such as advisory bodies (e.g. the newly formed International Council for Science and WBGU), will be required to strategically mediate between coal-face and political levels to further enhance enabling conditions.", "Finally, work is needed at public levels to increase demand for the co-production of action-oriented research (e.g. through engaging student projects in local communities or participatory forms of research) to help create a new social contract that provides greater support for action-oriented research [171,172].", "In conclusion, this paper has argued that while first-order modes of research are important, much greater emphasis is needed on second-order approaches that can accelerate learning and actions that lead to transformations towards a low-carbon, resilient and sustainable world.", "The paper has provided a framework for conceptualising such research and presented a set of ten mutually reinforcing essentials to guide its application.", "Currently, broader trends in society are unlikely to be sufficient for facilitating the massive upscaling of second-order transformation research we believe will be necessary to appropriately respond to climate change and many other contemporary global challenges.", "More deliberate and fundamental changes towards a greater appreciation of research for, or as, practice and research for societal improvement will be required.", "This, in turn, requires establishing enabling conditions, such as incentives, greater integration of research and practice, new forms of training, reconfiguring institutions, overcoming entrenched disciplines, and the legitimisation of diverse forms of knowledge and knowing.", "This needs to occur at all levels, including within research communities and in wider institutional and political systems and societal structures that influence the way knowledge is produced and used.", "Ultimately, this highlights that to accelerate the kinds of learning needed to enhance transformations for sustainability, transformations will also be needed in the way in which knowledge is produced and used." ]	[]
Building materials in eco-energy houses from Iraq and Iran	Builders from the Western part of Asia are trained to make buildings that can fulfil certain required functions while giving full consideration to all sites and environmental conditions. The research covers the zone between Iraq and Iran. The first investigated region is the "Mesopotamian Marshes" or Iraqi-Iran Marshes, a wetland zone situated in southern Iraq and partially in southwestern Iran. The other region is a desert district, which includes a prominent part of the southern and western parts of Iraq and part of Iran. The last is the centre city of Basra. The building materials were the most important building element that affected the conformation of vernacular habitats from the western part of Asia in general and the Iraq-Iran area in particular. In this study, we needed to focus on the effects of ecological and energy-efficiency processes in creating vernacular habitats and the selection of optimal building systems and materials in this part of the world, which can be an essential point for sustainable environmental building processes in the future. Reeds, clay, straw, bricks, and wood were the most popular building materials used by builders from this region. The impact of building material on the environment embodies the essential method implicitly significant in this research to effectively determine traditional building materials in the environment, in addition to comparative analysis. This presents an essential factor of our analysis, in addition to the impact of environments on building systems. The main target of this study is to benefit designers and building engineers in their pursuit to find optimal and competent solutions suitable for specific local microclimates using traditional methods in the design process that are sustainable and ecological.	[ "MATE" ]	[ "Ecological materials", "Energy efficiency", "Environmental profiles", "Vernacular habitat" ]	[ "2501" ]	[ "Vernacular habitats developed their individuality by tapping into nearby resources and exploiting them to confront the problems posed by a local environment.", "If rain is seasonal, the roof structure must also function efficiently in the dry season (Woolley, 2006).", "When it is warm, large, a high roof space continuum with the living area is practical.", "Temperature extremes call for thick insulating walls and roofs to maintain a comfortable internal environment (Almusaed, 2004).", "The most important question that needs to be answered is what is the relationship between the outer conditions of temperature, sunshine and rain and the inside thermal comfort.", "For different climatic sessions, it is necessary to take into account building, area, orientation, colours, etc.", "Although it is quite a complicated matter to theoretically solve these problems, it is possible to establish an empirical relationship between the exterior and inner environments for any building.", "In this way, the practical problem can be solved (Ginvoni Man, 1976).", "Throughout history, the human being has striven to erect shelters that fulfil basic needs relating to his survival, shelter that provides an environment favourable to his physical requirements.", "During his years of effort to achieve that environment, many local variations in dwellings have evolved (Wenzel, 1996).", "According to a UN document, the purpose of the habituation process is not to build houses but to ovulate a competent house.", "In other words, the house should provide the security of technical advice and be involved in acquiring small loans and cheap materials (Stulz, 1981).", "Climatic conditions compel the builder to find an efficient solution to serve the human being and the comfort of his existence.", "Building materials have an important impact on the home's functions and inhabitants.", "Environmental building materials add value to human life and reduce the negative impact on the environment drastically.", "In West Asia, climates, environmental elements with heavy buildings and shaded courtyards are common.", "These climates typically have a huge diurnal temperature range, and houses tend to employ an arrangement of these measures, with the particular mix being modulated to suit local micro-climatic characteristics.", "Selecting and specifying environmental building materials are often complicated processes (Jester, 1995).", "Builders from this region created their habitats taking into consideration the above-mentioned question, in an efficient manner and to fit human needs by adapting to local environmental parameters.", "Each building material displays the different physical characteristics; the structure requires rigid strength to support the dead weight of the roof and walls.", "If the situation dictates the range of fitting materials, together they command the form.", "West Asia's vast landmasses are diverse in geology and climate (Almusaed, 1997).", "This investigation has to find suitable solutions for building materials and then study different building materials from various sources and based on its characteristics of eco-energy house efficiency.", "The main lesson we can learn from this study is not only to further the conservation and repossession of the form of habitats already in existence, but also to rethink what can form new habitats for the future (Fig. 1).", "Climate is the main factor that directly affects the construction process and building materials in West Asia.", "This region is situated in the subtropics’ climate zone.", "It is affected by the subtropics’ high-pressure belt, which reaches the farthest north in the summer.", "The sky in West Asia is bright and glaring, even when thinly clouded.", "As one goes inland from the equator, seasonal differences in climate become more marked with a wetter wet season, and a drier winter period (Kukreja, 1978).", "There are many factors that affect building materials of traditional buildings from West Asia.", "Solar radiation deteriorates organic materials by initiating chemical reactions within the material and causing oxidation.", "Fig. 2 shows the monthly average temperature and sunshine hours in the West Asia Region.", "In this region, edifices have to be planned with great attention and thought, and with due respect for climate.", "Shade and protection from dust storms may have the highest priority in some areas, whereas in other areas, ventilation and the trapping of air streams and breeze are the primary goals.", "The effect of climate over the building materials is large; high temperatures, for example, can affect all types of materials in general.", "The speed of a chemical reaction doubles for every 10 °C increase in temperature (Nielsen, 2010).", "In addition, increasing levels of humidity can increase the deterioration physically and by creating an environment for harmful fungus and microbial growth as well as insect attacks.", "Changes in humidity also cause deterioration through changes in volume and stress within the material.", "AN environmental profile is achieved on the basis of the SBI (DK) calculation programme, BEAT 2000, which is a standardised technique for classifying and measuring the environmental effects related to building materials in their life cycles (11).", "It consists of seven parameters that cover all essential physical environmental impacts and effects.", "It is possible to examine an environmental profile indicator using the same indicator on another one.", "The first indicator is resource consumption, where the environmental impacts of resource benefits can be composed of a loss of resources and space consumption and risks of gas extraction or a cultivation process.", "The study will use three circumstances.", "The first is the ability of the material to engage in a recycling process, where it can provide for the immediate environment by reducing the use of new resources.", "Knowledge materials are a part of the renewable resource process, such as, in the building of plant materials, mainly wood, or non-renewable resources, such as mineral substances like brick, concrete and metals.", "The second indicator is the environmental impact of energy consumption.", "In this situation, the study will use three circumstances: renewable feedstock energy, the heat of combustion of renewable resources, where is wood is the principal base product; non-renewable feedstock energy, such as plastic; and renewable energy, such as energy from wind and hydropower production and biofuels.", "Last is non-renewable energy, such as coal, oil, gas.", "These energy sources are characterised by a limited quantity at disposal, and their use gives rise to, among other things, the greenhouse effect.", "The third indicator is a greenhouse effect, which shows the environmental impact of greenhouse-gas emissions.", "The anthropogenic greenhouse effect is due to cord gases (including carbon dioxide, methane and other) for the retention traps heat radiation that would otherwise radiate from the earth into space.", "In this way, they contribute to atmospheric warming.", "Acidification influence is a fourth indicator that shows the environmental impact of the emissions of acidifying compounds (mainly sulphur dioxide and nitrogen oxides), which attack plant foliage and acidify the soil.", "Nitrogen impact is a fifth indicator, which shows the environmental impact of the discharge of substances containing nitrogen or phosphorus.", "They can cause the growth of algae or plants to increase out of balance in relation to, and to the detriment of, the global ecosystem.", "The toxification effect is a sixth indicator of the environmental profile, showing the environmental impact of the emissions of substances that have acute and chronic toxic effects on humans.", "Disposability is the last indicator of environmental profiles and shows what happens to a building, structure or material once its life is over.", "In this situation, the study will use three circumstances: recycling/high-value recycling, the direct reuse of the material in its original form or reuse of the material innovatively through the production of new materials (Marsh, 2000).", "A low-worth recycling/incineration, the recycling of material for the production of brand new materials of lower quality, or burning the material with energy; and hazardous waste, where the material contains substances that must be disposed of according to law (Table 1).", "Comparisons are an instrument of inquiry.", "Materials related to environmental impacts can be assessed in relation to other factors such as energy consumption and the greenhouse effect, which is divided into the following:", "A material impact on the environment should not be observed in isolation from other environmental factors.", "The environmental impact of certain materials or structures can be compared to other factors (Dinesen, 1997).", "The environmental profile can compare the environmental impact per m2-floor areas of the house's acclimatisation needs and its building materials.", "The calculation of housing accumulation requires using the approach developed by SBI for the calculation programme BV95.", "The calculated cooling & heating requirements are then converted to gross energy consumption and CO2 emissions.", "A building is built to be occupied by humans.", "As such, it makes sense that architects, be they Classical, Renaissance, Modern, or contemporary, would use the human body as inspiration or a principle of design (Korydon, 2012).", "The building envelope is a man's barrier against external atmospheric conditions and their fluctuations.", "Builders from this zone have chosen three varied categories, in the building material's selecting process.", "These are mineral materials, organic materials and mixture material.", "This is the first building material utilised by human beings in the building process.", "It comes from renewable resources and can be extracted by humans easily; therefore, it is an ecological material with many good proprieties and can be successfully used in the construction process.", "It is a large part of human life (Brown, 1990).", "It is a popular building material because of it is specific environmental friendliness and great energy values.", "The main benefits of these materials are their cheapness, prevalence, and excellent proprieties of thermal and acoustical insulation.", "Reed is an organic building material; it has good resistance to water actions.", "It contains a high ratio of silicon substance, which leads to it being durable and flexible in technical action and structures; however, it is a highly flammable material.", "The high concentration of silica in the reeds makes this material unattractive for insects and other animals.", "Reed meets all the requirements to be an efficient insulator.", "The form of the stem allows it to serve all manners of functions (Elias, 1980).", "It is an excellent thermal and acoustic insulator material.", "The relative durability and flexibility in construction are the most important proprieties of reeds.", "The unique properties of reeds have been noted for a long time, in all regions of the world (Lauren, 2000).", "The influence of reeds on pathogenic bacteria is particularly intense.", "Reeds from the marshland region consist of several types of plants whose stems differ in thickness, flexibility and chemical composition.", "The most popular reeds used in the construction process are “Ihdri”, the regional name of this type of reed.", "The building of walls with reeds is much less labour intensive than that with other materials such as brick, adobe, or stone, and requires considerably less skill.", "Reed building is forgiving, encourages individual creativity, and leads to final structures that are climatically adapted and energy efficient.", "Reed does have some disadvantages.", "Its natural durability is lower than wood, and it must be treated with preservatives to last in exposed locations.", "Straw is a material with high thermal efficiency.", "It has an R-value substantially better than those of other traditional buildings, depending on the type of straw; it consists of barley that is 36% cellulose, 26% pentoses, 17% linen, wax, protein and ash – a composition very similar to wood.", "The straw has excellent insulation properties.", "Straw's biological functions include its axis for water.", "Therefore, straw bales are good at transporting moisture and thus regulate interior humidity, provided they bear the diffusion through open screen and coatings.", "Straw's weaknesses are risk of fire, insects or decay refraction caused by long-term, high moisture content (Gillv, 1970).", "Straw can provide greatly improved comfort, and dramatic energy savings compared to more expensive traditional building structures because they allow for smaller heating or cooling systems in conventional homes because of the increased insulation.", "Bale building is of particular value in severe environments where energy is costly.", "Wood is an organic material, a natural combination of cellulose fibres (that are strong in tension) embedded in a matrix that resists compression (Janssen, 1988).", "The principal components in wood are cellulose (40–50%) and hemicellulose (15–25%), with lignin (15–30%) as well as other material such as sugar, starch and protein.", "The technical properties of wood are highly dependent on the type and moisture content.", "It also contains varying other elements depending on the type, which defines its smell, colour and resistance to pest damage.", "There are two forms of wood, softwood and hardwood, which are subdivided into a number of different types (Edward and Joseph, 2011).", "Wood has a high caloric value (between 12.5 MJ/kg and 20.1 MJ/kg).", "Thermal re-utilisation of untreated and uncoated wood is un-problematic.", "Re-utilisation is possible if the wood is dismantled without damage.", "Untreated old wood conventionally can be used as raw material for a number of different applications (Woolley, 2006).", "The conversion of wood into a sustainable, renewable resource to be used as a main building material could be beneficial in various areas of the world.", "Mineral materials are made from non-renewable resources dug on land or at sea.", "They are homogeneous, naturally occurring solid inorganic materials with a clear crystal structure and a definite chemical composition (Damon, 1983).", "Builders from this zone have found good building materials from the local neighbourhood.", "Two essential building materials were selected in the building process: clay and brick, which are near the required scale of humans.", "Today, clay offers the only practical prospect for building the five hundred million houses that will be required in the coming years (Anil, 1981).", "A mass of clay has extraordinary formability and robustness.", "Excellent adhesion and bonding forces count as the main properties of clay.", "Brick is used as a principal building material from zone “I”.", "The typical house from Sumerian age was a one-story construction made from baked or sun-dried mud-brick.", "The family house included the following structure functions: a living room, kitchen, open courtyard, servant's residences, etc.", "A brick-building element has excellent thermal resistance (Barry, 1988).", "The material is very efficient in energy consumption and can be accepted as a bio-ecological material, especially in West Asia.", "Clay bricks are a more thermally inert material, and chemical reactions are relatively low.", "Thus, the effects of green houses are limited (Manand har, 1983).", "The recycling of clay bricks can be a difficult process due to the unavoidable mortar, render and plaster residue that tends to adhere to the bricks, particularly when such products have high cement content.", "However, older brick is easier to recycle because much of it was constructed using lime mortar (Manfred et al., 2006).", "This is one of the ancient building practices known.", "Clay is odourless, non-toxic and pleasant to work with in combination with straw, creating a soft to pulpy prepared mix of clay and vegetable fibres (straw) that can be used for filling the panels in timber-framed buildings or pressed in moulds for making clay bricks and boards.", "Ready-made mixes are now available on the market.", "The density of the mixture building material of clay and straw is 1200–1700 kg/m3.", "A mix of clay and straw is a heterogeneous material, with good thermal properties (Easton, 1996).", "Fig. 3 shows the environmental profile comparative analytical interpretation of environmental profiles of 1 m2 of different building materials.", "The straw–clay mix creates a unique, breathable combination of high insulation with thermal mass.", "The city is located in a region of the historic area of Sumer, the hometown of the fictional character Sinbad the Sailor, and a suggested place of the Garden of Eden.", "It held a significant position in the early regional history and was founded in 636 AD.", "It includes three different habitat arrangements.", "The first appeared in the north part of the city in the zone of marshes, Zone III.", "Another type appeared in the south part of the city in the desert area, Zone II.", "The last appeared the middle part of the city beside the big-city river Shat Al-Arab, Zone I, as shown in Fig. 4.", "From the practical experiences of the building process, specific habitat units from historical experiences were more related to the objective context (Solar Handbook, 1986).", "One of the most effective interventions of an old building from this region was respecting balances between many environmental factors to create a particular building style, which makes the balances between human life and the milieu condition optimal.", "The notion of ecological building is not an absolute (Almusaed, 1996; Marsh, 2000).", "It is, therefore, more correct to use the words more or less environmentally friendly, healthy or willing ecology (Doran and Cather, 2013).", "However, that is not sufficient in practical experience.", "Accordingly, old builders of the city and region took into account many other factors.", "One important factor was the implication of bio-climatic concepts in the building process", "Why a bio-climatic building concept?", "The climate has a primary influence on architectural form, not only in the challenges it poses to the builder but also in the materials it supports.", "In bio-climatic building conception, the edifice is converted to a subsystem of a vast environmental system that should be examined carefully.", "The holistic integration of subsystems in the system is critical.", "There are clear interpretations in native architecture (Houben and Guillaud, 1984).", "The temperature of the ground or walls can be substantially different from that of the neighbouring atmosphere.", "In bio-climatic buildings from Basra, the earth is valuable as a shelter, particularly in conjunction with landscaping.", "Specific houses From Basra", "Basra's traditional habitat has evolved within these constraints; it can be defined as architecture without architects: not that it is created without overall supervision, just that it is not in the hands of a sophisticated urban professional.", "Instead, it is vernacular in that it is the product of well-tried local craftsmen raised in the use of local materials to confront local social and environmental conditions.", "Glazed windows are not part of the Basra tradition.", "In the warmer regions, glass inhibits the flow of air in summer.", "Openings are sealed against the winter cold by opaque shutters or cloth screens.", "Rural housing often lacks window openings, making for a dark interior (Namdin, 1996).", "Building materials are convenient, readily available and can be made and used by the city builders to build their habitats.", "In all housing’ models, the allocation of habitat functions in the house resembles the shape of a tree, where the main building element is a courtyard, which is point “zero” in the house structure.", "The most common characteristic of the houses of the hot and humid regions is their openness.", "They are constructed to catch every breeze that occurs, for natural air currents are the best relief in humid climates [8-2].", "All habitat functions are allocated around the courtyard with a hierarchy approach.", "The most important is for functions of living, such as a living room.", "Next come service areas, such as kitchens.", "In this study, we investigated three different cases where old builders constructed something appropriate for the environmental, climatic and human requirements.", "A high temperature, “T”, due to the heat island phenomenon is a major risk factor that affects housing configurations and compositions in this zone.", "The recommendation is to create a habitat unit that adapts to extreme climate with a series of interferences such as significant thermal insulation, profound shadows on facades, and natural stream ventilations throughout the building functions and structures (Fig. 5).", "In Old Basra, a house is considered beautiful if it is capacious and if the situation is airy and exposed on all sides to the wind, especially to the northern breeze.", "A healthy house has its courtyards, basins of water, and wooden pieces on the front of the façade “Shanashil.”", "A handsome house is seen without terraces, with “Shanashil” on which the family may sleep during the night (Fathy, 1986).", "Most of the old Basra distinct habitats are decorated, usually to show invisible spirits and demons and to satisfy local traditions.", "Building materials have to be strictly respected (see Fig. 10).", "The habitat area is divided into two functional zones: a social area that includes the living room, kitchen, storage room, and bathroom and a private area that includes bedrooms.", "Communication between all operational areas occurs throughout the courtyard (Table 2).", "The habitat area of this edifice is ca. 125 m2: the social area is 30%, the private area is 45%, and the courtyard is 25%.", "The building materials used in the creation of the house are as follows:", "The roof is made of 10 cm of clay and straw, which improves the roof's resistances to waterproofing and high temperature; 3 cm of pressed straw; 10 cm of reeds; 5 cm of pressed straw and 10 cm of wood as a structural element.", "The floor is made of a 40 cm layer of clay and eventually sand as a healthy ground, and a 12 cm layer of brick.", "The exterior wall is combined with an inner wall with 24 cm of brick.", "The upper façade is made of wooden elements (Shanashil unit), with a thermal roll.", "The interior wall is made of 12 cm of brick.", "The estimated life cycle is 100 years.", "This is a category of heavy construction.", "Brick is the main material used in this construction.", "The impact on the environment by acclimatisation or building materials is limited.", "The houses are located in row buildings in front of a narrow street, placed directly by the old city river.", "The houses were built in the beginning of the last century.", "The results show that the release of CO2 is relative low; however, it is higher than other categories of houses.", "Fig. 6 shows the influence of the greenhouse effect distributed on habitat development and the environmental profile for 1 m2.", "The environmental impact of the materials is a representation of the results shown in the environmental profile of the same house's materials.", "A high humidity “H”, due to marsh water, is a major risk factor that affects housing configurations and compositions from this zone.", "A recommendation is to create a habitat unit with a building material resistant to water and humidity.", "The location is a historic site, from the Sumerian time.", "The housing configuration was founded for the preliminary time in the “Ur” city, where the initial settlement was founded (Ilay and Barry, 1998).", "UNESCO recognises the lacustrine region in the north of Basra as a monument of nature, representing one of the largest sites of unique ecosystem types in the world.", "There is a low density of people living in symbiosis with impressive flora and fauna full of rare species.", "In the north of Basra, on an “Artificial Island”, the house was built in water with a weak current, and the habitat unit is made of reeds and a mixed of clay and straw (Tobias et al., 2009).", "There are two ways to make the island suitable for the construction process: one is choosing good ground and the other is making it suitable for a particular case.", "Fig. 7 shows the different procedures for building an artificial island.", "The habitat unit area is 147 m2 (7 × 21) m:", "The habitat entrance is oriented towards the northwest, where the dominant wind originates.", "The frontal façades on the axis (northwest-southeast) are perforated to allow for permanent natural cross-ventilation (see Fig. 8).", "The minimum distance to the water must be approximately 10 m.", "The roof is always made of a vault form.", "The number of columns on the long side is as follows: seven pillars with the local name of Shabe, with 3 m between 2 pillars.", "The facade has four pillars: the roof pillars framing the entrance and the others located on the corners.", "The estimated life cycle is 25 years.", "The habitat site is created from many posts that include closely related strains of reeds between them.", "The height of the pillar (Shabe) can reach 10 m.", "The diameter of the pillar (Shabe) is approximately 70 cm at the base and 20 cm at the top (Fig. 9).", "The house is made of lightweight constructions.", "The principal building material applied is reed.", "Table 3 shows different material used in the composition and the percentage of building materials in layers.", "A demonstration shows that houses made of reeds are friendlier environmentally, with a positive impact on the environment and less energy consumption.", "A short life cycle material presents negative aspects to a building system.", "Fig. 10 shows the greenhouse effect for a habitat construction and the environmental profile for 1 m2.", "The sandy wind “W”, which is due to the Sahara Desert environment in the Arabian Peninsula, is a major risk factor that affects house configurations and compositions from this zone.", "Here, a blind wall is recommended.", "In many ways, the earth is the most prominent material used in natural buildings.", "Earth can be found everywhere, and, as a result, it used to be said that the majority of the world's population still lived in earth buildings (Keefe, 2005).", "Of course, concrete and bricks are made of material from the earth, but what will be discussed here are structures that are made of earth in its most natural state, with a minimum amount of treatment or processing (Wenzel, 1996).", "Strong prevailing sandy winds provoke a builder's response, resulting in lower, blind façades, flatter buildings offering less resistance and thus less prone to building damage and human discomfort.", "Experiences from the old building process show that malleable building materials can be employed when the climate is humid and are rigid when the climate is dry.", "Usually wet clay is used directly, mixed with Straw, and perhaps given more body by adding gravel or stone.", "This is prominent in areas of the world where earth buildings may have a relatively short life as part of a nomadic existence.", "When abandoned, they will not leave anything unpleasant behind (Wenzel, 1996).", "The extraction of clay, especially on a site where a house is due to be created, is a zero-carbon solution for construction, and clay and straw walls and floors can be used as a direct substitute for mass concrete (Marchell and Leary, 1974).", "The “Al-Zubeir House, Unit” building, in its architecture without architects, notes that inexperienced builders fitted their work to the local environment and topography.", "Basra's vast variety of environments and local microclimate assures a correspondingly wide range of a reply.", "The beauty of the “Al-Zubeir House Units” design derives less from self-conscious decorative attempts than from unadulterated, practical outlines produced by adapting regional material to be as economical as possible, resisting aggressive environmental elements and employing beneficial ones.", "Habitats from an Al-Zubeir district are truly vernacular, utilising only slightly altered material from its immediate surroundings.", "Rural, desert architecture is remarkable for its sculptural shapes achieved in clay.", "In this zone, edifices have to be planned with great attention and thought and with due respect for climate.", "Shade and protection from dust storms may have the highest priority in some areas, whereas in other regions, ventilation and the trapping of air currents and breeze are the primary considerations (Kukreja, 1978) (see Fig. 11).", "The courtyard space is an essential element in the habitat from zone III, where all habitat functions are allocated together and are oriented towards the courtyard.", "Intimacy is necessary in cultural life.", "All primary functions are oriented towards the house courtyard directly.", "The unit involves just one level (Fig. 12).", "The habitat area of this edifice is of ca. 450 m2, where the social area is 10%, the private area is 35%, and the courtyard is 55%.", "The building materials used in the conception of the house are as follows:", "The roof is made of the mixture of 10 cm of clay and straw, 3 cm of pressed straw, 10 cm of reeds, 5 cm of pressed straw and 10 cm of wood as a structural element", "The floor is made of a 30 cm layer of clay and eventually sand, as a solid base, and a 10 cm layer of clay and straw.", "The exterior wall is combined with an inner face layer of 3 cm special clay and straw, or 25 cm of clay and straw and the same inner face of 3 cm clay and straw.", "The interior wall is made of 15 cm clay and straw.", "The estimated life cycle is 50 years.", "Habitat shapes vary from a rectangular plan and extension to the north to a square one.", "The conglomeration of buildings creates a progression of private space, which is enclosed by a clay wall; therefore, a courtyard is created by this process (Jensen, 1997).", "The system was developed for individual utility, free of fixed rules.", "Each building is oriented in relation to its neighbours.", "The principal building material applied is clay and straw.", "Table 4 shows the different material used in house compositions and the percentage of materials in layers.", "The house material components are environmentally friendly and better employed in energy-efficiency features.", "Defining the qualities of an environment is neither easy nor absolute.", "They vary with time and change as people's expectations and education changes.", "Moreover, adapting to a new-architectural idea with a vernacular conception is difficult for many architects who have been educated in a culture that despises the aesthetics of what they see as ‘twee’ vernacular cottages.", "Incidentally, the aesthetic and functional housing opportunities remain innumerable and fun opportunities.", "Respect is given a high priority.", "Strategies to reduce environmental impacts can and should vary depending on the site.", "The selection of building materials is, overall, crucial because there is little difference in the material's environmental impacts and because they play a vital role both architecturally and in terms of the impact on the external environment near a residential use.", "For instance, the term “environmentally compatible” suggests that acceptable effects for humans and ecosystems can still be achieved with maximum amounts of emissions and limited contamination.", "This study discusses the requirements of building materials suitable for bio-climatic building in West Asia, where the meaning of the contemporary building category is an application of the bio-climatic concept, which is very complex.", "Everywhere, sustainable home thinking about the environmental impacts in the overall processes is implicit in houses.", "Basra had three different microclimates (a marshland district, desert region and regular hot climate in the city centre).", "This study tries to investigate an existing habitat in the Iraq–Iran area that has different microclimates.", "Vernacular building materials such as clay, brick, straw and wood, are the most popular building materials used in this region.", "The Ecology of Building Materials is a challenge regarding the possibilities for existing materials and the evaluation of new materials.", "Nevertheless, a complete ecological structure does not exist.", "However, a building can always be made more ecological and less environmentally impactful.", "The principal conclusion of the study shows that:", "The local microclimate directly influences the conformation of habitat.", "Therefore, the choice of building materials takes a high priority in the building process.", "Clay and straw, breeds, and brick are most frequently applied building materials.", "Natural materials were used efficiently in vernacular houses.", "It is clear that when insulating habitats; the interrelated environmental impact of the selection of insulation materials is not relevant compared to the savings in energy.", "Acclimatisation has a greater impact on the environment than building materials.", "Heavy materials (Bricks) used in the exterior or interior reduce energy requirements.", "In contrast, clay- and reed-based materials have a lower material-related environmental impact.", "To support the meaning of the building in the residual value of the primary fabric, maintenance and repairs must be carried out on all components corresponding to their specific renewal cycles.", "For future research, the life cycle of building materials by focusing on the experiences of vernacular houses from this zone should be increased the create a creative intervention where innovative material can be used in new buildings with an increasing life cycle of current materials.", "Brick, clay and wooden constructions should be improved to occupy a more considerable place in our residential buildings than they do today.", "The objective of the study is to assist architects and designers in selecting suitable solutions in terms of building materials.", "In addition to studying different building materials from various sources and their characteristic, it is necessary to adapt to traditional materials in modern, innovative applications and create a good solution in terms of building materials for West Asia specifically." ]	[ "Configuration of Scandinavian Habitat was constructed according to climatic act.", "Natural building materials were used efficiently in habitat components.", "Habitat functions were allocated according to climatic circumstances." ]
Strategic maritime container service design in oligopolistic markets	This paper considers the maritime container assignment problem in a market setting with two competing firms. Given a series of known, exogenous demands for service between pairs of ports, each company is free to design liner services connecting a subset of the ports and demand, subject to the size of their fleets and the potential for profit. The model is designed as a three-stage complete information game: in the first stage, the firms simultaneously invest in their fleet; in the second stage, they individually design their services and solve the route assignment problem with respect to the transport demand they expect to serve, given the fleet determined in the first stage; in the final stage, the firms compete in terms of freight rates on each origin-destination movement. The game is solved by backward induction. Numerical solutions are provided to characterize the equilibria of the game.	[ "ENGI", "SOCI" ]	[ "Container assignment model", "Duopoly", "Logistics", "Maritime network", "Monopoly", "Sequential games" ]	[ "2205", "3313" ]	[ "Over the last 50 years, containerization has grown to account nowadays for roughly 70% of total deep sea trade (by value) and it is now a key component of the global economy.", "The resulting standardization of freight handling processes has enabled quick and efficient cargo movements across transport modes and has relied on the concept of transshipment to provide global coverage.", "This involves the use of multiple services to establish connectivity between ports that are not otherwise served by direct maritime links.", "As such, the global liner shipping network has adopted a hub-and-spoke structure, whereby there is a strong incentive for liner services to include visits to heavily connected ports (such as Singapore, Rotterdam and Hong Kong) in their rotations.", "This structure, combined with the volatile and competitive nature of the ports and shipping markets, has led to intricate flows patterns that can often be difficult to predict.", "The development of accurate flow models would not only assist shipping lines in the deciding what services will be provided, but could also prove useful to other relevant stakeholders.", "These include port operators, governments and international organizations, who could use such models to allocate investments, perform strategic/operational planning, and design future policies.", "One of the earliest works to model maritime container flows is the Container World project (reviewed in Newton, 2008), while Perrin et al. (2008) developed one of the earlier macroscopic container assignment models.", "At the same time, much research has focused on the empty container management problem, which occurs due to the effects of trade imbalances, predominately (e.g., between Western and Asian markets).", "Previous work by Bell et al. (2011, 2013) focused on the development of a container flow assignment framework that acknowledges transshipment operations and capacity constraints in the ports and vessels involved.", "The resulting optimization algorithms remain linear in nature, are built around the frequency-based structure of liner shipping services and seek to minimize aggregate container travel durations or costs, respectively.", "Both techniques are capable of simultaneously addressing full and empty container flows – in the past the latter have mostly been examined in isolation and as part of the empty container repositioning problem (Song and Carter, 2009).", "Given their linear nature they have modest computational requirements, therefore making them particularly attractive for application in large problem settings that involve hundreds of ports and services.", "Following the seminal contributions by Agarwal and Ergun (2008) and Shintani et al. (2007), in the last few years a number of studies have jointly addressed the issues of network design and container flow assignment, taking into account cargo routing and empty container repositioning (e.g., Imai et al., 2009; Song and Dong, 2013; Mulder and Decker, 2014).", "Meng et al. (2014) provide an interesting overview of this fast-growing literature in the general context of studies on containership routing and scheduling.", "Brouer et al. (2014) also design a benchmark suite for liner shipping network design.", "However, all these studies keep the perspective of a single shipping line or alliance and do not deal with the consequences for freight rates or routes of competition between shipping lines or alliances.", "The aforementioned work provides important insights into the forces that drive the global flow of containers.", "However, the existing models still fail to take into account some crucial features of the global shipping market such as: the elasticity of freight demand with respect to the economic conditions (e.g., travel time and fees) that prevail in the shipping industry at any given moment; and, on the supply side, the effects of strategies adopted by groups of players (e.g., shipping lines or alliances) in the same shipping market.", "As regards the effects of competition on service network design, the economic theory of industrial organization highlights two possible outcomes (Tirole, 1988).", "If competing firms provide services of similar quality (e.g., service reliability) and features (e.g., planned delivery time), competition would take the form of strong price war, reducing profits and, in some cases, preventing the market from finding a stable configuration (or medium-term equilibrium).", "On the other hand, if competing shipping firms are able to determine some form of service differentiation they may reduce the strength of competition and, in this way, increase profits (which nevertheless will remain lower than in the monopolistic case).", "This case is potentially relevant for the shipping industry, given that firms are able to offer different service networks that, in turn, imply diverse delivery times and service features1.", "The policy implications of the two scenarios are quite different: if competition is strong, no regulation of the shipping sector is desirable, given that the market tends to determine the lowest possible prices for shippers; conversely, if competition is weak, prices and profits would tend to increase, and a regulatory remedy could be desirable if the barriers to market entry prevent new shipping lines filling unmet consumer demands.", "The model introduced in this paper seeks to address this gap in academic literature, by developing an algorithm that could be used to determine an optimal set of liner services, given the presence of a competing shipping firm.", "The resulting model has game-theoretic elements, whereby the choice of services to be offered by each shipping line or alliance reacts to the actions of its competitors.", "Container flow assignment is integral part of this process, as it is used to establish how the market would respond to the simultaneous provision of routes by competing parties.", "The types of service considered in the model follow established liner shipping trends, where vessel services comprise a looped sequence of port calls (commonly referred to as a port rotation).", "The resulting model takes into account revenues deriving from a demand for transport services that is distributed between firms in accordance to service costs.", "The latter include fees charged by the firm operating the chosen service as well as the opportunity cost of travel times.", "Empty container repositioning is retained in the container assignment model.", "The key actors of the model are two firms (i.e., shipping lines or alliances) that seek to maximize their weekly profits by operating in a given region with known transport demands for full containers among a set of ports.", "To meet this objective, they both aim to operate sets of liner services, each being a circular tour of ports with a given frequency and capacity.", "Respective fleet sizes constrain the services that each firm is capable of offering.", "The model acknowledges the practice of transshipment in the maritime industry, as the possibility exists for a firm to satisfy demand between two ports that are not served by the same tour.", "This is achieved by identifying two services that include the origin and destination port, respectively, that also intersect at some intermediate location.", "In modeling terms, this is achieved by simultaneously representing each service in three forms:", "as a sequence of port visits (in a closed loop form);", "as a sequence of links (journeys between two adjacent ports in the service);", "as a sequence of legs (a pair of any two ports in a service, between which travel is possible).", "In our analysis set N contains a series of possible container routes.", "Each route n consists of a set of links Ln.", "Each link l within Ln represents a trip between two consecutive ports in the same route.", "For each route n it is possible to obtain a set of legs An, representing every single container journey that could be made using this route.", "The relationships among ports, links and legs are illustrated in Fig. 1.", "Ideally these routes will intersect with at least some ports being served by several trips, therefore providing opportunities for transshipment of containers.", "This would be essential for the shipment of containers between two ports that are not directly connected.", "Furthermore, it might still be a viable option if direct routes exist, should they include large detours.", "Having said that, an ideal assignment algorithm would not merely compare journey times, but should also take into account the additional handling costs that are incurred in intermediate ports.", "An example of a multi-leg container trip with transshipment is provided in Fig. 2.", "Storage limitations in transshipment ports may impose constraints to the amount of containers that may be temporarily stored between journey legs.", "The model is designed as a complete-information sequential game, taking place over the following stages:", "Stage 1: The firms simultaneously invest in their fleet;", "Stage 2: The firms decide which routes they will operate and solve the flow assignment problem with respect to the transport demand that they expect to serve, given the fleet sizes determined in the first stage;", "Stage 3: The firms compete in terms of freight rates on each origin–destination movement, acknowledging the share of overall transport demand to be served by the competing party.", "The game is solved using backward induction (i.e., incorporating the solution of later stages in previous ones), relying on the concept of Subgame Perfect Equilibrium (Gibbons, 1992)2.", "The model is solved for two cases: monopoly and duopoly.", "The main difference between these cases is the expression for revenues that captures the monopolistic or competitive behavior of each firm, respectively, and is worked out as the solution of the third stage of the game.", "Given the complexity of the problem, we rely on numerical examples to characterize the equilibria of the sequential game.", "The contribution of the paper is both theoretical and methodological.", "First, we address the issue of the effect of competition on service provision in the shipping industry.", "From a methodological point of view, the main innovations are the use of game-theoretic concepts to address the issue of competition, while keeping the model tractable, as well as the introduction of innovation in the manipulation of variables to address the resulting mixed linear-integer program.", "Our theoretical and methodological setting considers a shipping industry where only one or two firms operate.", "However, the algorithm structure implemented in this study could be used as the basis for a future model that can analyze oligopolistic shipping industries where more than two firms operate in a single region.", "Furthermore, the monopolistic and duopolistic cases provide appropriate frameworks to analyze competition in the real-world regional shipping markets where we observe only one or two alliances (Ducruet and Nottenboom, 2012).", "The remaining of the paper is organized as follows.", "Section 2 describes the basic structure of the model; Sections 3 and 4 analyze the monopolistic and duopolistic cases; Section 5 draws conclusions.", "We consider a strategic model of service design with two competing firms (i.e., shipping lines or alliances); the list of all variables and parameters is in the Appendix.", "The geography and operational parameters of the ports sets are considered to be exogenous to the model.", "This includes the distances between port pairs as traversed by the services and the maximum throughput capacity MQPi for each port i ∈ I (where I is the set of existing ports or nodes).", "To keep the analysis as simple as possible, distances between any two ports connected by a link l ∈ L (where L is the set of all links included in the services considered) are expressed in terms of the exogenous sailing time (STLl).", "The functional structure of the maritime container shipping market is represented by the following sequential game:", "Each firm deploys its maximum transport capacity for each route n, that in turn determines firm's fixed costs in the following stages of the game;", "given firms’ capacity constraints and predictions7 of the other firm's service offer and the outcome of downstream (i.e., third stage) competition, each firm decides which services to operate and solves its container assignment problem;", "as an outcome of the second stage, container travel times and potential transport capacity limits (with respect to the potential demand)8 between each pair of ports are determined for each firm.", "At this point, firms determine their fees for full containers either while competing à la Bertrand9 between pair of ports that are served by both networks or operating as monopolist between pairs of ports that are served by their own network only.", "In this section we consider (as a benchmark) the case of a single firm that is free to accommodate the entire transport demand in all ports.", "As usual in complete-information sequential games, the solution has to be found by backward induction (i.e., incorporating the solution of later stages in previous ones).", "Flow conservation is maintained through constraints (3.1) and (3.2), for full and empty containers respectively.", "As empty containers are considered to be interchangeable, flow destinations are not important – as such the constraint definition iterates only across the set of ports i ∈ I.", "Each port has been assigned a maximum throughput parameter, whose value is linked to the port's ability to load and unload vessels quickly as well as the storage capacity of its yard.", "Eq. (3.5) ensures that this is not violated by active container flows for each port i.", "Whenever a vessel is traveling between two consecutive ports in a route n ∈ N, it might be carrying container flow that belongs to several legs a ∈ An.", "Eq. (3.6) is used to enforce an upper bound on the total volume of container flows to be allocated on legs that use any link l of the route.", "The Boolean parameter LLRal is used to indicate whether a link l is used by a leg a. Constraints (3.7) and (3.8) are used to enforce the demand or supply of container flows for each port i – this is used in conjunction with the flow conservation rules (3.1) and (3.2).", "The revenue for flows between a pair of ports r, s is determined by the relationship in (3.9), as discussed earlier in this section.", "Finally, (3.9) ensures that the actual flow of containers between a port pair r, s never exceeds the market demand MQFrs, while (3.10) ensures that a shipping line will not operate a combination of routes that necessitates the deployment of more vessels than the size of their fleet CFS.", "The distinction between empty and full containers throughout the above set of constraints demonstrates how the model accommodates the two types of flows using distinct decision variables.", "Should the need arise, the representation of container flows could be further decomposed into distinct container sizes and types (refrigerated, liquid-bulk etc.) with the introduction of additional demand parameters, decision variables and constraints, without otherwise changing the overall structure and premise of the problem.", "Given the solution of the second and third stages of the optimization, the total profit of the firm depends on the maximum capacity deployed on each route.", "Thus, the firm chooses the capacity deployment to maximize its profit.", "This model possesses a sufficient amount of operational detail that would facilitate its applications to realistic scenarios, as illustrated by the numerical example presented in the following section.", "Perhaps the most challenging step in the deployment of the above model would be the definition of the route, link and leg sets N, L and A, as well as the parameter arrays LRRln, LLRal and LRan that describe the relationships among them.", "A custom tool (Delos) was developed (using the C# programing language) to simplify this process.", "The key features of this tool include a geospatial data management interface that facilitates the visual definition of maritime transport networks, and a database that captures operational aspects of the maritime transport market environment (including parameters that relate to companies, services, ports and market demands).", "A screenshot of this software is provided in Fig. 3.", "The actual MILP model that was defined in the previous section was implemented using the OPL modeling language and solved using the CPLEX optimization engine.", "A custom C#/OPL interface was developed to link Delos with the final OPL model and manage the algorithm workflow.", "Given the solution of the second and third stage, the total profit of the firm depends on the capacity deployed on each route.", "Thus, the firm chooses the capacity deployments to maximize its profit, taking into account the financial feasibility of service provision and limitations imposed by the available fleet.", "At this point, it should be pointed out that the vessels would incur fixed ownership costs (CRC) regardless of whether the firm chooses to operate a combination of services that would utilize them – as such, there is a strong incentive to do so.", "To test the monopolistic version of the model we created a sample scenario focused on the Mediterranean feeder market (Fig. 3) and some of the busiest ports in the region.", "The largest potential container flow demands among ports in the region (Table 1) were extracted from the LINER-LIB benchmark dataset (Brouer et al., 2014).", "In our problem setting, these demands are conditional on the prices of the services offered and the delivery time realized by the network configuration that is selected by the firms.", "As these price and time values increase, it is more likely that shippers will become disinterested in the service.", "This threshold could be modified using the MCTrs parameter for each flow between ports r, s.", "A service network comprising seven weekly feeder services was designed using the Delos tool, featuring realistic travel times (Table 2).", "While each service only covers a subset of the ports encountered in Table 1, the overall network is structured in a way that any of the O–D flow pairs would be feasible using direct journeys, or transshipment.", "The number of services that visit each port is roughly proportional to the volume of container flows, and ranges between 1 and 4 calls.", "As such, transshipment is possible in 10 of the 23 ports considered.", "In this example, it is assumed that the firm seeks to operate in this market alone, seeking to serve potentially profitable flows using their small fleet of feeder vessels (see Table 3).", "The analysis is conducted on the basis of an iterative process, where the number of available vessels – i.e., the transport capacity to be deployed on different routes measured in terms of TEUs per week – is pre-determined.", "In the first iteration, we run the mixed linear integer program of the monopolist firm assuming, as our first guess, that the delivery time between any two ports is the average of all services potentially linking them weighted by the demand.", "The firm, in particular, chooses on which possible services to deploy the available vessels (or transport capacity).", "Having observed the solution of this stage, we correct the second-iteration guess of delivery time between any two ports taking into consideration only the services that are actually offered by the monopolistic firm.", "Thus, we run again the program for as many iterations as needed to observe convergence of solutions.", "Even though the model is represented as a MILP problem, individual instances were solved in less than 1200 ms using the CPLEX optimizer on a relatively modern computer workstation (Quad core Intel i7 2.8GHz processor and 16GB RAM).", "Problem instances were generated for all possible fleet compositions, with the results summarized in Table 4 – converged solutions were obtained in less than three iterations for all instances.", "The highest profit is obtained in Scenario D, where the firm is equipped with 4 vessels.", "In the case of larger fleet sizes, the firm serves exactly the same routes and container flows as in Scenario D, with its profit decreased by the ownership costs of the inactive vessels.", "Using the backward induction procedure, we determine that in the first stage of the game, and given the structure of the market and potential services, the firm should choose to operate only four vessels.", "A comprehensive diagram of container flows across service legs in Scenario D is provided in Fig. 4.", "The outcome of this process can be justified by the fact that further service provision would diminish the potential profit of the firm; given the limited set of potential services and the amount of transport service demanded (as defined within this problem instance), there was no requirement for more complicated transshipment arrangements than would have been necessary to serve port pairs without any direct service links.", "A different outcome would have been observed by expanding the set of potential services that the firm could select, thus making it more able to control the operating cost it faces to satisfy market demand.", "In this section we consider the case of two firms competing in the maritime container market.", "We solve the game relying on the concept of Subgame Perfect Equilibrium.", "Hence, we proceed by backward induction.", "The implication of the previous argument is that the revenue function for each firm is exactly as in the monopolistic case analyzed in Section 3.", "Let us now consider the equilibrium of type (b):", "The analysis is conducted on the basis of an iterative process.", "The algorithm, represented in Fig. 5, was implemented again using a combination of the Delos maritime network design tool (used for data management and algorithm flow control) and IBM OPL CPLEX.", "The duopolistic version of the algorithm was tested on the same market environment that was used earlier in this study, with the key difference being that in this case two symmetrical shipping firms being allowed to operate and compete.", "In this context, Firm 2 is referred to as the rival firm (represented with an asterisk in Section 4.1) and Firm 1 the other firm – both have the ability to allocate up to seven vessels to accommodate the various transport demands in the market.", "The algorithm would then allocate flows among firms, taking into account the effects of competition in the revenue functions as discussed in Section 4.1.", "A series of iterations is necessary to determine the convergence of the solutions of the mixed linear integer programs of the Firms 1 and 2 to the market equilibrium.", "The first iteration starts assuming a market share for the Firm 2 (stage S1 in Fig. 5); in particular, we assume that the rival firm behaves as a monopolist.", "Then, we run the mixed linear integer program of Firm 1 assuming – as first-iteration guess – that the delivery time between any two ports is the average of all services potentially linking them weighted by the demand (S2 in Fig. 5).", "Having observed the solution of the previous stage, we correct the second-iteration guess of delivery time between any two ports taking into consideration only the services that are actually offered by the Firm 1, and we run again the program (S3 in Fig. 5) for as many iterations as needed to observe convergence of solutions (S4 in Fig. 5).", "We are now in the position to run the mixed linear integer problem of the Firm 2, taking as given the market share determined for Firm 1 (S5 in Fig. 5), applying the same iterative procedure described for the Firm 1 (S6, S7, S8 in Fig. 5).", "Once we obtain the convergence of solutions of the program of Firm 2 (S8 in Fig. 5) we use the output market share as input of new iterations regarding the program of Firm 1 (S9 in Fig. 5).", "We run as many iterations of the described procedure as necessary to determine a stable market equilibrium (S10 in Fig. 5), that is defined by the fact that the solutions worked out by the mixed linear integer programs of Firm 1 and 2 produce market shares that are mutually consistent.", "A similar set of experiments as with the monopolistic case was carried out to test the behavior of the algorithm, where two firms of equal fleet size (ranging between 1 and 7 vessels for each case) were competing in the same market.", "No more than 10 steps where required to obtain converged solutions.", "Unique solutions were obtained for instances with fleet sizes below 5 vessels.", "As was the case with the monopolistic version of the algorithm, for larger fleet sizes, container flows were the same as Case E (5 vessels per firm), but with decreased profits (given the ownership costs of inactive vessels).", "The final market allocations were found to not be affected by the initial market share assumption for the competing firms.", "As seen in Table 5, the largest overall container flow is observed in Case D, with a total of 676 full TEUs/week.", "These were accompanied by equal, but reverse empty container flows across the ports served.", "It is worth remarking that while one of the potential outcomes of the competitive iteration process would have been an unstable assignment of the market share among the two firms, with an oscillating set of allocations for the firms, that prohibits the algorithm from terminating, such an outcome was not observed in any of the scenarios studied.", "The results show that the competing firms tend to choose different services and serve different markets (i.e., flows between pair of ports).", "Of particular interest in the instances attempted are Cases D and E.", "The former is the only case across the instances studied where both firms operate a common service (Service 1) in the converged solution.", "Even so, they do not seem to compete in servicing any of the flows.", "As highlighted in simpler contexts in the study of industrial organization economics (Tirole, 1988, Chapter 7), firms tend to invest in “product differentiation” to relax competition among them and, by this strategy, to increase their profits.", "In our model and this numerical example, the firms pursue such a service differentiation strategy by choosing services and container flow assignments that imply a complete separation of served markets.", "In the extreme Case E, Firm 2 opts to not enter in the market altogether, as all potentially profitable cargo flows have been absorbed by Firm 1.", "Instead, covering the ownership costs associated by their idle vessels is a least costly outcome.", "This behavior is also consistent with the practices of real firms during troughs in the market cycles, when many vessels are laid up, in anticipation of better market conditions in the future (Stopford, 2009).", "While it might not be possible to predict which of the two firms will have two lay up its vessels in this scenario, the results of this analysis might urge both to pursue a less aggressive strategy.", "Therefore, even though maximum possible individual gains diminish, extremely unfavorable individual outcomes would be eliminated.", "This paper presents a sequential game-theoretic model to analyze service network design, container assignment and service provision of shipping firms (or alliances) both when they operate in a set of ports as a monopolist and when they compete with a rival firm.", "The model takes into account exogenous demands for container transport among ports of origin and destination which reacts to the total cost of transport, including the travel fee that is paid to the shipping firm and the opportunity cost of time (e.g., depreciation of shipped commodities) for the shipper.", "Because of the complexity of the theoretical model, we rely on a numerical algorithm to characterize the equilibrium of the game.", "We find that:", "the monopoly firm does not cover all possible market demand, because of the high cost of available services (i.e., possible routes that it can activate) mainly linked to transshipment; moreover, the monopoly never satisfies all the existing demand in ports that are served through the chosen network;", "when a duopoly is considered, the scope for demand satisfaction improves; the firms tend to choose different service networks in order to curb the competitive pressure; only rarely do they opt to operate common routes – this happens once other options have been exhausted and under the pressure of vessel ownership and operation costs; again, the existing market demand is not fully satisfied, because of transshipment and network operation costs (e.g., container repositioning), as is the case also under monopoly.", "The introduction of competitive behavior adds a new level of complexity to container flow assignment and liner service network design problems, therefore bringing them closer to real life settings.", "Duopoly (and monopoly) is a good approximation of the behavior of the shipping market in several world regions.", "The model described in this paper can be used as a framework for future research addressing the case of oligopolistic shipping markets with multiple players.", "Several other issues need to be investigated in future research, such as what is the effect of widening the scope for service line selection and assignment.", "One potential research direction would be to explore the possibility that firms differentiate their networks and thus relax competition among themselves, and the possibility for each firm to optimize the cost structure of its network.", "The assessment of these issues in alternative set of ports can also shed some light on the role of the vessel capacity limit (and investment costs).", "The theoretical model and the numerical algorithm presented in this paper are sufficiently powerful to address these issues, so which will be explored as part of future research." ]	[ "Optimization of container flows across services offered by competing shipping lines.", "Game-theoretical approach that captures company decisions through a sequence of steps.", "Sequence of MILP problems are solved at each step of the game.", "Flow equilibrium is quickly reached through an iterative approach.", "Companies are able to directly compete or focus on distinct parts of the market." ]
Germline Development and Fertilization Mechanisms in Maize	Maize is the most important agricultural crop used for food, feed, and biofuel as well as a raw material for industrial products such as packaging material. To increase yield and to overcome hybridization barriers, studies of maize gamete development, the pollen tube journey, and fertilization mechanisms were initiated more than a century ago. In this review, we summarize and discuss our current understanding of the regulatory components for germline development including sporogenesis and gametogenesis, the progamic phase of pollen germination and pollen tube growth and guidance, as well as fertilization mechanisms consisting of pollen tube arrival and reception, sperm cell release, fusion with the female gametes, and egg cell activation. Mechanisms of asexual seed development are not considered here. While only a few molecular players involved in these processes have been described to date and the underlying mechanisms are far from being understood, maize now represents a spearhead of reproductive research for all grass species. Recent development of essentially improved transformation and gene-editing systems may boost research in this area in the near future.	[ "AGRI", "BIOC" ]	[ "fertilization", "gametophyte", "germline", "maize", "pollen tube", "progamic phase" ]	[ "1110", "1312" ]	[ "GLOSSARY", "Archesporial cell/archespore (AC): The cell or group of cells that give rise to the spore mother cells.", "In the female germline they directly differentiate into megaspore mother cell.", "In the male germline they proliferate to form a mass of pollen mother cells.", "Diclinous: Male and female reproductive structure in separate flowers.", "Ear: Inflorescence of female flowers generating a cob containing hundreds of kernels (seeds) after fertilization.", "Egg apparatus: A group of three cells located at the micropylar region of the embryo sac consisting of one egg and two synergid cells.", "Gamete: Male or female reproductive cell representing the end product of the germlines that unites with a gamete of the opposite sex to form a new organisms.", "In flowering plants sperm and egg cells are considered as gametes, while the central cell is debated as it only transiently generates an ‘organism’, the endosperm.", "Gametophyte: The haploid gamete-producing multicellular structure that begins after meiosis as a single celled haploid spore in flowering plants and ends after mitotic divisions as a pollen and embryo sac harboring male and female gametes, respectively.", "Germline: The cellular lineage that contains the gamete-producing cells culminating in the generation of gametes.", "In flowering plants the archespores are usually considered as origin of the germlines.", "Megaspore mother cell (MMC): Also called megasporocyte; a diploid cell, which undergoes meiosis forming a linear or T-shaped tetrad of four megaspores of which the three micropylar spores degenerate.", "Monoecious: Male and female inflorescences separated at the same plant.", "Pollen mother cell (PMC): Also called microsporocyte; a diploid cell forming a tetrad of four functional microspores by meiosis.", "Silk: Maize stigma containing numerous papilla hairs and two transmitting tracts associated with vascular stands.", "Spikelet: Reproductive structures in grasses containing the flowers (florets).", "In maize each spikelet generates a pair of florets: in the tassel all male florets are functional, while one of the two florets each aborts in the ear.", "Tassel: Inflorescence of male flowers at the apex of the stem of mature maize plants.", "Transmitting tract: Stylar tissue, which can be considered as a highway for pollen tube growth.", "In grasses it consists of small elongated cells and is each physically connected to a vascular strand.", "Maize (Zea mays L.) is a key resource for food, feed, and biofuel production (Godfray et al., 2010) and is thus the most widely grown agricultural crop, with a total production exceeding 1 billion tons since 2013 and further increasing (FAO report, release 8/12/2016).", "Its yield can be influenced by both external and internal factors, such as climate conditions, agronomic management, insect and pathogen infection, and fertility efficiency (Lobell et al., 2011; Jin et al., 2012).", "To ensure the success of fertility, which is the key premise of production and yield, maize plants need to produce functional gametophytes (embryo sacs and pollen) and ensure successful fertilization and seed development.", "Research to understand the mechanisms of germline development and the fertilization process in this species was thus already under way more than 20 years ago (Dumas and Mogensen, 1993).", "As maize is both monoecious and diclinous with male and female flowers separated on the same plant, the development of both germlines from archesporial cells is regulated differentially at the spatial and temporal level within bisexual inflorescence meristem primordia (Figure 1A) (Bonnett, 1940; Cheng et al., 1983; Dellaporta and Calderon-Urrea, 1993; Veit et al., 1993).", "Female gametophytes or embryo sacs originate from subepidermal cells of the L2 layer of ovule primordia.", "The archesporial cell differentiates directly into the megaspore mother cell (MMC), which undergoes meiosis, giving rise to one functional megaspore (a process called megasporogenesis) and during further megagametogenesis initially forms a seven-celled embryo sac containing the female gametes, egg, and central cell, respectively (Figure 1B–1G).", "Antipodal cells at the chalazal pole of the embryo sac then start to proliferate, forming a cell cluster of 20–40 cells (Diboll and Larson, 1966).", "The development of the male germline (Figure 1H–1P) is initiated in anthers from subepidermal cells, archesporial cells, which proliferate to form a mass of pollen mother cells (PMCs) that undergo meiosis (Bhatt et al., 2001).", "During the processes of microsporogenesis and microgametogenesis, tricellular haploid male gametophytes (pollen grains) are formed consisting of two spindle-shaped sperm cells engulfed by a large vegetative tube cell (Figure 1P).", "During the fertilization process, mature pollen grains are released from anthers and once a pollen grain lands on stigmatic papilla hair cells, it generates a pollen tube and undertakes a long journey toward the female gametophyte.", "In maize, pollen tubes grow up to 30 cm within 24 h to reach the ovary harboring a single ovule, where the two sperm cells are released to accomplish double fertilization with both egg and central cell, respectively (Miller, 1919; Bedinger, 1992).", "Guidance signals and mechanical structures of the female reproductive organs precisely regulate the directed growth of pollen tubes to ensure the success of fertilization and the production of seeds (Dresselhaus et al., 2011; Dresselhaus and Franklin-Tong, 2013).", "In this review, we provide an overview of the current knowledge of germline development and fertilization mechanisms using maize as a grass model plant.", "Transition from vegetative to reproductive growth in maize is associated with the initiation of tassel and ear primordia in floral meristems.", "Once tassel and ear primordia are established they further differentiate into spikelets, each generating a unisexual pair of flowers (florets).", "As described above, male and female florets in maize develop separately on the same plant to generate tassels and ears, respectively.", "At early stages of spikelet development, bisexual primordia initials are present in both developing tassels and ears, respectively (Cheng et al., 1983).", "Later on, the “wrong initials” in one primordium are arrested, while the other initials give rise to either male or female reproductive organs (Figure 1A).", "During this process, cellular vacuolization and the loss of organelle and cytoplasmic integrity can be observed (Cheng et al., 1983).", "Based on phenotypic analyses, several genes were found to regulate the sex-determination process in maize: mutation of TASSELSEED1,2,3,5 (TS1,2,3,5) caused feminization of tassels, as abortion of female primordia was defective in mutants.", "Similarly, TASSELSEED4 (TS4) and TASSELSSED6 (TS6) mutations also led to carpel development in tassel.", "TASSELSEED4 encodes a mir172 microRNA, which targets one of the APETALA2 floral homeotic transcription factor indeterminate spikelet1 (ids1) to regulate spikelet meristem determinacy.", "By contrast, DWARF1,2,3,5,8 (D1,2,3,5,8) and ANTHER EAR1 (AN1) activity is necessary to promote stamen abortion and feminization, since masculinized ear phenotypes have been observed in the corresponding mutants.", "As ANTHER EAR1 is a component of the gibberellin synthesis pathway and DWARFs are involved in both gibberellin biosynthesis and reception, it is reasonable to assume that gibberellin is also involved in stamen abortion as a hormone regulator (Fujimoto et al., 1988; Lebel-Hardenack and Grant, 1997; Calderon-Urrea and Dellaporta, 1999; Chuck et al., 2007).", "Subsequently, subepidermal or hypodermal cells in both male and female floret primordia generate archesporial cells.", "These are precursors of both PMCs and MMCs, respectively, which undergo meiosis.", "Archesporial cells are generally considered as the origin of the plant germline, ultimately resulting in the formation of gametes.", "Thus, defects in their formation process will strongly affect germline development.", "MALE STERILE CONVERTED ANTHER 1 (MSCA1) was the first gene identified in this process because its mutation disturbs male gamete development, generating anthers lacking mature pollen.", "Detailed analyses showed that stamen primordium initiation was normal in msca1, but archesporial cells could not be observed, indicating that transition from hypodermal to archesporial cells is blocked (Chaubal et al., 2003).", "At later stages of msca1 development, tapetum-expressed genes could not be detected and ectopic, nonfunctional vascular strands were found at the position usually occupied by developing pollen (Cigan et al., 2001).", "Development of female reproductive structures and other organs is less affected in msca1, although meristem size is reduced (Yang et al., 2015).", "This finding indicates that MSCA1 is required to induce male germline cell fate and meristem growth.", "MSCA1 encodes a glutaredoxin protein interacting with the TGA transcription factor FASCIATED EAR4 in meristems.", "Its detailed molecular role during germline development still remains to be elucidated, but it indicates that the microenvironment plays important roles during determination of germline cell fate.", "As MSCA1 encodes a glutaredoxin, a redox regulator that uses glutathione to reduce disulfide bridges of target proteins, it is reasonable to speculate that the redox status appears to have a critical effect on plant germline development (Tsukagoshi et al., 2010; Timofejeva et al., 2013).", "By decreasing the concentration of oxygen or H2O2, more germline cells can be observed and the msca1 mutant phenotype can be rescued.", "Moreover, an oxidizing environment inhibits their specification and causes ectopic differentiation (Kelliher and Walbot, 2012; Whipple, 2012).", "MULTIPLE ARCHESPORIAL CELLS 1 (MAC1) was identified as another key regulator for archesporial cell formation.", "Mutation of the gene caused several additional hypodermal cells to develop into archesporial cells in both male and female flowers.", "In female florets, an excess of archesporial cells led to the formation of additional embryo sacs.", "At male inflorescences archesporial cells of mac1 undergo excessive divisions and most fail to form microsporocytes (Sheridan et al., 1995, 1999).", "It was further demonstrated that MAC1, an ortholog of rice TAPETUM DETERMINANT-like protein homolog 1A (TDL1A), is secreted by newly specified archesporial cells toward surrounding maternal cells, preventing further archesporial cell proliferation (Wang et al., 2012).", "Although the induction of MAC1 secretion in developing ovules is still unknown and it is very likely that numerous additional factors including hormones could initiate and influence germline establishment, the identification of MSCA1 and MAC1 represents an important step in understanding the initiation of the germlines in maize.", "Once established from premeiotic archesporial cells via a lineage of sporogenous cells, PMCs undergo meiosis to give rise to haploid microspores and, thus, the haploid male generation of flowering plants.", "After two additional rounds of mitosis, mature tricelluar pollen (male gametophyte) forms in the anther locules (Chang and Neuffer, 1989).", "Similar to germline development in animals, archesporial cells of maize anthers are considered as “premeiotic” progenitor cells that undergo several rounds of mitotic division before entry into meiosis occurs.", "After mitotic division of archesporial cells is completed, their sporogenous progenitor cells enlarge and prepare for meiosis (Kelliher and Walbot, 2011).", "Initially archesporial cell formation and proliferation occurs in the four corners of stamen primordia (called microsporangia) within the subepidermal L2 layer enclosed by the outer L1 layer.", "Later, the L1 layer forms the epidermis and the L2 layer generates the sporogenous cell line and the primary parietal cell layer, which gives rise to endothecium and secondary parietal cell layer, respectively.", "The secondary parietal cell layer will further divide to form the middle layer and tapetum (Feng and Dickinson, 2007; Walbot and Egger, 2016).", "The various cell layers are supportive tissues for further male gametophyte development; thus any defect occurring during differentiation of these cell layers will influence the formation of male gametes (Walbot and Egger, 2016).", "During differentiation of these cell layers, several key regulators were found to be involved in cell type specification.", "OUTER CELL LAYER 4 (OCL4) encodes a plant-specific HD-ZIP IV transcription factor, which controls differentiation and maintenance of epidermal cell fate.", "Mutation of OCL4 showed partial male sterility with irregularly shaped, non-viable pollen grains caused by a nearly complete absence of starch accumulation.", "Detailed morphology analyses revealed that an extra subepidermal cell layer with endothecium characteristics can be detected.", "This extra cell layer likely represents the reason of poor anthesis and pollen dehiscence at maturity stage in the corresponding mutant (Vernoud et al., 2009).", "Timofejeva et al. (2013) described mutants with defects in the formation of middle layer and tapetum in maize.", "In ems63089 four to five somatic cell layers were generated instead of middle layer and tapetum, while the epidermis remained unaffected.", "At later stages meiosis in PMCs was arrested and degeneration occurred.", "In another mutation named mtm00-06, neither middle layer nor tapetal cells differentiate, resulting in small transparent anthers and degraded PMCs similar to the ems63089 mutant described above.", "Another similar mutation is tcl1 (tapetal cell layer1), where again middle layer and tapetum formation were defective and non-viable pollen was generated.", "These three mutants are not allelic and define three loci required for proper cell layer differentiation (Timofejeva et al., 2013).", "Up to now the molecular nature of the affected genes has not been elucidated, but the mutant phenotypes indicate that the surrounding sporophytic tissues of the anther strongly influence development of the male gametophyte.", "This hypothesis is further sustained by the analyses of other mutants.", "Normally a four-cell layer structure is required in the anther for proper male gametophyte development.", "Extra periclinal cell divisions in the middle layer were found in the ems72091 mutant, which leads to a five-layered structure.", "At the tetrad stage highly vacuolated cells in the middle layer and tapetum as well as degradation of microsporocytes can be observed in the mutant.", "On the other hand, a defect in tapetum formation was found in ems72063, ms 6015, and ms23 mutants.", "In ems72063 a five-layered structure was found after additional periclinal divisions of tapetal initials.", "A similar structure was also found in ms6015, but here the extra tapetal layer was generated by additional oblique divisions.", "In ms23, a tapetum bilayer was generated by an additional division of tapetal initials, which also leads to a five-layered anther structure affecting pollen development (Chaubal et al., 2000; Timofejeva et al., 2013).", "Again, the identity of the genes involved is not yet known.", "ms32 is another mutant generating a five-celled layer structure in the anther.", "MS32 encodes a basic helix-loop-helix transcription factor, an important regulator for both cell division and differentiation during anther development of maize.", "Mutation of MS32 leads to differentiation failures of tapetum precursor cells, and an extra tapetum layer is formed by additional periclinal divisions.", "Similarly to mutations described above, PMC development and differentiation was impaired in ms32 (Moon et al., 2013).", "Fully differentiated PMCs will then undergo meiosis to generate a tetrad of microspores, a process known as microsporogenesis (Figure 1H and 1I).", "Haploid microspores are released from tetrads by callase activity secreted from the tapetal cell layer (Figure 1J).", "The subsequent first mitotic division in microspores (pollen mitosis I, PMI) is asymmetric generating a larger vegetative cell containing a nucleus with active chromatin and a small generative cell with more compacted chromatin (Figure 1M and 1N).", "The generative cell of the bicellular immature pollen undergoes a second pollen mitosis (PMII) forming two sperm cells (Figure 1O).", "The mature pollen grain is tricellular and contains a thick cell wall and a porus, allowing the pollen tube to exit the grain (Figure 1P) (Chang and Neuffer, 1989; Bedinger, 1992).", "The molecular mechanism of meiosis initiation is well studied in animals and fungi, but little is known about it in plants.", "All meiotic processes in maize require the activity of plant-specific AMEIOTIC1.", "In the ameiotic1 mutant, transition to meiosis and progression through early stages of meiotic prophase were disrupted, and premeiotic cells entered mitosis instead of meiosis (Staiger and Cande, 1992; Pawlowski et al., 2009).", "Two to three synchronous mitotic divisions occurred before chromatin was degraded (Golubovskaya, 1989; Golubovskaya et al., 1997).", "During initiation of meiosis, AMEIOTIC1 protein accumulates in the nucleus and binds to chromatin in early meiotic prophase I, regulating leptotene–zygotene progression (Pawlowski et al., 2009).", "It will now be exciting to elucidate the mechanism that triggers the accumulation of AMEIOTIC1 in the PMC nucleus to accomplish the transition to meiosis.", "Another mutant, maize sterile 8 (ms8), caused pleiotropic defects during male gamete development, resulting in smaller anthers exhibiting meiotic failure.", "MAIZE STERILE 8 (MS8) encodes a putative β-1,3-galactosyltransferase showing strongest expression at the meiotic stage in immature anthers.", "Unlike AMEIOTIC1, MS8 did not directly influence meiosis of PMCs.", "A detailed phenotype analysis revealed that in the ms8 mutant, morphology and composition of epidermis and tapetum were altered, leading to numerous smaller epidermal cells and fewer but larger tapetal cells.", "Callose remodeling around the PMC was also influenced, ultimately leading to meiosis arrest (Wang et al., 2010, 2013).", "The outcome of PMC meiosis is the formation of a tetrad.", "To obtain functional microspores, the callosic wall of the tetrad needs to be degraded in a process mediated by tapetum-secreted β-glucanase (Stieglitz, 1977; Suen et al., 2003).", "After four unicellular microspores have been released from each tetrad, PMI results in an asymmetric cell division generating a bigger vegetative cell (VC) and a smaller generative cell (GC).", "The GC moves to the opposite pole of the VC nucleus and is engulfed by the VC.", "PMII of the GC will give rise to two sperm cells further maturing into the tricellular pollen grain.", "Following PMI, cell wall formation is initiated and an intine pollen cell wall layer is generated by the VC, while an outer exine layer is formed by surrounding tapetal cells.", "During dehydration and maturation, additional lipids and proteins are deposited onto the surface of the exine from degenerating tapetal cells, forming the pollen coat (Bedinger and Fowler, 2009).", "Tapetum-expressed MS45 appears to be important for cell wall deposition after tetrad release.", "Mutation of MS45 results in a male-sterile phenotype due to poorly formed pollen cell wall (Cigan et al., 2001).", "A gene required for asymmetric PMI division is MAB1, whose deficiency mutant leads to the formation of two vegetative nuclei generating pollen lacking GCs.", "MAB1 encodes a MATH-BTB domain protein, a component of a Cullin-E3 ligase (Juranić et al., 2012).", "It will now be exciting to identify its targets, which likely need to be degraded to allow microspores to properly progress to the bicellular stage containing both functional GC and VC.", "Genes involved in the basic cellular activity are also contributing to pollen development.", "Maize fuzzy tassel mutant, for example, leads to male sterility due to a mutation in dicer-like 1, which encodes a key enzyme for microRNA biogenesis.", "Stamen morphology is affected in the mutant and anthers contain a mixture of unicellular, bicellular, and tricellular pollen, which indicates that dicer-like 1 is involved in different developmental steps of anther maturation (Field and Thompson, 2016).", "At anthesis when pollen is shed, mature pollen grains are desiccated and in a state of physiological dormancy until they come into contact with a receptive stigma (Fonseca and Westgate, 2005).", "Until now, little is known about the molecular mechanisms of pollen dehydration and maturation.", "Previous research showed that the amount of ubiquitin and ubiquitinated proteins was severely reduced during pollen maturation.", "This dramatic loss of ubiquitin was temporally correlated with male gametophyte development, as the decrease could be observed after initiation of microspore mitosis (Callis and Bedinger, 1994).", "However, the biological meaning of this reduction remained unclear.", "More systematic approaches are now necessary to identify more players to aid our understanding of the various differentiation and maturation processes associated with microsporogenesis and microgametogenesis in maize and other grasses.", "A maize ear contains about 400–500 ovaries each containing a single ovule (Dumas and Mogensen, 1993).", "In contrast to the male germline, the female germline is immediately initiated from a single subepidermal ovular archesporial cell that differentiates into an MMC.", "During processes known as megasporogenesis and megagametogenesis, it undergoes meiosis, forming a linear or T-shaped tetrad of four megaspores.", "The apical (micropylar) three megaspores degenerate and the remaining functional megaspore undergoes three rapid synchronous cycles of free-nuclear mitotic divisions to generate a haploid bipolar eight-nucleated immature female gametophyte (FG) or embryo sac.", "Cellularization takes place generating a seven-celled FG containing two female gametes, an egg and a central cell, as well as accessory cells (two synergids and three antipodals at both FG poles).", "During further maturation, the FG enlarges and antipodals proliferate, generating a cluster of 20–40 cells (Huang and Sheridan, 1994; Vollbrecht and Hake, 1995) (Figure 1B–1G).", "Similar to most flowering plants, development of maize embryo sacs is according to the Polygonum type (Sprunck and Gross-Hardt, 2011) and involves many basic cellular processes critical for megagametogenesis such as polar vacuole formation, nuclei positioning and migration, cellularization, and cell fate determination.", "Oriented mitosis and defined nuclear positioning of daughter nuclei are key features during the development of Polygonum-type FGs.", "By studying FG mutants with abnormally positioned or extra nuclei in maize (Evans, 2007; Srilunchang et al., 2010), it was suggested that nuclei acquire their fate according to their spatial positioning along the micropylar–chalazal axis, indicating that cell specification in this process is determined by positional information (Tekleyohans et al., 2016).", "The most well studied gene in maize FG development is INDETERMINATE GAMETOPHYTE1 (IG1).", "IG1 encodes a LATERAL ORGAN BOUNDARIES (LOB) domain transcriptional regulator, and its mutant leads to the production of haploid embryos of paternal origin (Kermicle, 1969).", "Further cytological analysis revealed that mutation of ig1 affects the number and position of nuclei in the embryo sac.", "Extra egg cells and central cells have been found, and often central cells have contained extra polar nuclei (Lin, 1978; Guo et al., 2004; Evans, 2007).", "Expression studies showed that IG1 was asymmetrically distributed in the embryo sac with the highest amount in antipodal cells (Evans, 2007).", "An abnormal microtubule pattern was observed in ig1, likely associated with unsynchronized and abnormal nuclear movements and division (Huang and Sheridan, 1994, 1996).", "baseless1 is another mutation that was found to affect central cell development by altering the position of the two polar nuclei.", "In wild-type plants, the two polar nuclei of a central cell are always positioned in close proximity to the egg cell along the longitudinal axis of the embryo sac.", "In baseless1, the position of central cell polar nuclei was shifted from the center toward the sides abgerminal of the central cell, typically next to the egg cell near the midline of the central cell (Gutierrez-Marcos et al., 2006).", "Similar to baseless1, misplaced polar nuclei and kernel defect was also found in baseless2 (bsl2), no legacy1 (nol1), heirless1 (hrl1), superbase1 (sba1), and maternally reduced endosperm1 (mrn1) mutants (Chettoor et al., 2016).", "As the corresponding gene loci were not yet identified, there is no further information available about the underlying molecular mechanisms.", "However, the defects appear to have no effect on fertilization efficiency, indicating that the position of polar nuclei is not interfering with gamete and gamete nuclei fusion.", "In Arabidopsis it was proposed that an auxin gradient along the micropylar–chalazal axis of the immature FG specifies cell identity during cellularization and further maturation of the FG (Pagnussat et al., 2009).", "By analyzing the auxin response using the DR5 reporter, an obvious auxin gradient could not be detected in the developing maize embryo sac (Lituiev et al., 2013).", "A strong auxin response was, however, observed in the epidermal cells surrounding the MMC, and thus may influence its differentiation at the beginning of the female germline and the onset of meiosis.", "Later, an auxin response was observed in proliferating antipodal cells, which might be involved in regulating and maintaining central cell fate or by providing positional information.", "Another regulatory factor for maize embryo sac development is ZmEAL1.", "ZmEAL1 belongs to a small grass-specific EA1-box protein family (Uebler et al., 2015) and appears to be secreted from the egg cell during FG development at the chalazal pole toward the central cell.", "Phenotype analysis of ZmEAL1-RNAi plants showed the formation of multiple central cells likely originating from antipodal cells.", "ZmEAL1 acts upstream of IG1, indicating that the egg cell is critical for preventing antipodal cells from adopting central cell fate (Krohn et al., 2012).", "SUMOylation/DSULylation also seems to play an important role in FG development and cell specification.", "SUMOylation is a reversible post-translational modification operated by small ubiquitin-related modifiers (SUMOs), which lead to changes in protein stability and activity.", "In maize, a diSUMO-like protein called ZmDSUL was identified with two head-to-tail SUMO-like domains.", "Transcripts of ZmDSUL were found in the developing FG and the egg apparatus, and also in the zygote.", "Downregulation of ZmDSUL activity revealed that ZmDSUL is required for nuclei segregation and positioning as well as cell specification during FG maturation (Srilunchang et al., 2010).", "In summary, positional information, especially of the various FG nuclei, is most critical for cell fate determination and it will now be exciting, but also a challenge, to identify the molecular mechanisms initiating migration of nuclei, cell fate determination, and its maintenance during FG maturation.", "In contrast to animals and evolutionarily older plants such as mosses and ferns, sperm cells of angiosperms are immobile, and transportation is achieved via a pollen tube delivering two sperm cells to the ovule to accomplish fertilization.", "After landing on the stigma (silk), maize pollen grains adhere at papilla hairs, hydrate, and germinate to generate pollen tubes carrying sperm cells.", "Compatible pollen tubes are able to penetrate the intercellular space of stigmatic hair cells and grow inside the silk toward one of the two transmitting tracts, which are each attached to vascular stands.", "Although the length of a maize silk could be more than 30 cm, pollen tubes are capable of reaching ovules within 24 h, indicating that their growth speed exceeds 1 cm per hour (Miller, 1919; Bedinger, 1992).", "The long journey of the tube needs guidance and nutrient supply to reach the ovule.", "Similar to animal reproductive systems, strong competition exists between pollen tubes, and the fastest wins the race and releases its sperm cell cargo for double fertilization.", "As a sporophytic receptive tissue, papilla hair cells of the maize stigma are responsible for holding pollen grains and promoting their germination.", "Within 5 min after capture, pollen grains start to germinate and tubes become visible (Bedinger, 1992).", "Although the structure of pollen grains and the stigma surface are well described (Shivanna et al., 1982), little is known about the physiological and molecular mechanism of maize pollen adhesion.", "In general, plant stigmata are classified into two categories: (1) wet stigmata with surface secretion of proteins, lipids, and pigments, and (2) dry stigmata, which are characterized by an intact surface of papilla cells covered by cell wall material (Edlund et al., 2004).", "Grasses including maize generate dry papilla hair cells.", "The first experimental data to indicate that unknown proteins at the surface of papilla hair cells may play important roles during adhesion concerned the efficiency of pollen attachment, which was demonstrated to be reduced once surface proteins were removed or masked (Heslop-Harrison and Heslop-Harrison, 1981); however, the exact interaction mechanism between pollen grain and hair cells is still unknown.", "Moreover, the adhesion process is highly unspecific, as even pollen grains from various plant species including Arabidopsis can adhere to maize papilla hair cells and germinate (Lausser et al., 2010).", "This finding also suggests that pollen grain adhesion and hydration do not represent a crossing barrier in maize.", "Water content of mature pollen grain is relatively low, as pollen grains are metabolically quiescent and highly desiccated (Buitink et al., 2000).", "The mechanism underlying water and nutrient flow from papilla hair cells to pollen grains is unclear.", "Aquaporin proteins at the stigmatic papilla cells were suggested to play important roles as a water channel (Dixit et al., 2001), but until now—to our knowledge—there is not a single report about the function of aquaporin channels and other proteins in maize papilla cells.", "There is therefore still a long way to go before the adhesion and hydration mechanism is understood.", "Pollen germination and pollen tube growth toward and inside the transmitting tract are complex processes involving many interactions between the male gametophyte (pollen tube) and the female sporophytic tissues of the stigma, style, and ovule.", "Similar to adhesion and hydration, pollen germination on maize silk hairs is rather unspecific, as incompatible pollen of maize and other plant species are capable of germinating, although in some cases with a low germination ratio (Lausser et al., 2010).", "After germination, pollen tubes need to penetrate the intercellular space between papilla hair cells (Figure 2) and navigate their way toward the transmitting tract.", "The tract can be regarded as a highway for pollen tubes growing at maximum speed.", "Alien pollen tubes from non-grass species are not capable of penetration, while incompatible tubes of certain maize genotypes or related species may penetrate but are not able to find the transmitting tract or arrest growth inside of the silk.", "It was discussed that without nutrient support from maternal tissues, incompatible pollen tubes arrest after consuming their own energy (Lausser et al., 2010).", "Based on this observation, it was postulated that a recognition mechanism exists that allows compatible tubes to penetrate the silk, to be guided toward the transmitting tract by unknown attractants, and to support its growth by supplying nutrients and paving the way toward the ovule.", "These processes altogether likely represent crossing barriers in maize.", "But how do pollen tubes find their way and manage to soften the maternal tissues and drive the sporophytic cells apart?", "POLLEN EXTENSIN-LIKE 1 (PEX1), expressed exclusively in maize pollen, was the first discovered gene with a putative role in this process.", "It encodes a protein with two distinct domains, an extension like domain and an N-terminal putative globular domain (Rubinstein et al., 1995a).", "Localized to the pollen tube wall, PEX1 was proposed to be involved in pollen tube growth inside of maternal tissues and as extensin could provide structural support to keep pollen tube cell wall integrity by covalently cross-linking and, thus, stabilizing the cell wall during growth (Rubinstein et al., 1995b).", "In contrast to PEX1, which facilities pollen tube growth by strengthening the pollen tube cell wall, there exists another class of genes named expansins, which function through losing cell wall integrity of maternal tissue.", "In the maize genome, a total of 88 expansin genes were annotated and classified into three subfamilies.", "Sixteen of these were found to be specifically expressed in pollen with putative functions for facilitating pollen tube penetration and in vivo growth (Zhang et al., 2014).", "EXPB1 encodes β-expansin, which was speculated to loosen the cell wall of maize papilla cells and style to facilitate pollen tube penetration and growth.", "Phenotype analysis of an EXPB1 transposon insertion line showed that downregulation of EXPB1 interfered with neither pollen viability nor in vitro pollen tube growth.", "However, pollination with large amounts of pollen resulted in a lower transmission ratio of mutated pollen; pollen tubes frequently twisted and grew around papilla hair cells before penetration, which indicated difficulties for penetration, in vivo growth, and/or fertilization (Valdivia et al., 2007, 2009; Marowa et al., 2016).", "Group I allergens are the major allergens in maize pollen, but little is known about their biological function.", "It was reported that group I allergens are structurally related to expansins and are also able to facilitate the loosening of cell wall material during pollen tube invasion into maternal tissue (Cosgrove et al., 1997).", "Pollen coat proteins are also involved in this process.", "Xylanase and β-glucanase are active enzymes synthesized in tapetum cells during anther development and were transferred to the pollen coat during anther maturation.", "The function of these enzymes is similar to that of expansins: they are supposed to be secreted from the pollen tube to hydrolyze the cell wall of maternal tissue during pollen tube penetration and facilitate its growth inside the transmitting tract (Suen et al., 2003).", "In other species, guidance molecules have been found in the transmitting tract to guide and facilitate the growth of pollen tube in maternal tissues.", "In lily, plantacyanin is generated in the stigma as a diffusible chemotropic factor, which was able to lead pollen tube reorientation in vitro (Kim et al., 2003).", "The function of plantacyanin in pollen tube guidance in the transmitting tract was further supported by functional analysis in Arabidopsis, as its overexpression caused disrupted pollen tube guidance in the style (Dong et al., 2005).", "Up to now there has been no proof for the existence of similar guidance signals in maize.", "A comparison of pollen tube growth from the stigmatic papilla hairs cells toward the transmitting tract between maize and its close relative Tripsacum dactyloides indicated, however, that such molecules may exist, as 100% maize pollen tubes successfully targeted maize transmitting tracts, while this number was strongly reduced when T. dactyloides was used as a pollen donor (Lausser et al., 2010).", "In summary, our knowledge about the progamic phase in maize and other grasses is still in its infancy, which is somewhat surprising considering that the molecular understanding of these processes could help to overcome hybridization barriers between genotypes and even between grass species to improve, for example, the gene pool for plant breeding.", "After a long and competitive journey, pollen tubes finally reach their destination, the ovule.", "The maize ovule is deeply embedded inside the maternal tissues of the ovary, enclosed by glumes and husk.", "After leaving transmitting tracts, pollen tubes are mechanically directed on the surface of the inner integument toward the micropylar region of the ovule.", "They compete with each other until the fastest one targets the micropylar nucellus region and penetrates several nucellar cell layers, and finally reaches the FG (Lausser et al., 2010).", "After entering, the tip of the pollen tube will rupture to release two sperm cells to execute double fertilization, culminating in the fusion of two gamete pairs: egg cell and sperm cell, as well as central cell and sperm cell (Dresselhaus et al., 2016).", "Pollen tube arrival to gamete fusion was reported to be achieved in less than 1 h, while the fusion of male and female nuclei (karyogamy) takes an additional 5 h in the case of the egg cell and 3 h for the central cell (Mól et al., 1994).", "After a long journey through the transmitting tract, pollen tubes finally reach the micropylar opening of the ovule.", "Ovules lacking FGs guide pollen tubes until reaching this region (Lausser et al., 2010), indicating that growth and guidance are almost completely under sporophytic control.", "However, a short-range attraction signal is required to lure pollen tubes through five to six layers of nucellus cells.", "The first identified pollen tube attractant secreted from the FG was ZmEA1 (Zea mays EGG APPARATUS1).", "The analysis of ZmEA1-RNAi plants revealed that pollen tubes are able to reach the micropylar region of the ovule but fail to penetrate and grow toward the egg apparatus of the FG (Márton et al., 2005).", "ZmEA1 is exclusively expressed in the egg apparatus consisting of egg cell and synergid cells.", "Synthetic ZmEA1 could attract maize pollen tubes in vitro and arrest their growth at higher concentration.", "Moreover, ectopic expression of ZmEA1 in Arabidopsis synergid cells enabled its ovules to attract in vitro germinated maize pollen tubes (Márton et al., 2012).", "These findings demonstrated the attraction ability of ZmEA1 for maize pollen tubes and suggested that the egg-apparatus-controlled hybridization barrier can be overcome even between unrelated plant species.", "Via an improved ligand–receptor labeling assay, it was further demonstrated that ZmEA1 interacts in vitro with the subapical tip region of pollen tubes in a species-specific manner.", "Internalization and degradation of ZmEA1 occurs quickly after pollen tube interaction, indicating that pollen tubes remain sensitive to attractants while navigating through the nucellar cell layers of the micropyle (Uebler et al., 2014).", "To date, ZmEA1 represents the only pollen tube guidance molecule in maize.", "The observation that the mutant phenotype is similar to that of ovules lacking FGs suggests that it may represent the sole micropylar pollen tube attractant in this species.", "Homologous genes were also identified in other grasses (Uebler et al., 2015), indicating that the guidance mechanism might be conserved.", "It will now be exciting to identify the pollen tube receptor (complex) and the downstream signaling machinery leading to the pollen tube growth behavior described above.", "To accomplish their mission as a delivery system, pollen tubes need to rupture to release their sperm cell cargo after penetration into the FG.", "Mutant analysis in other species revealed that pollen tube burst needs to be triggered by certain factors such as the receptor-like kinase FERONIA (FER) signaling pathway in Arabidopsis, otherwise pollen tube overgrowth occurs inside of the ovule around the FG (Escobar-Restrepo et al., 2007).", "Similar phenotypes have not yet been described systematically in maize, likely because of technical difficulties associated with the visualization of pollen tube growth deep in the maternal tissues of the ovary.", "However, homologs of FER are also expressed during reproduction in maize (T. Dresselhaus and coworkers, unpublished results) and studies of RNAi lines of ZmES4 in maize showed an overgrowth phenotype indicating that some components of the molecular reception machinery might be conserved.", "ZmES4 (Zea mays Embryo Sac 4) encodes a small defensin-like cysteine-rich protein (DEFL), which accumulates in the secretory zone of the synergid cells surrounding the filiform apparatus before fertilization (Amien et al., 2010).", "ZmES4 belongs to a small FG-specific gene family, of which all four members are predominately expressed in synergid cells, egg cells, and central cells, with lower expression in antipodal cells (Cordts et al., 2001).", "Around 7–8 h after fertilization, the signal of ZmES4 was no longer detectable in the degenerated synergid cell, suggesting that ZmES4 was quickly inactivated and its protein actively degraded after fertilization.", "Downregulation of ZmES4 with RNAi technology indicated that ZmES4 is involved in pollen tube burst: pollen tubes were capable of penetrating micropylar nucellus cell layers, but rupture was not observed in vivo.", "In vitro analysis with synthetic mature ZmES4 protein revealed that ZmES4 is able to induce pollen tube burst in a species-specific manner through interacting with a potassium channel, KZM1.", "This leads to membrane depolarization and ion flux, culminating in water uptake, likely inducing pollen tube burst due to high osmotic pressure inside of pollen tube.", "The species-specific or preferential activity of ZmES4 was speculated to represent a prezygotic hybridization barrier, and thus may act as a component of reproductive isolation in this species (Amien et al., 2010).", "Pectin methylesterases (PMEs), which play a critical role in cell wall stability and integrity (Micheli, 2001), appear to be also involved in pollen tube burst.", "As a fast-growing cell, the pollen tube secrets large amount of PMEs to the apoplast to regulate cell wall stiffness.", "The activity of PMEs is regulated by PME inhibitor (PMEI), which accumulates at the very tip of pollen tube and likely also at the micropylar region of the embryo sac.", "Applying recombinant ZmPMEI1 to in vitro growing pollen tubes destabilizes its cell wall and causes pollen tube rupture in a concentration-dependent manner.", "The burst pattern is different from that of ZmES4, as it happens in the subapical region instead of the extreme tip.", "These findings indicate that embryo sac secreted ZmPMEI1 might destabilize the tip membrane after pollen tube penetration and then act in concert with ZmES4 to release sperm cells for fertilization (Woriedh et al., 2013).", "Similar to Arabidopsis, it is very likely that many more players are involved in sensing the arrival of pollen tubes and releasing the sperm cell cargo such as receptor complexes, components of downstream signaling pathways leading, for example, to calcium spiking, reactive oxygen species production, and modification of transporter activities (Dresselhaus et al., 2016).", "In contrast to Arabidopsis, the maize embryo sac is surrounded by many more maternal cell layers of the ovule and ovary, and thus it is still not possible to investigate the fertilization process directly in vivo.", "For the study of gamete fusion in maize, isolation methods were established to obtain functional male and female gametes for in vitro fertilization.", "Egg and central cells can be manually isolated from ovules after enzymatic digestion and microdissection (Wagner et al., 1989; Kranz et al., 1991), while sperm cells can be collected from pollen grains after osmotic treatment or pH shock followed by gradient centrifugation (Dupuis et al., 1987).", "The establishment of these isolation procedures made in vitro gamete fusion possible.", "Electromediated gamete fusion was first established to generate in vitro zygotes (Kranz et al., 1991).", "However, with this method not only egg cell could fuse with sperm cells; occasionally synergid cells, and non-gametic cells such as leaf mesophyll protoplasts, could also fuse with sperm cells.", "This observation indicates that gamete fusion via electrofusion is very limited for understanding and describing the molecular mechanisms involved in gamete adhesion and fusion.", "This applies also to calcium-mediated cell fusion.", "In a medium containing 5 mM calcium chloride, sperm cells were able to fuse with egg cells after adhering for several minutes without external stimuli (Fauré et al., 1994).", "Although this method also has limitations, the fusion ratio of sperm cells with egg cells is significantly higher compared with sperm cell–mesophyll protoplast fusion, indicating some specificity.", "Fusion of sperm cell and egg cell occurred within 10 s after adhering.", "Notably, the fertilized egg cell was not able to fuse with an additional sperm cell, indicating the presence of a polyspermy block in maize (Fauré et al., 1994; Spielman and Scott, 2008).", "Cell wall material was observed shortly after in vitro fusion and may represent a physical barrier preventing supernumerary sperm cells from adhesion and/or fusion (Kranz et al., 1995).", "The first study on calcium spiking during gametes fusion was also reported in maize.", "It was shown that the gamete adhesion step lasted for several minutes before fusion occurred.", "During this time, cytosolic calcium variation was not observed.", "After triggering fusion, a calcium wave appeared in the fertilized egg cell for up to 30 min (Digonnet et al., 1997).", "This calcium elevation was suggested to be necessary and sufficient to activate the egg cell.", "It was further reported that sperm cell incorporation needs calcium influx, as sperm incorporation could be prevented after treatment using the calcium channel inhibitor gadolinium chloride (Antoine et al., 2001).", "It should be noted, however, that gamete fusion in Arabidopsis triggers only short calcium elevations (Denninger et al., 2014; Hamamura et al., 2014), indicating that the reported long-lasting calcium wave might be correlated to the calcium fusion system and does not reflect the in vivo situation.", "To circumvent the problem of measuring calcium oscillation while using it simultaneously to achieve cell fusion, researchers established a BSA-mediated in vitro gamete fusion system.", "At optimal BSA concentration, gamete fusion usually occurred 7–10 min after adhesion (Peng et al., 2005).", "However, despite these improvements, experimental methods are still very limited and little progress has been made during the past 10 years to understand gamete adhesion, fusion, and activation in maize.", "This is in strong contrast to Arabidopsis, where a number of genes have been identified recently as being involved, for example, in gamete attachment (GEX2), gamete (sperm cell) activation (EC1), gamete fusion (HAP2/GCS1), and, likely, karyogamy (GEX1) (for review see Dresselhaus et al., 2016).", "Homologs of these genes exist in maize (J. Chen and T. Dresselhaus, unpublished results).", "Their functional analyses will elucidate to what extent fertilization mechanisms are conserved in plants and whether the knowledge generated can be used to overcome hybridization barriers to improve the gene pool for modern plant breeding.", "In this review, we focused on the regulatory mechanism of both male and FG development and the various processes associated with fertilization in maize.", "Compared with our understanding of these processes in the model plant Arabidopsis, much less is known about them in maize and other grasses, mainly due to technical difficulties associated, for example, with much thicker tissues and additional cell layers, and limitations in transformation, but also the availability of proper greenhouses that are necessary for the study of reproduction in this important plant species.", "In conclusion, it is surprising to realize that although germline development and fertilization mechanisms have been studied for over a century in maize, the underlying regulatory mechanisms have remained largely unclear and only a few molecular players have been identified to date.", "Considering that yield in maize and other grass crops strongly depends on functional gametes, successful fertilization, and seed development, one would expect that more research efforts should have been made by both academia and the plant breeding industry to understand the underlying molecular mechanisms, which are likely applicable also to other C4 grasses such as sorghum and sugar cane but also to C3 grasses such as rice, wheat, or barley.", "However, recent development of efficient gene-editing methods in maize, especially the CRISPR/Cas9 system (Char et al., 2016; Svitashev et al., 2016), and significantly increased transformation efficiency of maize using even leaves as target tissue and ecotypes that could not previously be transformed as recently reported (Lowe et al., 2016) may strongly contribute to boosting research in this area and lead to the identification of many more molecular players, which will help understand germline development and fertilization in maize and other grasses.", "Funding by the Alexander von Humboldt Foundation to L.-Z.Z. and the German Research Council (DFG) via Collaborative Research Center SFB924 to T.D. is greatly acknowledged." ]	[]
Differences in biomarker and behavioral responses to native and chemically dispersed crude and refined fossil oils in zebrafish early life stages	Petroleum products including crude oils and refined distillates are unique environmental pollutants consisting of thousands of compounds with varying physical-chemical properties and resulting toxicity for aquatic biota. Hence, for a reliable risk assessment individual petroleum product toxicity profiles are needed. Furthermore, the influence of oil spill response strategies like the application of chemical dispersants has to be implemented. The present study addressed the toxicity of water-accommodated fractions (WAFs) of two different oil types on fish early life stages on different biological organization levels in the laboratory model species Danio rerio. Experiments with a 3rd generation dispersant used in loading rated resembling the exposure in experiments with chemically dispersed oils were included, enabling a direct comparability of results. This approach is of high importance as especially the investigation of dispersant toxicity in relevant exposure concentrations is rather scarce. Zebrafish embryos were exposed to different WAFs shortly after and up to 120 hour post fertilization (hpf). Besides phenotypic effects including edema and spine deformations, reduced responses to dark stimuli, increased CYP1A activity and marginal AChE inhibition were observed in sublethal effect concentrations. Both oil types had varying strength of toxicity, which did not correlate with corresponding chemical analysis of target PAHs. Chemically dispersed oils induced stronger acute toxicity in zebrafish embryos compared to native (initial) oil exposure, which was further reflected by very low exposure concentrations for biomarker endpoints. Based on a comparison to the dispersant alone, a higher toxicity of dispersed oils was related to a combination of dispersant toxicity and an elevated crude oil compound bioavailability, due to dispersion-related partitioning kinetics. In contrast to LEWAF and CEWAF neither typical morphological effects nor mechanism-specific toxicity were observed for the dispersant alone, indicating narcosis as the responsible cause of effects.	[ "ENVI" ]	[ "AChE", "Dispersant toxicity", "EROD", "Embryo toxicity", "Oil spill assessment", "Oil toxicity" ]	[ "2304", "2305", "2310", "2311" ]	[ "With steadily increasing oil production, consumption, transportation and rapidly moving exploration (Fritt-Rasmussen et al., 2018; Shafiee and Topal, 2008) huge and small diffuse oil spills still pose a high risk for the aquatic environment.", "In order to accomplish a comprehensive risk assessment of the individual situation, important knowledge about regional ecology, the key oil compounds as well as the ecotoxicological risk of the spilled oil is required (Baker, 1995; Taylor et al., 2018).", "Petroleum products such as crude oils and refined distillates or blended mixtures of fuels each are unique environmental pollutants consisting of thousands of compounds with varying physical-chemical properties (Singer et al., 2000).", "As a consequence, the variety of characteristics might lead to a range of potential exposure scenarios and resulting strength of toxicity for aquatic biota.", "Against the background of a rapid decision making after an oil spill, the unique characteristics highlight the importance of individual petroleum product toxicity profiling.", "Especially embryonic and larval stages of developing fish have been found to be highly sensitive towards the exposure to water-accommodated fractions (WAF) (de Soysa et al., 2012; Jung et al., 2013; Xu et al., 2016).", "It has been concluded that the phenotypical adverse effects caused by oil components in fish embryos are markedly conserved across several freshwater and marine laboratory as well as endemic species of oil-impacted areas (Hodson, 2017; Incardona, 2017; Incardona et al., 2013).", "Frequently reported common adverse effects in fish embryos include craniofacial deformations (de Soysa et al., 2012; Hansen et al., 2019), cardiotoxicity (Brette et al., 2014; Incardona et al., 2013; Jung et al., 2013; Khursigara et al., 2017; Pauka et al., 2011; Philibert et al., 2016) and impaired swimming behavior patterns (Hicken et al., 2011; Mager et al., 2017; Philibert et al., 2016).", "Especially the measurement of biomarker activities in fish early life stages can be sensitively indicative for active defense mechanisms or severe damage caused by oil components.", "With respect to the toxicity of PAHs and co-planar structures especially the phase I oxidation during biotransformation, catalyzed by CYP enzymes is a reliable and highly sensitive biomarker and measured as the EROD activity (Sanni et al., 2017; Van der Oost et al., 2003).", "Also neurotoxicity is emerging as an important endpoint in ecotoxicological risk assessment with biomarker measurements (in this study AChE inhibition) and swimming behavioral assays as suitable screening tools already at early life stages (Legradi et al., 2018).", "The combination of different biomarker measurements can improve the knowledge on toxicity mechanisms and indicate adverse effects even before visible at phenotypical level.", "In the context of oil spill toxicity assessment, alterations in the exposure scenarios caused by different oil spill response strategies have to be considered.", "The deployment of chemical dispersants is one important measure for wide-area response.", "Hence, especially in the case of a larger oil spill, it is quite likely that the exposure situation gets altered with the introduction of another chemical mixture.", "Through reducing the interfacial tensions and breaking down the oil slick, dispersants minimize the risk of smothering seabirds and coastal shorelines.", "However, besides being a chemical burden by itself, this response action results in the enrichment of the water column with dissolved and particulate crude oil compounds, thus potentially increasing the exposure of the pelagic community towards potentially toxic oil compounds (Dupuis and Ucán-Marín, 2015; Ramachandran et al., 2004).", "Even though chemically dispersed petroleum products had been addressed intensively, the knowledge of toxic effects attributed to the dispersant itself being a complex mixture of surfactants and hydrocarbon solvents in relevant exposure concentrations is rather scarce and conclusions about the benefit of dispersant use were contradictory (Prince, 2015).", "Against this background, the present study addressed the toxic impact of two different oil types on early life stages of zebrafish (Danio rerio).", "The zebrafish is a well-established laboratory teleost model species in ecotoxicology and has already been proven a valid test organism in the context of oil spill research (de Soysa et al., 2012; Incardona et al., 2013; Pauka et al., 2011).", "By investigating morphological effects and mechanism-specific endpoints (biomarker activity, behavior changes) at low and simultaneously more environmentally relevant exposure concentrations, the study in detail focused on a) establishing individual petroleum toxicity profiles due to individual chemical composition and b) the dispersant role in chemically dispersed petroleum products by including experiments with a 3rd generation dispersant alone in loading rates resembling the exposure in the experiments with chemically dispersed crude oil, thus enabling a direct comparability of results.", "Through comparing effects of chemically dispersed oil and dispersant alone it was intended to understand whether and to what extent the dispersant contributes to acute fish embryo toxicity.", "The present study focused on two different petroleum products of varying processing degree.", "As a crude oil a naphthenic North Sea crude oil (NNS) characterized by low viscosity and high proportion of low molecular weight saturates and aromatics was selected.", "Additionally, a distillate marine gas oil (MGO), representing a commonly used fossil fuel, was used.", "The MGO is supplemented with the green dye Dyeguard Green MC25 produced by John Hogg Technical Solutions.", "Both oil types were tested as native (initial) samples without additional artificial weathering or any other treatment.", "Representative for a typical oil spill response technique, the 3rd generation dispersants Finasol OSR 51® and 52® (Total Special Fluids, France) were included.", "Finasol OSR 51® was combined with the NNS, while chemically dispersion of MGO was realized using Finasol OSR 52®.", "In order to investigate the influence of dispersants on petroleum product toxicity, Finasol OSR 51® was additionally investigated as representative for an exposure to dispersant alone.", "Dispersants and specific oil combinations were selected based on recommendations with respect to oil type treatment.", "An overview of the different approaches can be found in Table 1.", "In general, low-energy water-accommodated fractions (LEWAF) for native (initial) petroleum product alone, chemically enhanced water-accommodated fractions (CEWAF) for dispersed petroleum product and high energy water-accommodated fractions (HEWAF) for dispersant alone were prepared according to Singer et al. (2000).", "Briefly, WAFs were prepared in aspirator glass flasks (500 mL) by application of oil (LEWAF), a dispersant oil mixture (CEWAF; dispersant-to-oil-ratio (DOR) 1:10) or dispersant alone (HEWAF) on the surface of fish medium at an oil/dispersant-to-water ratio of 1:50 (LEWAF), 1:200 (CEWAF) or 1:2000 (HEWAF).", "The LEWAF setup was carefully stirred avoiding a vortex while for the CEWAF and HEWAF setup stirring speed was adjusted to create a 25% vortex of the water column.", "WAFs were incubated stirring at 10 °C for 40 h followed by 1 h settling time.", "Dilution series were prepared in fish medium from the 100% stock solutions that had been drained off carefully, and were warmed up to 26 °C before embryos were exposed to the samples.", "Artificial fish medium was prepared, aerated, warmed up and adjusted to a pH of 7.5 ± 0.5 one day before using.", "This study utilized wildtype zebrafish of the WestAquarium strain (Bad Lauterburg, Germany) from the facilities of the Institute for Environmental Research, RWTH Aachen University.", "The breeding groups consisted of 100–120 adult zebrafish from 1 to 3 years of age.", "Zebrafish were kept in 170 L tanks in a flow-through system with a water exchange rate of 40% per week and a biological filter and UV light as cleaning steps.", "Fishes were fed twice a day with dry flakes (TetraMin®, Tetra GmbH, Melle, Germany) and larvae of Artemia spec (JBL GmbH & Co.", "KG, Neuhofen, Germany).", "A constant day-night rhythm (14:10) and temperature (26 ± 1 °C) was maintained.", "Spawning took place from 30 min after the onset of light.", "Fertilized eggs were collected 2 h after the onset of spawning.", "Shortly after fertilization embryos were divided in groups of 5 individual and transferred into 10 mL air sealed glass vials with sparsely head space containing the exposure solutions.", "The glass vials have been proven to minimize the evaporation of volatile WAF compounds in pretests.", "For the fish embryo acute toxicity test (FET) 20 embryos per treatment dilution were exposed to WAF samples according to the OECD guideline 236 (OECD, 2013).", "To investigate the swimming behavior 16 (control) and 20 (treatment) individual embryos were used per treatment dilution, while for 7-Ethoxyresorufin-O-deethylase (EROD) and acetylcholinesterase (AChE) biomarker measurements a pool of 40 embryos was necessary for each exposure concentration.", "WAF exposure dilutions for the FET were selected based on range finding tests in order to identify a clear concentration-response relationship spanning an effect range from 0 to 100%.", "Exposure concentrations for EROD and AChE measurements were in the range of sublethal effect concentrations inducing partial to no morphological effects in order to avoid secondary acute toxic effects masking biomarker responses of embryonic defense mechanisms.", "In detail, the highest exposure concentrations were around EC50, followed by exposure concentrations around EC10 and below EC10.", "CEWAF from both oil types showed stronger fluctuation around the steep curve and hence exposure concentrations were adjusted to a range covering slightly increased (NNS) or decreased (MGO) maximum exposure concentrations.", "Embryos were incubated at 26 °C using a semi-static approach with medium exchange every 24 h and a constant day/night rhythm (14:10).", "WAFs for medium exchange were prepared fresh daily.", "All zebrafish experiments of the present study included negative (fish medium, all endpoints) and positive (3,4-dichloranlilin (4 mg L−1), FET) controls to verify test validity and basal biomarker responses.", "The prolonged fish acute embryo toxicity test up to a maximum of 120 hour post fertilization (hpf) was performed according to OECD guideline 236 (2013) with minor modifications with respect to sample type specifications (details described below).", "All experiments were terminated with the final measurement shortly before 120 hpf.", "Zebrafish embryos and larvae below 120 hpf are not protected animal stages according to EU Directive 2010/63/EU (European Parliament and Council of the European Union, 2010) (see also: (Strähle et al., 2012), TierSchG (Tierschutzgesetz) and the respective regulation TierSchVerV (Tierschutz-Versuchstierverordnung).", "After termination, larvae were euthanized by prolonged immersion in a benzocaine ethanol solution.", "Observation of embryonic development was carried out throughout the exposure period every 24 h and lethal and sublethal effects according to criteria defined in the OECD guideline 236 were documented.", "Additionally, medium pH was measured every 24 h.", "An experiment was classified valid if no >10% of negative control and at least 30% of positive control eggs showed lethal effects according to the OECD 236 guideline.", "All data presented in the present study (3 independent experiments) were valid according to these criteria.", "In order to identify sublethal effect concentrations, a 4-parameter non-linear regression model (GraphPad Prism v 6, San Diego, USA) was used to fit a concentration-response curve to the data.", "Top and bottom were fixed to 0 and 100, respectively.", "Equation: Y = 100 / (1 + 10^((logEC50-X) ∗ HillSlope)).", "Larval swimming behavior alterations were investigated using 96 hpf zebrafish larvae and a light/dark transition test.", "16 (control) to 20 (WAFs) zebrafish larvae per treatment concentration were transferred individually alongside 200 μL exposure solution into wells of a 96-well plate.", "They were exposed to 2 cycles of alternating light (10 min) and dark (4 min) periods after an initial acclimatization period of 10 min in light conditions.", "The swimming performance of control and WAF-treated larvae in sublethal effect concentrations was tracked during the experiment.", "Experiments were conducted using a DanioVision observation chamber and EthoVision tracking software (Noldus, The Netherlands).", "Data processing was conducted in spreadsheets (Microsoft Excel 2016).", "In detail, the mean distance moved of 16–20 larvae per treatment and 1 min time bin was used for the evaluation of swimming patterns.", "In total, 3 independent experiments were performed and hence data were plotted and analyzed as mean (3 experiments) of mean (20 individuals per experiment) distance moved per 4 min interval in order to ensure a comparability of light and dark phase.", "Graphs were plotted using the software GraphPad Prism (version 6, San Diego, USA).", "Biomarker activity in unexposed and WAF-treated hatched zebrafish larvae was investigated at 96 and 120 hpf.", "In total, 40 embryos per treatment concentration were pooled after the exposure, anesthetized using a saturated benzocaine solution and washed twice with cold phosphate buffer saline (PBS, Sigma-Aldrich).", "Anesthetized larvae were quickly transferred to 1.5 mL tubes and excess solution was replaced with 700 μL of phosphate buffer (1.8 L 0.1 M Na2HPO4 adjusted with 0.5 L 0.1 M NaH2PO4 to pH 7.8).", "Larvae were immediately shock frozen in liquid nitrogen and stored at −80 °C until further use.", "For measurement of enzyme activity larvae in buffer were carefully thawed on ice and homogenized for 10 s using an electric dispersing device (VDI 12, VWR International GmbH, Germany).", "Afterwards, homogenates were centrifuged for 15 min at 4 °C and 10,000g. Subsequently, the supernatant was instantly transferred to new tubes and placed on ice.", "Both EROD and AChE were measured successively in order to avoid re-freezing and thawing of the samples.", "The investigation of phase I metabolism induction as a biomarker of exposure to PAHs and similar co-planar structures was conducted by means of the fish embryo 7-ethoxyresorufin-O-deethylase (FE-EROD) assay.", "The test was performed according to the protocol described by Schiwy et al. (2015) with modifications regarding egg number and quantification method (kinetic measurement) as described in Meyer-Alert et al. (2019).", "Sample supernatants of treated and control larvae were tested in triplicates on a 96-well plate.", "The substrate 7-ethoxyresorufin (2.4 μM) was added to each well and subsequently incubated for 10 min at 26 °C in darkness.", "Shortly before the kinetic measurement of fluorescence for 25 min (step 1: kinetic cycles: 15, interval time: 20 s, step 2: kinetic cycle: 30, interval time: 40s) in a microplate reader (Infinite® M 200, Tecan Group, Switzerland), NADPH (3.35 mM) was added to initiate the enzymatic reaction.", "Substrate deethylation was determined by measuring the formed resorufin at 540 nm excitation and 590 nm emission wavelength.", "Quantification of EROD activity was performed based on the resorufin calibration series (500–2 nM) and expressed in pmol resorufin mg−1 min−1.", "Data processing was done in spreadsheets (Microsoft Excel) with specific enzymatic activity normalized to the mean activity of the untreated control.", "Normalization was performed in order to guarantee comparability within the study and with previous studies.", "Data were statistically analyzed for normal distribution (Shapiro-Wilk test) and equal variance (Levene test) and then further investigated for statistical significant difference to untreated control using the SigmaPlot software package (v. 12.5, Systat Software, 2007).", "Data fulfilling both criteria were analyzed using One-way Analysis Of Variance (ANOVA) with Dunnett's post-hoc test (p < 0.05).", "Non-parametric Kruskal-Wallis ANOVA on ranks with Dunn's post-hoc test was used for data that were not normally distributed and/or not of equal variance.", "As a surrogate endpoint for neurotoxicity the acetylcholinesterase (AChE) activity was measured according to the initial protocol established by Ellman et al. (1961) with modifications described by Velki et al. (2017) regarding adaptions to a 96-well plate format.", "7.5 μL sample supernatant and assay reagents (180 μL sodium phosphate buffer (0.1 M, pH 7.8), 10 μL 5,5′-dithiobis-2-nitrobenzoic acid (DTNB 1.6 mM), 10 μL acetylcholine iodide (156 mM)) were added in triplicates to a 96-well plate.", "The increase in absorbance was immediately measured in triplicates at 412 nm for 10 min in 10 s intervals using a microplate reader (Infinite® M 200, Tecan Group, Switzerland).", "Resulting data were controlled for linearity in absorbance increase (R2 ≥ 0.98) and minimum increase of absorbance over time (Δt3min ≥ 0.1).", "Only data fulfilling the control criteria were used for further evaluation.", "Enzymatic activity was calculated as nmol AChE min−1 mg−1.", "For the calculations the molar extinction coefficient of 13,600 M−1 cm−1 was used.", "Data processing including normalization was performed as described for the EROD activity.", "In parallel to enzyme activity measurements, protein in whole-specimen homogenates was determined.", "A Bicinchoninic Acid kit (Sigma Aldrich GmbH, Germany) was used according to the manufacturer's instructions.", "Protein quantification was carried out with a dilution series of bovine serum albumin (BSA) as an external standard (0.31–1 mg mL−1).", "For protein measurement the sample supernatants were diluted 1:2.", "Solid phase micro extraction (SPME) as described in detail in Potter and Pawliszyn (1994) was used to quantify a set of 18 target PAHs in native LEWAFS of both oil types.", "Details can be found in the SI (Section 1).", "Native and chemically dispersed WAFs from both oil types induced a concentration-related increase in sublethal and lethal effects (Fig. 1) in the prolonged fish embryo acute toxicity test (FET).", "While zebrafish embryo exposure to undiluted LEWAF stocks (1:50) of both oil types led to 100% mortality in 120 hpf larvae, the CEWAF stocks (1:200) were markedly more toxic to zebrafish embryos.", "Dilutions of 12.5% (NNS) and 5% (MGO) still induced 100% mortality at the test end.", "In a direct comparison of both oil types, a higher embryotoxicity was observed for the distillate in both LEWAF and CEWAF approaches (Fig. 1).", "Calculated EC50 values of MGO with 25.2% (LEWAF) and 1.1% (CEWAF) of stock were below the EC50 values resulting from NNS exposure (33% for LEWAF and 2.0% for CEWAF, Table 2).", "In order to ensure a direct comparability of dispersant HEWAF to the (NNS) CEWAF approach, the resulting EC50 value was expressed as % of 1:200 CEWAF stock containing corresponding dispersant amounts (DOR: 1:10).", "Similar to LEWAF and CEWAF the exposure to the dispersant alone did induce concentration-related embryotoxicity.", "With 50% of zebrafish larvae at 120 hpf showing malformations at exposure dilutions of 4.4% of stock (EC50), the HEWAF toxicity was in a comparable range but still below the acute toxicity induced by the corresponding CEWAF (2.0%).", "Importantly, the embryotoxicity of the dispersant HEWAF, in contrast to oil LEWAF and CEWAF, was strongly dependent on the embryonic developmental stages.", "Embryos that were protected by the chorion did not show any harmful effect even for the highest exposure concentrations.", "After hatching set in around 72 hpf, these normal developed embryos died until the final inspection at 120 hpf (coagulation).", "In general, most prominent sublethal morphological effects on LEWAF- and CEWAF-treated embryos were heart deformations, pericardial and yolk sack edema associated with bradycardia as well as spine deformations independent of the oil type (SI, Fig. 1A–C).", "In contrast, no cardiovascular effects were observed for the HEWAF treatment, but hatched larvae showed deformations of caudal fins, mainly manifested in nodes occurring in fin ray structures (SI, Section 2, Fig. 1D).", "Fin deformations of HEWAF-exposed embryos were observed for up to 60% of embryos at test end.", "Interestingly, while hatching success was not affected for MGO-exposed zebrafish embryos, NNS caused significantly reduced hatching rates in sublethal exposure dilutions at 120 hpf (Fig. 2).", "Also HEWAF-exposed larvae showed a tendency towards hatching delay for 72 and 96 hpf embryos compared to the untreated control.", "In general, the chemically dispersed oils (CEWAF) showed the trend to stronger EROD induction compared to the native oils (LEWAF, Fig. 3).", "Zebrafish embryos exposed to LEWAFs of both oil types did show marginally increased EROD activities up to 2-fold normalized to the untreated control with the exception of larvae at 120 hpf exposed to MGO LEWAF, reaching a maximum induction of 3.1-fold.", "In contrast, while for the crude oil CEWAF no changes in EROD activity were detected in 96 hpf larvae, a concentration-related increase in CYP4501A induction up to a maximum of 4.7-fold at the highest exposure concentration (3.13% of stock) was found in 120 hpf larvae.", "Independent of the investigated developmental stages, larvae exposed to MGO CEWAF showed the highest EROD activities for all exposure concentrations (up to 5.9-fold of control).", "Both oil type CEWAFs induced increased EROD activities compared to control groups already at the lowest exposure concentrations that were not found to induce morphological effects during embryonic development (<EC10).", "Overall, MGO exposure resulted in higher EROD induction independent of test approaches using native or chemically dispersed oil WAFs.", "The dispersant Finasol OSR 51®, which was tested in concentration ranges identical to CEWAF loading rates, did not increase EROD activities (Fig. 3).", "In comparison to the control groups, none of the treatments gave a statistically significant difference (One-Way ANOVA with Dunnett's post hoc test, p < 0.05).", "No clear concentration-related AChE inhibition was observed (Fig. 4), and also no significant difference could be detected (One-Way ANOVA with Dunnett's post hoc test, p < 0.05).", "However, sublethal exposure dilutions of crude oil (12.5 and 25% of stock) resulted in decreased AChE activities at 96 hpf (50% of NC).", "All remaining exposure concentrations, independent of oil type or developmental stage, led to AChE activities corresponding to their unexposed control.", "Additionally, also the dispersant Finasol OSR 51® did not show AChE inhibition.", "Unexposed control larvae did respond to the photo-alternating cycles in a normal manner, with high activity during dark and low activity during light phases (Fig. 5).", "In contrast, native and chemically dispersed MGO treatments led to a constantly low baseline swimming activity independent of the light/dark transitions in all exposure concentrations.", "Larvae exposed to NNS crude oil showed similar behavior of no response to dark stimulus.", "Detailed locomotion patterns of NNS treatments will be published and described within a separate study.", "No differences in response to the dark stimulus were observed between LEWAF and CEWAF exposure groups.", "The most prominent target PAHs in undiluted WAFs from both oil types were naphthalene, phenanthrene and fluorene.", "Naphthalene was found in concentrations >290 μg L−1 with higher concentrations in the NNS, while fluorene and phenanthrene were detected in the low μg L−1 range.", "All remaining target PAHs were in the low ng L−1.", "Details can be found in the SI (Table 1).", "Morphological effects including cardiotoxicity and spine deformations were in compliance with previous studies focusing on WAF-induced acute toxicity in early life stages of zebrafish (de Soysa et al., 2012; Incardona et al., 2005; Incardona et al., 2013; Jung et al., 2013; Pauka et al., 2011; Perrichon et al., 2016) as well as other species (Jung et al., 2015; Khursigara et al., 2017; Martin et al., 2014; Rodrigues et al., 2010; Xu et al., 2017) for a variety of other crude and refined petroleum products compared to the present study.", "Overall, a higher toxicity of the distillate compared to the crude oil was observed.", "This, however, did not correlate with the chemical profile of target PAHs.", "With the exception of higher naphthalene concentration in NNS crude oil, the concentrations of other target PAHs were only marginally increased in MGO LEWAF (see SI, Table 1).", "The lack of effect explanation from the present chemical analysis, mainly based on the US-EPA priority PAHs, indicates that either PAH-derivates or other uncharacterized WAF compounds were responsible for the differences in embryotoxicity, which was concluded also in previous studies (Andersson and Achten, 2015; Meador and Nahrgang, 2019).", "Especially alkylated PAHs and crude oil fractions containing alkylated congeners have been found to induce much stronger embryotoxicity than the mother compounds (Adams et al., 2014; Incardona, 2017).", "Furthermore, it has to be considered that the MGO is supplemented with a green dye.", "The toxicity of this additive could not be addressed in the present study and hence a contribution of the dye towards increased toxicity cannot be excluded.", "Phenotypical adverse effects have been attributed mainly to tricyclic PAHs and their alkylated derivates, which act via AhR-dependent and independent mechanisms - in the latter case often through interruptions of calcium signaling (Brette et al., 2014; Brette et al., 2017; Hodson, 2017; Incardona, 2017; Sørhus et al., 2016).", "In contrast, it was recently suggested that crude oil toxicity might be linked to narcosis instead of receptor-mediated pathways (Meador and Nahrgang, 2019).", "With focus on molecular processes in oil exposed fish larvae based on whole transcriptional or microarray analyses, it becomes obvious that WAFs, being complex pollutant mixtures, initiate multiple and simultaneously interacting molecular mechanisms (Hook et al., 2010; Xu et al., 2016), and that the pathways of crude oil toxicity remain partially unknown.", "Overall, despite identical phenotypical effects the different strength in toxicity caused by the two oil types emphasizes again the importance of individual ecotoxicological profiles in the context of a reliable risk assessment.", "In general, it has to be mentioned that the present experimental setup with respect to WAF stock solutions and chronic embryo exposure displayed a worst case scenario.", "The present concentrations of dissolved oil compounds exceeded concentrations found in the environment after an oil spill.", "Mean total PAH concentrations in the water column during the Deepwater Horizon Blowout were, for example, mainly <10 μg L−1 (Echols et al., 2015), which is 10 times below the effective concentrations found for the WAF investigated in the present study.", "Additionally, as already discussed previously, chronic exposure without a recovery phase is not representative for open water conditions (Bejarano et al., 2014).", "However, also short-time exposure towards crude oil WAF resulted in adverse effects, indicating the time window around hatching to be the most sensitive (Mager et al., 2017).", "The fact that biomarker responses were altered already at more realistic sublethal exposure concentrations (around EC10) further supports the relevance of the present results.", "Results from FET indicate a contribution of the dispersant to the higher toxicity of crude oil CEWAF compared to LEWAF.", "In compliance to some WAF treatments the dispersant alone did induce a trend towards a hatching delay in zebrafish embryos.", "This effect seems to be a general response to dispersant exposure, as recently similar patterns in hatching delay have been observed for zebrafish embryos exposed to a different chemical dispersant (Li et al., 2018).", "However, the increased adverse effects in embryos cannot exclusively be explained by the dispersant itself as the HEWAF exposure resulted in higher effect concentrations than the CEWAF exposure.", "Hence, a combination of dispersant toxicity and an elevated crude oil compound bioavailability, due to increased partitioning kinetics, is most likely responsible for the high CEWAF toxicity.", "Dispersant application leads to the formation of small droplets with high surface to volume ratio (Prince, 2015), resulting in increased dissolved fractions in the water column.", "Increased relative PAH concentrations in aqueous solution of WAFs after the application of a dispersant have been observed in several studies (Cohen et al., 2001; Couillard et al., 2005; Hook and Osborn, 2012).", "Importantly, the increase of dissolved oil compounds indicates that the initial WAF system was not in a stable state of equilibrium, in which droplets should not change PAH partitioning.", "However, also the influence of droplets in micro- or even nano-scale should be considered as a route of exposure (Hansen et al., 2019).", "Interestingly, HEWAF-exposed embryos of the present study did not show typical adverse morphological effects (blue sac disease) during embryonic development as oil exposed embryos and lethal effects became apparent only after larvae were hatched and no longer protected by the chorion.", "Additionally, in contrast to CEWAF and LEWAF treatments no mechanism-specific bioactivity via CYP1A induction was observed.", "These results might indicate narcosis (baseline toxicity) via membrane disruption as the predominant cause of effect of the dispersant.", "In contrast, even though narcosis was suggested as an important toxicity mechanism identical mode of actions have been concluded in a previous study since highly similar gene expression profiles were identified for oil and dispersant treatments (Hook and Osborn, 2012).", "Biphasic biotransformation predominantly occurs in vertebrate livers and efficiently transforms lipophilic xenobiotics into polar hydrophilic metabolites that can be eliminated via excretion (Schlenk et al., 2008).", "The EROD activity has been found to be indicative for petroleum product fish exposure in several studies across a variety of developmental stages (Oliveira et al., 2007; Perrichon et al., 2016; Sanni et al., 2017; Van der Oost et al., 2003; Whyte et al., 2000).", "The biotransformation capacity of fish to efficiently metabolize and eliminate PAHs, depending on physical-chemical properties or the mixture composition, is particularly high (Logan, 2007; Van der Oost et al., 2003) relativizing the risk of pollution indicated by CYP activity biomarker.", "Nonetheless, especially during early organogenesis a quite limited capacity for metabolism is described, and hence a higher bioconcentration of dissolved PAHs in embryos is expected (Incardona, 2017; Jung et al., 2015).", "Furthermore, biotransformation can generate bio-activated metabolites (e.g. DNA-adducts) that can elicit a variety of effects if fish.", "EROD activities observed in the present study were in a comparable range found in zebrafish larvae (Pauka et al., 2011; Perrichon et al., 2016) and adults (Arukwe et al., 2008) exposed to a set of fresh and weathered crude oils.", "The EROD activity in zebrafish larvae was measured in the present study during in the most sensitive time windows of post-hatching development (96 and 120 hpf) within the limits of animal free testing, as has already been shown before (Bräunig et al., 2015; Kais et al., 2017).", "Even though cyp450 gene expression has been determined in early embryonic stages prior to hatching, post-translational modifications were suggested to impact the enzyme activity leading to low responses on protein level (Meyer-Alert et al., 2018) within the first 2–3 days of development.", "Interestingly, the distillate induced stronger EROD activity compared to the crude oil even in comparable WAF exposure dilutions.", "The potential to induce CYP1A in fish for the most prominent PAHs namely naphthalene, phenanthrene and fluorene varies enormously throughout literature.", "While those PAHs were described as weak CYP1A inducer when tested as single compounds (Barron et al., 2004; Billiard et al., 2004; Hawkins et al., 2002), other studies reported high CYP1A activity in zebrafish embryos exposed to complex sediment extracts with phenanthrene being one of the dominant PAHs (Kais et al., 2017).", "Results from several studies focusing on modes of action in fish embryo toxicity further expanded the complexity of potential molecular mechanisms by demonstrating AhR-dependent and -independent CYP1A-dependent and -independent mechanisms (Incardona, 2017; Incardona et al., 2005; Incardona et al., 2006; Scott et al., 2011).", "Finally, from previous and present results it can be concluded that oil WAFs represent a complex mixture of weak and strong CYP1A inducer acting additively, synergistically or even antagonistically via different pathways.", "Neurotoxicity in early life stages of fish is emerging as an important endpoint in ecotoxicological risk assessment with biomarker measurements (AChE inhibition) and behavioral assays as suitable screening tools (Legradi et al., 2018).", "In zebrafish, the brain and the nervous system are nearly fully developed around 96 hpf (e.g. reviewed in (Legradi et al., 2015)).", "The AChE expression and activity in zebrafish increases within the first days of embryonic and larval development (Bertrand et al., 2001; Yen et al., 2011) and is largely inhibited by, for example, neuroactive pesticides in 96–120 hpf larvae (Velki et al., 2017).", "In some studies crude oil compounds such as PAHs have been found to inhibit AChE in fish based on in vitro studies with purified AChE (Jett et al., 1999; Kang and Fang, 1997) or AChE biomarker measurements in adult fish tissue (Akaishi et al., 2004), while other studies failed to determine PAH (Tang et al., 2003) or oil contamination-related inhibitory (Jung et al., 2011) response.", "In concordance with the present study whole specimen-homogenates of zebrafish embryos showed reduced AChE activity after the exposure towards sediment extracts, highly contaminated with PAHs (Kais et al., 2015).", "Stronger AChE inhibitory effects in the crude oil compared to the distillate might be related to higher biotransformation and detoxification in MGO-treated embryos as indicated by the CYP1A activity.", "Also more bioavailable compounds in NNS that interact with AChE cannot be excluded based on the limited chemical analysis of the present study.", "Changes in swimming behavior patterns can further indicate a neurotoxic potential of complex mixtures.", "Petroleum product WAF-exposed larvae had shown reduced sensitivity towards touch stimulus (de Soysa et al., 2012).", "Furthermore, in compliance with the present study, reduced swimming activities have been observed for oil-exposed larvae and juveniles in locomotor and light dark/transition tests (Mager et al., 2014; Perrichon et al., 2016).", "However, at least one previous study has found no effects on swimming behavior for Arabian light crude oil (Perrichon et al., 2016).", "Importantly, previous studies have further shown that impaired swimming activity can manifest as delayed effects in fish that were originally exposed to crude oil WAFs during an early time window of embryonic development, even after a prolonged time window without exposure (Hicken et al., 2011; Mager et al., 2014).", "However, the mode of actions behind behavioral changes can be attributed to several pathways.", "They might not exclusively be related to neuroactive effects, but rather a secondary effect due to several other physiological events.", "Deformations in the muscular system (de Soysa et al., 2012) as well as cardiotoxicity-based reduction in swimming activity (Hicken et al., 2011) or changes in brain monoamine contents (Vignet et al., 2017) have been suggested to explain behavioral disruptions.", "Furthermore, recent results indicate impairments of normal eye development, which might explain lacking response to light/dark alterations simply by interrupted visual perception.", "In this context, significant down-regulation of genes involved in functional eye development (Xu et al., 2017; Xu et al., 2016) and histological changes including reduced eye size or disruption and apoptosis in retinal cell layers (Huang et al., 2013; Kawaguchi et al., 2012) have been reported for fish larvae exposed to crude oil WAFs or PAHs.", "In summary, the present findings were in compliance with findings for other fish species and oil types indicating that at least most petroleum products are able to induce a common set of adverse effects in fish early life stages.", "In terms of oil spill risk assessment, the present results further complement the knowledge about possible environmental impacts of oil contaminations and help evaluating their severity based on the determined effect concentrations.", "However, crude oil toxicity in fish seems to be diverse.", "Further studies should focus on the modes of action of those complex mixtures on different biological organization levels.", "Additionally, future work should include more insight into the toxicity as well as the mode of actions of dispersants in relevant exposure concentrations.", "For the extrapolation of the present results to endemic species of oil-spill affected regions in the context of oil spill risk assessment it has to be considered that response sensitivities can significantly vary among different species (Incardona, 2017; Jung et al., 2015; Perrichon et al., 2016).", "Also the extrapolation to later developmental stages including juveniles and adults is limited due to potential changes in metabolism or tissue-specific CYP1A activity.", "Another aspect that urgently needs to be addressed in more detail is the role of oil weathering due to accompanying changes in physical-chemical properties as well as exposure scenarios and toxicity, since the weathering starts immediately after the spill.", "Overall, each oil spill displays a unique situation for response strategies and data including the results obtained from the present study can contribute to a better understanding of oil toxicity." ]	[ "Oil-specific (sub-)lethal effect concentrations for oil spill risk assessment were established.", "Oil types induced different strength of embryo toxicity not correlated with common PAHs.", "Sublethal exposure concentrations resulted in CYP1A induction but no AChE inhibition.", "Larvae showed strong behavioral impairment already at low exposure concentrations.", "Dispersant contributes to higher toxicity of chemically dispersed oils compared to native oils." ]
Model reformulations for Work and Heat Exchange Network (WHEN) synthesis problems	The Duran-Grossmann model can deal with heat integration problems with variable process streams. Work and Heat Exchange Networks (WHENs) represent an extension of Heat Exchange Networks. In WHEN problems, the identities of streams (hot/cold) are regarded as variables. The original Duran-Grossmann model has been extended and applied to WHENs without knowing the identity of streams a priori. In the original Duran-Grossmann model, the max operator is a challenge for solving the model. This paper analyzes four ways to reformulate the Duran-Grossmann model. Smooth Approximation, Explicit Disjunctions, Direct Disjunctions and Intermediate Temperature strategy are reviewed and compared. The Extended Duran-Grossmann model for WHEN problems consists of both binary variables and non-smooth functions. The Extended Duran-Grossmann model can be reformulated in similar ways. In this study, the performance of different reformulations of the Extended Duran-Grossmann model for WHEN problems are compared based on a small case study in this paper.	[ "CENG", "COMP" ]	[ "Duran-Grossmann model", "MINLP", "Reformulations", "Work and heat exchange networks", "disjunctive programming" ]	[ "1500", "1706" ]	[ "Heat integration has been widely used to save hot/cold utilities because thermal energy contributes significantly to the total cost of a process (Huang and Karimi, 2013).", "The classical heat integration techniques, such as pinch technology (Klemeš and Kravanja, 2013), can only deal with the heat integration problem with known stream data.", "If heat integration and process optimization are performed simultaneously, i.e. heat integration considering variable process streams, more benefits can be achieved.", "Duran and Grossmann proposed a mathematical model for simultaneous process optimization and heat integration (Duran and Grossmann, 1986).", "The Duran-Grossmann model is a powerful tool to solve the heat integration problem with variable process streams.", "This paper has been cited more than 350 times by the end of 2018.", "Their model has been successfully applied to organic Rankine cycle systems recovering low-temperature waste heat (Yu et al., 2017a), processes for liquefaction of natural gas (Wechsung et al., 2011), optimal reactor network synthesis (Lakshmanan and Biegler, 1996), and fuel cell systems (Marechal et al., 2005).", "To improve the performance of the model, several reformulations are proposed in previous studies, which will be reviewed and compared in this study.", "The new topic (see e.g. Yu et al., 2018a) referred to as Work and Heat Exchange Networks (WHENs) arise if pressure manipulations are considered while designing Heat Exchanger Networks (HENs).", "There are many potential applications of WHENs theory, such as a novel process for offshore liquefaction of natural gas (Aspelund and Gundersen, 2009), effluent gas recovery (Liao et al., 2017), process integration in carbon capture processes (Fu and Gundersen, 2016), and optimal distillation column integration (Nair et al., 2018).", "More applications can be found in the literature (Yu et al., 2018b).", "More generally speaking, not only temperatures but also pressures have to meet some specifications in a system.", "Pressure specifications for process streams make the problem more challenging compared with conventional HENs.", "Holiastos and Manousiouthakis (2002) proposed a mathematical model minimizing hot/cold/work utility cost for HENs.", "Here “work utility” refers to the generation or consumption of work.", "Aspelund et al. (2007) proposed a manual methodology referred to as Extended Pinch Analysis and Design (ExPAnD), where traditional Pinch Analysis is extended with pressure considerations and Exergy Analysis.", "Marmolejo-Correa and Gundersen (2012) proposed a methodology combining Exergy and Pinch Analyses to design a Reverse Brayton cycle for the liquefaction of natural gas.", "Based on this study, Marmolejo-Correa and Gundersen (2013) developed a novel diagram for exergy and energy targeting for a heat recovery system subject to changes in both temperature and pressure.", "This method is particularly suitable for low temperature systems such as LNG processes.", "Fu and Gundersen (2015a) presented a systematic graphical design procedure for the integration of compressors in HENs above ambient temperature.", "Similarly, Fu and Gundersen (2015b) integrated compressors into heat exchanger networks below ambient temperature.", "Four theorems were proposed and used as the basis for the design methodology.", "Fu and Gundersen also integrated expanders into heat exchanger networks above (Fu and Gundersen, 2015c) and below (Fu and Gundersen, 2015d) ambient temperature.", "Wechsung et al. (2011) combined Pinch Analysis, Exergy Analysis, and Mathematical Programming to synthesize HENs below ambient temperature with compression and expansion of process streams.", "The WHENs problem involves both heat integration and work integration.", "The Duran-Grossmann model can be extended to solve WHEN problems.", "Since the thermodynamic path and the identity (hot/cold) of process streams are unknown in WHENs, classical heat integration methods cannot be applied.", "In addition, the identity of streams can also temporarily change in WHENs.", "This paper is a more comprehensive version of Yu et al. (2018c) that extended the Duran-Grossmann model to WHEN problems, where the identities of streams are unknown a priori.", "The present paper adds yet another reformulation based on a so-called Intermediate Temperature strategy.", "The various reformulations of the original Duran-Grossmann model are applied to the Extended Duran-Grossmann model for WHEN synthesis problems.", "There are four different reformulations for the Extended Duran-Grossmann model presented in the literature.", "This study investigates the different reformulations and their computational expenses.", "DG.1 and DG.2 are the equality and inequality constraints for the industrial process.", "The vector ω denotes process parameters such as pressure, temperature, or parameters in cost correlations.", "DG.3 and DG.4 are used to assign the inlet temperature of each stream to potential pinch candidates.", "It should be noticed that only cold stream inlet temperatures are modified to consider the effect of the Heat Recovery Approach Temperature (HRAT).", "DG.5 and DG.6 denote the total hot stream heat load and total cold stream heat load above each pinch candidate temperature.", "DG.7 and DG.8 aim at identifying the correct pinch point, which features the maximum heat deficit among all the pinch candidates.", "DG.9 and DG.10 are energy balances for the system.", "The Duran-Grossmann model incorporates max operators, which result in non-differentiabilities at Tp.", "Max operators are challenging for deterministic solvers and have to be removed before solving the model.", "The original Duran-Grossmann model has proven to be powerful in process design.", "Thus, interest has increased in the Process System Engineering (PSE) field to find ways to reformulate the model.", "Four different reformulations have been found in the literature, and these are presented and used in the Extended Duran-Grossmann model for WHEN synthesis.", "The four reformulations are the following: (1) Smooth Approximation, (2) Explicit Disjunction, (3) Direct Disjunction, and (4) an Intermediate Temperature strategy.", "Here, ɛ is a small constant, typically between 10−3 and 10−6.", "However, this reformulation may encounter problems when dealing with isothermal streams.", "In addition, the performance of the approximation depends on the value of the small constant, which may cause numerical conditioning problem if chosen improperly (Grossmann et al., 1998).", "The small parameter is close to zero, and the Smooth Approximation can sometimes be ill-conditioned.", "To remove the max operator in the original Duran-Grossmann model, Grossmann et al. (1998) proposed a disjunctive reformulation.", "This reformulation can even handle isothermal streams in a system.", "The key idea of the disjunctive formulation is the explicit treatment of three possibilities for process stream temperatures: a process stream is totally above, totally below or across the pinch candidate temperature, as shown in Fig. 1.", "When a stream is totally above the pinch candidate temperature, both the inlet and outlet temperatures are greater than the pinch candidate temperature.", "When a stream is totally below the pinch candidate temperature, both the inlet and outlet temperatures are below the pinch candidate temperature.", "These two statements are valid regardless of the streams being hot or cold.", "However, if the stream is across a pinch candidate temperature, the constraints are different for hot and cold streams.", "For hot streams, the inlet temperature is greater than the pinch candidate temperature, and the outlet temperature is less than the pinch candidate temperature.", "In contrast, different constraints apply to cold streams.", "To avoid the use of max operators, intermediate variables are introduced to calculate the correct heat load of hot and cold streams respectively, as shown in the Eq. (2).", "Recently, Quirante et al. (2017) proposed another novel and robust disjunctive reformulation.", "This method reformulates the max operator from a pure mathematical point of view without any physical insight regarding the heat integration background.", "We refer to this reformulation as Direct Disjunction in this study.", "This reformulation has fewer Boolean variables compared with the Explicit Disjunction (Grossmann et al., 1998), thus shows better relaxation gaps and reduced number of equations.", "The four reformulations are proposed in the following chronological order: Smooth Approximation (Balakrishna and Biegler, 1992), Explicit Disjunction (Grossmann et al., 1998), Intermediate Temperature strategy (Anantharaman et al., 2014) and Direct Disjunction (Quirante et al., 2017).", "Smooth Approximation has the following advantages: no binary variables are needed, and it is computationally efficient.", "However, the reformulation has difficulty when handling isothermal streams and intermediate utilities.", "In addition, the Smooth Approximation parameter has to be chosen properly, otherwise numerical issues could arise.", "To overcome the limitations of Smooth Approximation, Explicit Disjunction, which is capable of handling isothermal streams and multiple utilities, is proposed.", "However, 3 Boolean variables are introduced for each pair of streams and pinch candidates.", "The number of binaries are increasing rapidly with the scale of the problem.", "Therefore, it becomes challenging to solve the model if the problem size is large.", "Motivated by this challenge, Direct Disjunction, which only needs 2 Boolean variables for each pair of streams and pinch candidates, provides a better reformulation of the original Duran-Grossmann model.", "Direct Disjunction should perform much better than Explicit Disjunction, especially for medium or large-scale problems.", "The Intermediate Temperature strategy introduces a new continuous variable to avoid using max operators.", "One binary variable to activate/deactivate the corresponding constraints has to be introduced.", "The different reformulations are subject to the trade-off between continuous variables and binary variables.", "In this study, we mainly focus on the application of the Duran-Grossmann model for Work and Heat Exchange Networks (WHENs).", "The Duran-Grossmann model has been successfully extended to WHEN problems (Yu et al., 2018e).", "A brief introduction to the WHENs problem is presented here.", "The WHENs problem can be stated as follows: Given a set of process streams with supply and target state (temperature, pressure), as well as hot, cold and power utilities; the objective is to design a network consisting of heat transfer equipment such as heat exchangers, heaters and coolers, and pressure manipulation equipment such as expanders, compressors, pumps and valves with minimum Exergy Consumption or minimum Total Annualized Cost.", "In the WHENs problem, a process stream whose target pressure is greater than the supply pressure is called a work sink stream (WSK).", "Opposite, a work source stream (WSR) can be defined as a process stream whose target pressure is less than the supply pressure.", "Any process stream can be heated, cooled or simply not changed before pressure manipulation.", "Fig. 2 illustrates the superstructure of a stream in the category of WSK.", "Detailed information about the superstructure is available in Yu et al. (2018e).", "For the Extended Duran-Grossmann model, the max operators can be replaced by Smooth Approximations as well.", "It is similar to the reformulation for the original Duran-Grossmann model as discussed in Section 2.1.", "However, binary variables are involved in the Smooth Approximation reformulation in this case.", "The detailed model is omitted in this section since it is straightforward.", "Here, R is a valid upper bound to relax the constraints for the binary variables denoting stream identities.", "The value of R can be estimated based on temperatures of the process streams.", "After the reformulation, the Extended Duran-Grossmann model becomes a disjunctive model, which can be transformed into a Mixed Integer Non-Linear Programming (MINLP) problem by the Big-M method or the convex hull method (Türkay and Grossmann, 1996).", "In this study, LogMIP (Vecchietti and Grossmann, 2004), a specially designed program for disjunctive programming, is adopted as the solver.", "Users can freely choose the Big-M method or convex hull method in the GAMS environment, which facilities the modeling and solution substantially.", "Compared with Explicit Disjunction, only two Boolean variables are needed for each pair of streams and pinch candidates.", "With the above disjunctions, the model can easily be implemented in the GAMS environment along with other equations related to the process.", "The Extended Duran-Grossmann model can determine the optimal split ratio of stream C2 and the optimal temperature(s) of stream C1 before compression.", "For this case study, all the reformulations are able to find the global optimum.", "The detailed results concerning stream C1 are listed in Table 2.", "Without considering pressure manipulation, the original pinch temperatures are 120/100 °C for hot and cold streams respectively.", "In the optimal configuration, stream C1 is split into two sub-streams with heat capacity flowrates being 1.47 kW/°C and 1.53 kW/°C respectively.", "A new pinch is created and located at 300/280 °C.", "It can be seen that part of C1 (sub-stream C1_S1) is heated to the new pinch temperature 280 °C before compression.", "The other part is cooled down to ambient temperature before compression.", "The optimized superstructure of C1 is shown in Fig. 3.", "The results are consistent with the study by Fu and Gundersen (2015a).", "The overall system performance under optimal conditions are summarized in Table 3.", "The Composite Curves and the Grand Composite Curve are shown in Fig. 4.", "The hot utility demand is zero because the compression heat of C1_S2 can be fully utilized to heat cold streams in the system above the pinch.", "Stream C1 is compressed at the new pinch temperature and at ambient temperature.", "In contrast, compression at the original pinch temperature or the supply temperature are not good options from the perspective of exergy consumption.", "The compression of C1_S1 has the similar effect as a heat pump.", "After compression, the sub-stream C1_S2 becomes a hot stream in the system that will reduce hot utility.", "The hot and cold Composite Curves are closer to each other and there are two pinch points in the GCC.", "This demonstrates that an efficient heat exchanger network can be derived with the optimized superstructure for stream C1.", "Even though all the reformulations can reach the same global optimum as discussed above, the computational expense shows big differences for the different reformulations.", "For the Explicit Disjunction and the Direct Disjunction reformulations, the disjunctive programming models can be reformulated into MINLP models by the Big-M or convex hull methods with LogMIP as the solver.", "In essence, the LogMIP solver calls other MINLP algorithms to solve the disjunctive model.", "For this small-sized problem, BARON (Tawarmalani and Sahinidis, 2004) is adopted as the MINLP solver.", "Table 4 shows the computational performance of each reformulation.", "It is clear that Smooth Approximation performs much better than the other reformulations for this case study.", "The Smooth Approximation reformulation has fewer continuous variables and significantly fewer binary variables.", "The computation time is also considerably less than for the other three reformulations.", "The Direct Disjunction model has more disjunctions and continuous variables but fewer binary variables compared with the Explicit Disjunction model.", "The advantage of the Direct Disjunction reformulation is that it can easily be extended to cases with isothermal streams and multiple utilities.", "However, in the WHENs problem, phase change process streams are difficult to handle in a general way.", "Such streams need special attention in WHEN problems.", "The convex hull reformulation performs slightly better than the Big-M method for both Explicit and Direct Disjunction.", "It is clear that the intermediate temperature strategy has much fewer binary variables compared with the other two disjunctive reformulations.", "However, the performance of intermediate the temperature strategy is not satisfactory.", "This reformulation can only find an upper bound of the objective function within the CPU time limitation of 4000 s.", "The optimality gap is still very large when the solver terminates.", "The Intermediate Temperature strategy shows slow convergence properties.", "This case study is a simple and small size problem and only non-isothermal streams are considered.", "For large-scale problems, the intermediate temperature strategy may fail to get the optimal results using global solvers, such as BARON.", "It has been reported that the Smooth Approximation might cause numerical problems when isothermal streams or multiple utilities are involved.", "For cases without isothermal streams, Smooth Approximation performs better than other reformulations, as shown in this study.", "Each reformulation has its own merits and flaws.", "Which reformulation is better depends on the problem size and the stream properties involved in the system.", "For large-scale problems, BARON could be unable to reach the global optimum within rational time.", "Decomposition algorithms are likely to improve the computational efficiency of the Extended Duran-Grossmann model.", "In this paper, the Duran-Grossmann model for heat integration is extended to solve Work and Heat Exchange Network problems.", "To solve the model efficiently, four reformulations of the original Duran-Grossmann model are reviewed and applied to the WHENs problem.", "In the Extended Duran-Grossmann model, even stream identities are variables in addition to temperature and heat capacity flowrate for the process streams.", "The Extended Duran-Grossmann model can get the same results as a manual procedure for WHENs based on Pinch Analysis and Thermodynamics.", "Each reformulation has its own advantages and disadvantages.", "For small-scale WHEN problems, all the reformulations can find the global optimum.", "However, the computational efforts and results of different reformulations have been compared.", "For the case study, the results show that the Smooth Approximation outperforms the other three reformulations.", "However, this case study represents a small size problem.", "Large-scale problems should be tested in future work.", "Isothermal process streams and multiple utilities should also be considered.", "The small parameterɛin the Smooth Approximation and the Big-M value in the disjunctive programming models have significant influence on the solution of the problems.", "In summary, the reformulations of the Extended Duran-Grossmann model can only deal with small to moderate scale problems.", "Since there is a large number of binary variables in the model, it is quite computationally expensive for large-scale problems with the four reformulations evaluated in this study.", "In the future, other reformulations or decomposition algorithms should be developed for the Extended Duran-Grossmann model in order to solve more complex WHEN problems in PSE field." ]	[]
Adjuvant effect of TLR7 agonist adsorbed on aluminum hydroxide (AS37): A phase I randomized, dose escalation study of an AS37-adjuvanted meningococcal C conjugated vaccine	An adjuvant system (AS37) has been developed containing a synthetic toll-like receptor agonist (TLR7a). We conducted a phase I randomized, observer-blind, dose-escalation study to assess the safety and immunogenicity of an investigational AS37-adjuvanted meningococcus C (MenC) conjugate vaccine in healthy adults (NCT02639351). A control group received a licensed MenC conjugate alum-adjuvanted vaccine. Eighty participants were randomized to receive one dose of control or investigational vaccine containing AS37 (TLR7a dose 12.5, 25, 50, 100 μg). All vaccines were well tolerated, apart from in the TLR7a 100 μg dose group, which had three reports (18.8%) of severe systemic adverse events. Four weeks after vaccination, human complement serum bactericidal assay seroresponse rates against MenC were 56–81% in all groups, and ELISA seroresponses were ≥81% for all AS37-adjuvanted vaccine groups (100% in 50 and 100 μg dose groups) and 88% in the control group. Antibody responses were maintained at six months after vaccination.	[ "IMMU", "MEDI" ]	[]	[ "2403", "2723" ]	[ "Adjuvants are required to improve the immunogenicity of vaccines containing purified antigens and to facilitate the development of new vaccines [1].", "An objective of many adjuvant discovery and development programs is to elicit more effective engagement of T-helper cells to optimize the quality, breadth and durability of antibody responses or to induce effector CD4+ or CD8+ T cells to kill intracellular pathogens [2,3].", "One approach is to use agonists for toll-like receptors (TLRs) that activate innate immune cascades, mainly on antigen-presenting cells, and facilitate the generation of T-helper cell responses [4].", "A new class of adjuvants, small molecule immune potentiators (SMIPs), has been identified that target TLRs [5].", "TLR7/8 SMIPs include imidazoquinolines and benzazepines; most research has been on imidazoquinolines, such as imiquimod and resiquimod, which are potent cytokine inducers and have beneficial effects in the topical treatment of various skin disorders [6–8].", "Imiquimod also significantly improved immunogenicity against an intradermal influenza vaccine when applied as a topical skin adjuvant [9].", "There have however been reports of systemic adverse events (AEs) associated with topical imidazoquinoline use [10–14].", "New formulations were needed to retain TLR7/8 adjuvants at the injection site and augment immune activation without inducing systemic cytokine release [6].", "There have been promising developments in TLR7/8 delivery systems, including encapsulating nanoparticles [15,16] and lipid conjugation [17,18].", "Another approach has been adsorption to aluminum hydroxide (alum) adjuvants.", "This includes a SMIP adjuvant system (AS37) containing a synthetic TLR7 agonist, TLR7a, with a benzonaphthyridine chemical scaffold, adsorbed to alum.", "Adjuvants based on insoluble salts of aluminum have been shown to be well tolerated and effective components of human vaccines over many decades of use [19].", "Pre-clinical studies of AS37 demonstrated a superior capacity in eliciting an effective immune response when compared to alum-adjuvanted controls in animal models using several target pathogens [5,20–22].", "This included a significantly higher persistence of protective antibody titers against Neisseria meningitidis serogroup C (MenC) 8 months after the last immunization in mice [21].", "The adsorption of TLR7a to alum also significantly improved expansion of the memory B cell compartment in mice [23].", "We now present results from a first-time-in-human study of AS37 to assess the safety, reactogenicity, and immunogenicity profiles of an investigational AS37-adjuvanted MenC-CRM197 conjugate vaccine (AS37-MenC-CRM197) compared to those of the licensed MenC-CRM197 conjugate vaccine (Menjugate, GSK) in healthy adults.", "Menjugate has well established safety and immunogenicity profiles, and contains alum as adjuvant [24].", "The alum dose was kept constant in each study group, enabling an assessment of the impact of different AS37 doses in the investigational vaccine groups.", "This was a phase I, randomized, observer blind, adjuvant dose escalation study performed at a single center in Germany between March 2016 and August 2017 (ClinicalTrials.gov identifier: NCT02639351).", "Healthy adults aged 18–45 years were enrolled, as determined by medical history, physical examination, and the results of screening tests.", "Inclusion and exclusion criteria are listed in the Supplementary Methods.", "The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice.", "The protocols and associated documents were reviewed and approved by an independent ethics committee.", "All participants provided written informed consent before study entry.", "An independent data monitoring committee (IDMC) was appointed to oversee safety of the study participants.", "Participants were randomized to receive a single intramuscular vaccination of either the licensed alum-adjuvanted MenC-CRM197 conjugate vaccine (Menjugate; control) or the investigational vaccine, AS37-MenC-CRM197.", "For the dose escalation of AS37 (specifically, TLR7a), the participants were enrolled into one of four cohorts in a step-wise manner, with 20 participants (four receiving control vaccine and 16 the investigational vaccine) per cohort.", "Within each cohort, participants were randomized using an electronic data capture system in a 1:4 ratio to receive either the control MenC-CRM197 vaccine (four groups of four participants) or the investigational vaccine, with each cohort receiving increasing doses of TLR7a (12.5, 25, 50, or 100 μg; four groups of 16 participants).", "In each cohort, the first five participants (i.e., the first 1:4 randomization block) were vaccinated at a rate of one participant per day.", "Enrolment was then paused for review of the safety results up to Day 14 by the IDMC.", "The remaining 15 participants were then enrolled.", "The IDMC also reviewed safety results obtained through Day 29 before enrolment of the next cohort.", "Predefined criteria were in place to halt the study if there were safety concerns, based on Food and Drug Administration guidelines [25].", "The study was conducted in an observer-blind manner, i.e. vaccine recipients and those responsible for the evaluation of any study endpoint were blinded to the administered vaccines and vaccines were prepared and administered by authorized medical personnel who did not participate in any of the study clinical evaluations or assays.", "A 0.5 mL single dose of the control vaccine contained MenC oligosaccharides (10 μg) conjugated to the protein carrier CRM197 (12.5–25.0 μg).", "Before administration, the diluent, containing alum, sodium chloride, and water for injection, was added to the lyophilized vaccine, containing mannitol and sodium phosphate.", "A 0.5 mL single dose of investigational vaccine had the same contents as the control vaccine with the addition in the diluent of TLR7a (12.5, 25, 50, or 100 μg), histidine (0.1–0.8 mg), and tris(hydroxymethyl)aminomethane (30–243 μg).", "The preparation and physicochemical characterization of AS37 (containing TLR7a adsorbed to alum) was described previously [26]; for all formulations, TLR7a was completely adsorbed to alum.", "The vaccines were injected in the deltoid muscle of the non-dominant arm.", "The primary safety objective was to assess the safety of the investigational vaccine as compared to that of the control MenC-CRM197 vaccine.", "Solicited local (induration, pain, erythema, and swelling) and systemic (arthralgia, chills, diarrhea, fatigue, fever, headache, loss of appetite, myalgia, nausea, rash, urticaria, and vomiting) AEs were recorded directly into the clinical database on the day of vaccination (Day 1, after the first 30 min following vaccination) and on diary cards for the following 13 days (to Day 14).", "Fever was defined as temperature ≥38.0 °C.", "Unsolicited AEs, serious AEs (SAEs), AEs of special interest, new onset chronic disease (NOCD), medically-attended AEs, and AEs leading to withdrawal were recorded throughout the study.", "AEs of special interest were as defined by the Committee for Medicinal Products for Human Use of the European Medicines Agency [27] and included anaphylaxis, Bell's palsy, convulsion, demyelination, encephalitis, Guillain-Barré syndrome, neuritis, and vasculitis.", "NOCD included conditions such as auto-immune disorders, allergies, type 1 diabetes, and asthma.", "The intensity of each solicited symptom or AE was graded as mild, moderate, or severe.", "Severe induration, erythema, or swelling was defined as a diameter >100 mm, severe temperature as >39.5 °C, and all other severe AEs as preventing normal activities.", "Hematological and biochemical parameters, measured before and 24 h after vaccination, and at Days 8 and 29 were graded 0–4 [25].", "Abnormal laboratory findings that were judged by the investigator to be clinically significant were recorded as an AE or SAE.", "An exploratory safety analysis was also conducted to assess plasma concentrations of TLR7a up to 72 h after vaccination, measured by liquid chromatography/tandem mass spectrometry, from participants who signed a separate informed consent form.", "Blood samples were taken before vaccination, at Days 8 and 29, and six months after vaccination (Day 181) for evaluation of the primary and secondary immunogenicity endpoints.", "Additional blood specimens were collected for assessment of exploratory endpoints before vaccination and at Days 1, 4, 8, 29, and 181 from participants who signed a separate informed consent form.", "Immune responses were evaluated by human complement serum bactericidal assay (hSBA) directed against MenC [28] and by enzyme-linked immunosorbent assay (ELISA) to MenC polysaccharide [29].", "An hSBA seroresponse was defined as a post-vaccination hSBA titer ≥8 for participants with a pre-vaccination hSBA titer <4 or an increase of at least four times baseline hSBA level for those with pre-vaccination hSBA titer ≥4.", "An ELISA seroresponse was defined as an increase in antibody concentration of at least four times baseline concentration for participants with pre-vaccination concentration ≥0.06 μg/mL or post-vaccination concentration ≥0.12 μg/mL for those with pre-vaccination concentration <0.06 μg/mL.", "As this was a first-time-in-human study and because of the primary safety objective for the study, sample size was based on the probability of observing at least one participant with a given event, assuming a given frequency of AEs following vaccination.", "With 16 participants per cohort receiving the active vaccine, AEs with frequency ≥15% could be detected with at least 90% probability and AEs with frequency ≥10% could be detected with 81% probability.", "With four participants per cohort receiving the control vaccine, AEs with frequency ≥30% could be detected with at least 80% probability.", "Safety analyses were conducted on all exposed participants (total vaccinated cohort).", "The incidence of AEs per study group was calculated.", "The immunogenicity analysis was performed on the per-protocol set (PPS) for immunogenicity, including participants who received the study vaccine correctly and complied with study procedures.", "Geometric mean titers (GMTs) measured by hSBA, antibody geometric mean concentrations (GMCs) measured by ELISA, and seroresponse rates were estimated using two-sided 95% Clopper-Pearson confidence intervals (CIs).", "For the immunogenicity endpoints, the logarithmically (base 10) transformed antibody titers were modelled using an analysis of covariance model with a qualitative factor for the AS37 dose (0, 12.5, 25, 50, or 100 μg) and log (base 10) pre-vaccination titer as a covariate.", "The adjusted GMT and the two-sided 95% CIs of the GMT were estimated based on this model, as was the ratio of GMTs and corresponding CIs.", "All analyses were performed using SAS software version 9.3.", "Eighty healthy adults aged 18–45 years were enrolled and vaccinated in the study, and were included in the safety analyses and PPS for immunogenicity.", "The demographic characteristics of the participants were similar across the five study groups (Table 1).", "All participants were Caucasian (European heritage), apart from one participant in the AS37-50 μg dose group, whose ethnic background was reported as unknown.", "The study vaccine formulations containing AS37 with TLR7a dose 12.5–50 μg were well tolerated, with no clear trend in the incidence of AEs (solicited or unsolicited) in relation to AS37 dose escalation.", "All participants in each group reported at least one solicited local AE (all reported injection site pain and a maximum of two participants per group reported other solicited local AEs; Fig. 1) and 44–75% reported at least one solicited systemic AE (Table 2).", "The most commonly reported solicited local AE in each group was pain and the most common solicited systemic AEs were headache and fatigue (Fig. 1).", "There was one report of severe pain in the AS37–100 μg group; all other local solicited AEs were mild to moderate in severity (Supplementary Table 1).", "In the AS37-100 μg group, there were three reports of severe solicited systemic AEs (18.8%), including one report each of headache and nausea and one report of fever with temperature >40.0 °C on Days 5 and 6 that resolved by Day 11.", "In the other groups, one severe solicited systemic AE was reported (severe chills in AS37-25 μg group).", "The number of participants experiencing unsolicited AEs was comparable between the study vaccine groups and the control MenC-CRM197 vaccine group (Table 2).", "Most unsolicited AEs were reported by one participant each; only nasopharyngitis was reported by more than two participants in at least one group.", "One case of intermittent, mild dizziness (in AS37-25 μg group), which was considered as possibly or probably related to the study vaccine, was reported from Day 1 to Day 14 of the study.", "There were no reports of an AE leading to withdrawal from the study, NOCD, or AEs of special interest.", "One SAE (joint injury in the control MenC-CRM197 vaccine group) was reported, which was not considered related to vaccine administration.", "No clinically relevant laboratory abnormalities were observed during the study and none were reported as AEs.", "The plasma concentration-time profile showed that levels of TLR7a slightly increased with dose, peaking at levels below 100 ng/L at the 8 h time point (Table 3).", "Thereafter, TLR7a concentrations decreased slowly up to the 72 h time point.", "The immune response to vaccination was measured by hSBA directed against MenC and by ELISA to MenC polysaccharide.", "All vaccines induced an immune response measured by hSBA (Table 4).", "The seroresponse rate measured by hSBA was 69–81% for AS37-adjuvanted vaccine groups at Day 29, which was comparable to that in the control MenC-CRM197 group (75%).", "There was no apparent dose-response relationship with increasing TLR7a dose, with broad and overlapping 95% CIs for GMTs and GMT ratios (baseline: Day 29) among the study groups.", "The seroresponse rates were maintained at comparable titers in all groups at the six months post-vaccination time point (Table 4).", "Antibody GMCs measured by ELISA showed a trend for higher MenC specific antibody titers in the groups receiving AS37 than in the control MenC-CRM197 group, although 95% CIs overlapped (Table 5).", "Seroresponses at Day 29 measured by ELISA were 100% in the AS37-50 μg and 100 μg dose groups, 94% and 81% in the AS37-12.5 μg and AS37-25 μg dose groups, respectively, and 88% in the control MenC-CRM197 group (Table 5).", "Comparable responses were observed at Day 181.", "This is the first report of the safety, reactogenicity, and immunogenicity of a vaccine including a novel SMIP adjuvant system (AS37) containing TLR7a adsorbed to alum, administered to healthy adults.", "The objective driving the development of AS37 was to achieve a vaccine adjuvant system with strong immunopotentiating properties and minimal side effects.", "The safety and superior immunogenicity of AS37 over alum was demonstrated in pre-clinical studies [5,20–23].", "Additionally, a recent comparative study evaluating the same TLR7 agonist molecule in nonhuman primates showed consistent adjuvanticity properties regardless of it being adsorbed to alum or coformulated with a lipidic nanoemulsion [30].", "In the assessment of the safety and immunopotentiating properties of this novel adjuvant in humans, Menjugate was selected as control because of the extensive safety data available for this vaccine [24] and because it is also adsorbed to alum.", "Since the alum dose was constant in all groups, this enabled evaluation of the effects of TLR7a dose escalation in groups receiving the investigational AS37-MenC-CRM197 vaccine.", "The investigational vaccine formulations containing AS37 with TLR7a dose 12.5–50 μg had acceptable reactogenicity and safety profiles, which were comparable to those of the control MenC-CRM197 vaccine.", "In the AS37-100 μg group, there were three reports of severe solicited systemic AEs.", "No clinically-significant abnormal laboratory values were reported and no participants withdrew from the study because of an AE.", "One SAE (joint injury) was reported that was not considered related to vaccine administration.", "TLR7a plasma levels slightly increased with adjuvant dose to a maximum level below 100 ng/L, suggesting limited systemic exposure to the SMIP, as expected from preclinical observations from rat and dog toxicokinetic models of TLR7 (unpublished results).", "For the 12.5–50 μg formulations, there was no clear trend in the occurrence of AEs (solicited or unsolicited) relative to adjuvant dose escalation.", "However, we cannot exclude the possibility that, with the highest dose formulation, the TLR7a plasma level may have contributed to the 18.8% rate of severe solicited systemic AEs in this group.", "All vaccines induced an antibody immune response four weeks post-vaccination measured by hSBA, with no apparent dose-response relationship with increasing TLR7a dose.", "GMT and GMT ratio 95% CIs were large and overlapped among the control MenC-CRM197 and AS37-adjuvanted vaccine groups.", "There was a trend for higher MenC specific antibody concentrations by ELISA in the groups receiving AS37, with highest concentrations in the AS37-50 μg and AS37-100 μg dose groups, but again 95% CIs overlapped.", "Although the study was not powered to compare immunoresponse between groups, the ELISA assessment of antibody quantity may have revealed slight differences among groups that were not measurable with a less sensitive functional assay, such as hSBA.", "The seroresponse rate measured by hSBA was up to 81% four weeks after vaccination for the AS37-adjuvanted vaccine groups, which was comparable to the rate with the control MenC-CRM197 vaccine.", "For seroresponse measured by ELISA, rates were high in all groups four weeks post-vaccination, reaching 100% in the AS37-50 μg and AS37-100 μg dose groups and 88% in the control group.", "Seroresponse rates measured by both assays persisted in all groups at six months post-vaccination.", "There was no evidence of a faster seroresponse in the AS37-adjuvanted vaccine groups than in the control group one week after vaccination.", "Up to now, it has been difficult to demonstrate an added value of adjuvants in potentiating the immune response to glycoconjugates [21].", "Interpretation of our immunoresponse observations should take into account that the control MenC-CRM197 vaccine has strong immunogenicity [24], as confirmed by results in our study, and both the control and investigational vaccine formulations contain alum in the adjuvant.", "Moreover, this study was conducted in young, healthy individuals, who typically have a robust immune response to the MenC vaccine [31] and therefore do not require a vaccine with additional adjuvantation.", "These factors are likely to have made it difficult to detect an improved immunoresponse with the inclusion of TLR7a in the investigational vaccine.", "In conclusion, this phase I study of healthy adults demonstrated that investigational MenC-CRM197 vaccine formulations containing AS37 with TLR7a dose 12.5–50 μg have acceptable safety and reactogenicity profiles.", "All vaccine formulations induced immune responses that were maintained for six months.", "Despite the difficulty in showing additional enhancement in immunoresponse from AS37, the trend observed by ELISA is promising.", "Larger studies are required to confirm the immunopotentiating properties of this adjuvant candidate in different antigen models, and to investigate any association between systemic markers and the safety and immunogenicity of the vaccine.", "Future studies should also assess the advantages of this approach in relation to other TLR7/8 delivery systems, which are at earlier stages of development, such as encapsulating nanoparticles [15,16] and lipid conjugation [17,18].", "This study was sponsored by Novartis Vaccines Division.", "On 2 March 2015, Novartis non-influenza vaccines business was acquired by the GSK group of companies.", "GlaxoSmithKline Biologicals SA supported all costs associated with the development and publishing of the manuscript.", "Anonymized individual participant data and study documents can be requested for further research from www.clinicalstudydatarequest.com.", "AGL, UDO, DTOH, GDG, ES, OF, and DM are employees of the GSK group of companies.", "UDO and OF hold restricted shares in the GSK group of companies.", "JO was an employee of the GSK group of companies and SB was a consultant employed by the GSK group of companies at the time of study conduct.", "TK and TF received fees through their institution for the conduct of the study.", "Menjugate is a trademark owned by the GSK group of companies." ]	[ "The adjuvant system, AS37, was tested in an investigational vaccine.", "Vaccine formulations containing TLR7a doses 12.5–50 μg had acceptable safety.", "Safety was comparable to that of a licensed vaccine containing aluminum hydroxide.", "There was no trend in the incidence of adverse events with TLR7a doses 12.5–50 μg.", "All formulations induced immune responses that were maintained for 6 months." ]
MYB fusions and CD markers as tools for authentication and purification of cancer stem cells from salivary adenoid cystic carcinoma	Cancer stem cells (CSC) are considered the major cause of aggressive tumor behavior, recurrence, metastases, and resistance to radiation, making them an attractive therapeutic target. However, isolation of CSC from tumor tissue and their characterization are challenging due to uncertainty about their molecular markers and conditions for their propagation. Adenoid cystic carcinoma (ACC), which arises predominantly in the salivary glands, is a slow-growing but relentless tumor that frequently invades nerves and metastasizes. New effective treatment approaches for ACC have not emerged over the last 40 years. Previously, based on a highly conserved SOX10 gene signature that we identified in the majority of ACC tumors, we suggested the existence in ACC of SOX10+ cells with neural stem properties and corroborated this hypothesis via isolation from ACC tissue a novel population of CSC, termed ACC-CSC. These cells activated NOTCH1 signaling and co-expressed SOX10 and other ACC-intrinsic neural crest stem cell markers with CD133, a CSC cell surface marker, suggesting that ACC is driven by a previously uncharacterized population of SOX10+/CD133+ cells with neural stem cell properties. Here, we authenticated ACC identity of our primary cultures by demonstrating that most of them harbor MYB-NFIB fusions, which are found in 86% of ACC. We demonstrated using CyTOF, a novel mass cytometry technology, that these cells express high β-catenin and STAT3 levels and are marked by CD24 and CD44. Finally, to streamline development of ACC cell lines, we developed RT-PCR tests for distinguishing mouse and human cells and used immunomagnetic cell sorting to eliminate mouse cells from long-term cell cultures. Overall, this study describes a new population of CSC that activates signaling pathways associated with poor prognosis, validates their ACC identity, and optimizes approaches that can be used for purification of ACC-CSC and generation of cell lines.	[ "BIOC" ]	[]	[ "1307", "1309" ]	[ "Adenoid cystic carcinoma (ACC) is a deadly cancer: with a prevalence rate of 1224 cases, 918 patients die from ACC in the U.S. every year (http://www.accoi.org/faq/acc-statistics/).", "ACC is treated by surgery with or without radiation, but only 40% of patients survive 15 years owing to intrinsic radiation resistance of ACC cells and their propensity to metastasize, relapse, and spread along nerves (Singh et al., 2015; Oplatek et al., 2010).", "The recurrence rate is high (53%) owing mostly to neural invasion, radio-resistance, and hematologic metastases (Shingaki et al., 1986).", "Aggressive ACC behavior suggests that it may be driven by cancer stem cells (CSC).", "CSC possess properties of normal stem cells and are widely associated with invasion, recurrence, metastases, and resistance to cytotoxic therapies (Beck and Blanpain, 2013; Tysnes, 2010; Pattabiraman and Weinberg, 2014).", "Their identification in ACC will advance understanding of molecular etiology and cell of origin, improving diagnostics, predicting disease outcome, and developing effective therapies.", "However, characterization of CSC is controversial when it is based solely on CD markers, whose expression is not stem cell-selective (Jaggupilli and Elkord, 2012).", "In addition, CSC isolated from cell cultures are often not representative of tumor tissue and therefore lack clinical value (Liu et al., 2014; Madjd et al., 2016; Leon et al., 2016).", "With the goal to identify clinically relevant CSC in ACC, we performed gene expression profiling of surgically resected tumor specimens to identify stem cell signaling and associated selective markers.", "This analysis demonstrated that most of ACC specimens selectively express SOX10, a marker of neural crest cells and oligonedroglial progenitors (Nonaka et al., 2008; Gorris et al., 2015), providing a clue to how CSC can be identified and isolated from ACC tissue.", "Indeed, in line with a special role of SOX10 in this cancer, we identified in the majority of ACC the expression of a highly conserved SOX10 gene signature that contained a cluster of neural stem cell drivers and markers, such as NOTCH1, MAP2, GPM6B, and FABP7, as well as genes/proteins involved in WNT and NOTCH signaling (Ivanov et al., 2013a; Ivanov et al., 2013b).", "These findings suggested that SOX10 expression delineates activation of a neural stem cell program in ACC and marks a previously unrecognized population of cells with neural stem cell properties.", "The creation and maintenance of subcutaneous patient-derived xenografts (PDX) from fresh or cryopreserved ACC tissue (Moskaluk et al., 2011) provided a renewable source of ACC cells for validation of our CSC hypothesis.", "As we previously demonstrated, these PDX models reproduced ACC morphology and maintained the SOX10 gene signature (Ivanov et al., 2013a; Moskaluk et al., 2011).", "To isolate SOX10+ CSC from grafted ACC tissue, we used a ROCK inhibitor-based cell culture protocol (Liu et al., 2012) and profiled our cell cultures for expression of SOX10 and other ACC-intrinsic stem cells markers as well as for expression of CD133, a CSC cell surface marker.", "In line with our hypothesis, we isolated from patient-derived xenografts (PDX) and clinical ACC specimens a new population of SOX10+/CD133+ cancer stem cells, which we named ACC-CSC.", "These cells co-expressed SOX10 with activated NOTCH1 and met basic CSC criteria by producing spheroids in vitro and generating aggressive ACC-like tumors in nude mice.", "In addition, ACC-CSC activated stem cell signaling with involvement of SOX10 and NOTCH1 (Panaccione et al., 2016).", "In this study, we authenticate cultured ACC-CSC using ACC-intrinsic MYB-NFIB fusions, investigate their signaling pathways, and test new tools, markers, and assays for their purification.", "Detailed description of ROCK inhibitor-based cell culture is available from (Liu et al., 2012).", "Briefly, primary cultures were grown in F + Y Medium (3:1 (v/v) Dulbecco's modified Eagle's medium: Ham's F12 nutrient mixture with 7.5% fetal bovine serum (all from Invitrogen), 0.4 μg/mL hydrocortisone (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 8.6 ng/mL cholera toxin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Invitrogen), and 10 μM/L ROCK-inhibitor (Y-27632, SelleckChem) mixed in a 4:1 ratio with conditioned media from 3T3-J2 cells.", "To create conditioned media, irradiated (30 Gy) 3T3-J2 cells were incubated in a T-150 flask supplemented with 30 mL of DMEM media for 4 days.", "Media was filter-sterilized and then mixed in a 1:4 ratio with F + Y media.", "Generation and analysis of all PDX models but ACCX33 have been described in our previous studies (Moskaluk et al., 2011; Ivanov et al., 2013b).", "Briefly, fresh tissue cut into small (~ 1 mm3) pieces were implanted subcutaneously in the shoulder area of 4–6 weeks old female nude mice (NCr nu/nu) purchased from NCI-Frederick.", "Animals were operated and maintained in accordance with the Institutional Animal Care and Use Committee guidelines.", "ACCX33 was created from a clinical tumor specimen, ACC33, described in (Panaccione et al., 2016).", "Nude mice were purchased from Frederick National Laboratory of Cancer Research (Frederick, MD).", "Human breast carcinoma MX-1 cells were purchased from Frederick National Laboratory of Cancer Research and grown with ROCK inhibitor as in (Liu et al., 2012).", "293FT cells were purchased from Thermo Fisher Scientific (Witham, MA).", "Orthotopic tumor models were generated via injection of 104 ACC-CSC cells mixed with Matrigel (Corning, Tewksbury, MA) at a 1:1 ratio into the salivary glands of nude mice.", "For end-point RT-PCR (28 cycles), we used SuperScript III One Step RT-PCR kit (ThermoFisher, Waltham, MA) in combination with Bio-Rad Imaging system.", "We designed and used the following primers: mTusc2F: 5′-CCTCAGTGTTAACTGGTATTGGCGGA and mTusc2R: 5′-CAACTTGAGGGCAACTGGTTGAAATCA-3′ (for mouse RNA detection); hPPIAF: 5′- TGAGGGTAGGAGTCAAGATCAGC-3′ and hPPIAR: 5′-AGAGGCTCTATATGCTACAAGCAGTACC-3′ (for human RNA detection); B2: 5′- CGGGTGCTCTTACCC ACTGA-3′ (for single-primer mouse RNA detection), and -Alu-2: 5′-ACATGGTGAAACCCCGTCTCTAC-3' (for single-primer human RNA detection).", "To detect ACC-CSC, we used the following human-specific primers: hCD133F: 5′-GAAACTGCTTGAGCATCAGGATACTC-3′; hCD133R: 5′-CAGATCCAACATGAACTCCTGAAGC-3′; hCD24F: 5′-GTGGTGCGATCTCAGATCAGTGTA-3′, hCD24R: 5′-CTGTGGCCATATTAGATTACTGGAAC-3′; and hCD44F: 5′-TCTCCCACTTGGCA AGTCCTTTG-3′, hCD44R: 5′-TCCGTTGACAATGGCCAAGG-3′; hJAG1F: 5′-CCGACACGGTCTCGGATCA-3′, and hJAG1R: 5′-TTCACGGTCTCAATGGTGAACCAACAA-3′.", "To confirm MYB-NFIB fusions in PDX models cultured ACC cells, we used the following primers: ACCX11FUSF: 5′-GTAGAAGATCTGCAGGATGTGATCAAACAGG-3′; ACCX11FUSR: 5′-CCTCCTAGCCTT CGTTGGTG-3′; ACCX14FUSF: 5′- GAATATTCTTACAAGCTCCG-3′, ACCX14FUSR: 5′-CCT CCTAGCCTTCGTTGGTG-3′; and ACCX33FUSR: 5′-GAAGCTTTTGGGTTGAGACAATGGG-3′.", "To confirm human MYB expression in cultured ACC cells, we used the following primers: hMYBF: 5′-AAAGCGCCTCGCCAGCAAG-3′ and hMYBR: 5′- CCTCCTAGCCTTCGTTGGTG-3′.", "To validate activation of β-catenin and STAT3, we used the following primers: TCF7L2F: 5′-TGATTTCCTTCAAAGACGAGGG-3′ and TCF7L2R: 5′-CTTAAAGAGCCCTCCATCTTGC-3′; MYCF: 5′-AGCGACTCTGAGGAGGAACAA-3′ and MYCR: 5′-CTCAGCCAAGGTTGTGAGGT-3′.", "Trypsinized cells were sorted using CD24 and CD133 Micro-bead kits, and depletion of mouse cells from ACC cultures was done with Mouse Cell Depletion kit (#130-104-694), all available from Miltenyi Biotec (Cambridge, MA).", "Cultured ACC cells were resuspended in MACS buffer (phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA) and incubated for 15 min with magnetic bead- (CD133 & mouse cell depletion) or biotin-conjugated (CD24) antibodies.", "The CD24 kit subsequently washes and incubates the cells with a magnetic bead-conjugated anti-biotin antibody.", "Cells are then passed through a column encapsulated within a magnetic field using gravity flow, capturing bead-conjugated cells within the column.", "The column is washed three times with MACS buffer before removing the column from the magnet and cells are gently flushed from the column with MACS buffer.", "Immunofluorescent staining was performed as described in (Panaccione et al., 2016).", "CyTOF analysis was performed on CyTOF2 mass-spectrometer (Yale School of Medicine) with CD133-PE antibodies from Miltenyi Biotec (San Diego, CA) and heavy metal-tagged antibodies 165Ho-PE, 173Yb-STAT3, and 147Sm-β-catenin (Fluidigm, San-Francisco, CA).", "Cells were fixed, permeabilized, and stained for surface and intracellular targets using Fluidigm protocols.", "Immunoblot analysis was performed with β-catenin, STAT3, p-STAT3, GAPDH, and anti-rabbit IgG, HRP-linked antibodies available from Cell Signaling (Beverly, MA).", "We recently generated 6 primary ACC cultures from patient-derived xenograft (PDX) models as well as from a surgically obtained specimen using a ROCK inhibitor-based approach to cell culture (Liu et al., 2012).", "Short tandem repeat (STR) analysis that we performed at that time confirmed origin of ACC cultures from corresponding tumors, and expression in these cells of ACC-intrinsic markers SOX10, FABP7, etc. strongly suggested their ACC identity (Panaccione et al., 2016).", "Here, we asked if ACC cell cultures harbor MYB-NFIB fusions, which are ACC-intrinsic genetic aberrations found in 86% of ACC patients and considered to be primary ACC-driving events (Brill et al., 2011; Drier et al., 2016).", "To identify these fusions and sequence them, we first performed targeted RNA sequencing of PDX models using the FusionPlex Solid Tumor panel and kit (ArcherDX, Boulder, CO).", "This technology uses anchored multiplex PCR to detect previously described and novel gene fusions, SNPs, and InDels in selected genes.", "Adapters with molecular barcodes are used for quantitative multiplex data analysis, read deduplication, and accurate mutation calling.", "This analysis revealed MYB fusions in PDX models ACCX11, 14, 33, and 5M1 and in one additional model, ACCX9.", "In 4 of 5 models, we identified canonical MYB-NFIB fusions between exons 12 or 14 of MYB (NM_005375) and exon 9 of NFIB (NM_005596) (Fig. 1).", "Similar MYB-NFIB fusions have been previously described by other research teams (Persson et al., 2009; Mitani et al., 2010).", "In ACCX33, we found a previously uncharacterized fusion that joined exon 14 of MYB with a long non-coding RNA gene from chromosome 6q24.3, RP11-545I5.3 (GenBank: AL356599, pos.", "75864-76334) (Fig. 1).", "We next designed PCR primers and confirmed MYB fusion breakpoints in PDX models and cell cultures using RT-PCR and Sanger sequencing.", "Overall, these analyses confirmed ACC identity of our PDX models and corresponding cell cultures validating them as authentic tools for studies aimed at the role of MYB fusions in ACC-CSC propagation.", "We previously demonstrated that SOX10, NOTCH1, and their common target FABP7 play essential roles in propagation of CD133+ ACC-CSC and that NOTCH signaling is involved in their resistance to radiation (Panaccione et al., 2016).", "In addition to NOTCH, other signaling pathways, such as WNT/β-catenin and STAT3, are known to stimulate CSC survival (Matsui, 2016).", "To find out if WNT/β-catenin and STAT3 are engaged in ACC-CSC, we used CyTOF, a novel time-of-flight mass spectrometry-based technology for multiplex cytometry and single cell analysis to find out if these pathways are activated in ACC-CSC, which are marked by CD133 expression.", "CyTOF analysis of cultured Accx11 cells co-stained with CD133-PE antibodies, secondary antibody against PE, as well as β-catenin and human STAT3 metal-tagged antibodies demonstrated that the majority of CD133-positive Accx11 cells, or ACC-CSC, express high levels of β-catenin, and that most of β-catenin-positive cells are also enriched with STAT3.", "Specificity of CD133-PE antibodies used in CyTOF was validated on 293-FT cells that don't express CD133 (Fig. 2A).", "Activation of STAT3 and β-catenin in ACC-CSC was confirmed by Western blot that showed higher levels of these proteins in CD133-expressing Accx11 and MX-1 than in CD133-negative 293FT cells (Fig. 2B).", "We also performed real-time PCR that demonstrated increased levels of a major β-catenin effector, TCF7L2 (Cadigan and Waterman, 2012), and STAT3 target MYC (Banerjee and Resat, 2016) supporting our CyTOF data (Fig. 2C).", "These findings were in line with the recently reported cooperation between WNT/β-catenin and STAT3 signaling in CSC maintenance (Chen et al., 2017).", "To further purify ACC-CSC for their comprehensive characterization and cell line derivation, we used the most robustly growing ACC cell culture, Accx11, which spontaneously formed spheroids and developed into long-term cell culture (P > 30).", "Based on previously published reports on spheroids isolated from other cancer cell cultures (Lee et al., 2015; Christensen et al., 2010), we hypothesized that they are composed entirely of CD133 + cancer stem cells and can be used for ACC-CSC purification.", "In line with this expectation, we demonstrated via immunofluorescent staining on bulk Accx11 cells that spheroids express CD133 and NOTCH1, two ACC-intrinsic stemness markers (Fig. 3A) and confirmed that floating spheroids collected from culture media express higher levels of CD133 than bulk cells (Fig. 3B, C).", "In order to obtain pure ACC-CSC cultures, media containing unadhered spheroids was collected from plates containing bulk cells.", "Spheroids were allowed to settle at the bottom of 15 mL conical tubes for 15 min.", "After aspirating the media, the cells were washed with PBS and this was repeated twice more.", "The remaining spheroids were transfered into fresh plates with media.", "After several days, spheroids adhered to the surface and produced neural stem-like cells that developed processes reminiscent of axons and growth cones (Fig. 3D).", "Further passaging produced transparent semi-adherent cells with variable morphology, axon-like protrusions, and tendency to pile up and form spheroids (Suppl. Fig. 1).", "To find out if these cells are tumorigenic, we injected 104 cells into the salivary glands of nude mice.", "Confirming their tumor-initiating properties, spheroid cells produced ACC-like tumors in all four mice used in this experiment (Fig. 3E).", "To further purify ACC-CSC for their comprehensive characterization and cell line derivation, we used the most robustly growing ACC cell culture, Accx11, which spontaneously formed spheroids and developed into long-term cell culture (P > 30).", "Based on previously published reports on spheroids isolated from other cancer cell cultures (Lee et al., 2015; Christensen et al., 2010), we hypothesized that they are composed entirely of CD133+ cancer stem cells and can be used for ACC-CSC purification.", "In line with this expectation, we demonstrated via immunofluorescent staining of bulk Accx11 cells that spheroids express CD133 and NOTCH1, two ACC-intrinsic stemness markers (Fig. 3A), and confirmed that floating spheroids collected from culture media express higher levels of CD133 than bulk cells (Fig. 3B, C).", "In order to obtain pure ACC-CSC cultures, media containing unadhered spheroids was collected from plates containing bulk cells.", "Spheroids were allowed to settle at the bottom of 15 mL conical tubes for 15 min.", "After aspirating the media, the cells were washed twice with PBS.", "The remaining spheroids were transfered into fresh plates with media.", "After several days, spheroids adhered to the surface and produced neural stem-like cells that developed processes reminiscent of axons and growth cones (Fig. 3D).", "Further passaging produced transparent semi-adherent cells with variable morphology, axon-like protrusions, and tendency to pile up and form spheroids (Suppl. Fig. 1).", "To find out if these cells are tumorigenic, we injected 104 cells into the salivary glands of nude mice.", "Confirming their tumor-initiating properties, spheroid cells produced ACC-like tumors in all four mice used in this experiment (Fig. 3E).", "In our previous study, we demonstrated that ACC-CSC can be purified via Mitenyi magnetic beads bound to CD133 antibodies and that purified CD133+ cells as well as spheroids selectively express SOX10 with activated NOTCH1 and initiate tumor growth in nude mice (Panaccione et al., 2016).", "To more comprehensively characterize ACC-CSC CD markers, we asked if CD24 and CD44 are also expressed in these cells.", "In our end-point RT-PCR experiments with primers specific for human CD24 and CD44 transcripts, we demonstrated that cultured Accx11 cells expressed both cell surface markers.", "In this experiment, Accx11 spheroids and MACS-purified CD133+ cells showed higher CD24 and CD44 levels than the CD133-negative cell fraction suggesting that ACC-CSC selectively express CD24 and CD44 in addition to CD133 (Fig. 4A).", "To confirm CD24 and CD44 expression in ACC tumors, we analyzed our expression array data generated on surgically resected ACC specimens (Ivanov et al., 2013a).", "In this dataset, we observed high levels of CD24 and CD44 in the majority of ACC tumors (Fig. 4B).", "We then used MACS to produce CD133- and CD24-sorted Accx11 cells and analyzed these fractions for CD24/CD133 expression.", "Remarkably, CD24+ Accx11 cells were mostly CD133-positive and vice versa suggesting that a large proportion of ACC-CSC have the double-positive CD24+/CD133+ phenotype (Fig. 5A).", "Based on all these data, we concluded that CD24, CD44, and CD133 are all clinically informative markers for tracking CSC in ACC tissue, PDX models, and primary cell cultures that we created.", "These cell surface markers may help to further purify ACC-CSC via MACS or FACS as well as investigate their heterogeneity, signaling status, and relation to other tumor cells using CyTOF analysis (Han et al., 2015).", "We noticed that at late passages (P > 10) mouse cells introduced in ACC cell cultures from feeder layer or PDX models may overgrow human ACC cells (our unpublished observations).", "To detect mouse cell contamination of ACC cultures, we designed a simple RT-PCR test based on primate- and mouse-specific Alu and B2 repeats, respectively.", "We also used mouse- and human-specific primers for ubiquitously expressed genes TUSC2 and PPIA, respectively.", "All these primers were validated on primary Accx11 culture as well as on mouse fibroblasts and human breast cancer cells MX-1.", "This analysis identified mouse RNA contamination in the original (bulk) Accx11 cell culture but not in the negative control, MX-1 (Suppl. Fig. 2).", "We noticed that at late passages (P > 10) mouse cells introduced in ACC cell cultures from feeder layer or PDX models may overgrow human ACC cells (our unpublished observations).", "To detect mouse cell contamination of ACC cultures, we designed a simple RT-PCR test based on primate- and mouse-specific Alu-2 and B2 repeats, respectively.", "We also used mouse- and human-specific primers for ubiquitously expressed genes TUSC2 and PPIA, respectively.", "All these primers were validated on primary Accx11 culture as well as on mouse fibroblasts and human breast cancer cells MX-1.", "This analysis identified mouse RNA contamination in the original (bulk) Accx11 cell culture but not in the negative control, MX-1 (Suppl. Fig. 2).", "The same test was then used to compare efficiencies of different approaches to mouse cell elimination.", "While MACS CD sorting significantly diminished the signals from mouse RNA, it failed to completely eliminate mouse cells most likely due to non-specific binding to magnetic beads or cell clumping (Fig. 5A).", "Seeking more efficient approaches, we analyzed cells after purification by: 1) collection of floating spheroids produced by bulk Accx11 cells, 2) development of clonal populations of CD133+ cells, and 3) depletion of mouse cells with a MACS Miltenyi kit.", "Following these purification techniques, mouse cells were below the limits of PCR detection (Figs. 5B and C).", "The third approach, however, is the most optimal as it maintains tumor heterogeneity and therefore may be better for experimental use.", "Development of targeted therapies for ACC has been stalled by lack of reliable cell lines/cultures and approaches for CSC isolation.", "Unfortunately, most ACC cell lines generated 20–30 years ago and shared between laboratories turned out to be grossly contaminated or misidentified (Phuchareon et al., 2009; PLoS One, 2014).", "These cell lines are now blacklisted (http://iclac.org/databases/cross-contaminations/).", "However, of > 100 manuscripts that relied on these cell lines only two have been retracted (Oral Oncol., 2012), and new studies with same cell lines have been recently published (Sumida et al., 2016; Maruyama et al., 2010; Wu et al., 2015; Fang et al., 2015; Sun et al., 2015; Sumida et al., 2013; Wang et al., 2016).", "While the most plausible explanation for the ACC cell line identity crisis is lack of proper authentication (Yu et al., 2015), ACC cells are also notoriously difficult to culture as they rapidly undergo senescence or die in regular cell culture media (our unpublished observations).", "In this study, we continue characterization of a novel population of CSC, ACC-CSC, that we recently purified from ACC using a ROCK inhibitor and mouse cell feeder layer-based approach (Panaccione et al., 2016).", "To our knowledge, this report is the first study to detect MYB fusions in ACC cultures, despite the fact that MYB-NFIB fusions have been described in 86% of ACC patients (Brill et al., 2011).", "In a fraction of ACC tumors, MYB fusions with other genes, such as TGFBR3 and RAD51B, as well as MYBL1 fusions with NFIB have been also reported (Drier et al., 2016; Mitani et al., 2016).", "Here, we identified a novel MYB fusion with RP11-54515.3, an lncRNA gene from chromosome 6q24.3.", "Intriguingly, this lncRNA is a cis natural antisense transcript (cis-NAT) for SHPRH, an E3 ubiquitin ligase.", "While exact functions of cis-NAT are not known, they may inhibit expression of corresponding sense transcripts via transcription interference or post-transcriptionally (Rosikiewicz and Makalowska, 2016).", "Interestingly, SHPRH functions as a tumor suppressor protein that induces degradation of β-catenin, a CSC driver, and has been recently recognized as a target of axitinib, a small molecule inhibitor of WNT/β-catenin signaling (Qu et al., 2016).", "It may be hypothesized therefore that the MYB-RP11-54515.3 fusion may inhibit SHPRH translation and stimulate ACC-CSC via WNT/β-catenin signaling.", "This hypothesis will be validated on Accx33 cells that harbor this fusion.", "Overall, generation of ACC cells with oncogenically activated MYB provided long-awaited tools for studies focused on the MYB role in ACC.", "Our next goal was to perform a more detailed characterization of ACC-CSC to identify therapeutically amenable pathways of their propagation.", "To this end, we used CyTOF, an innovative flow cytometry technology, which is based on time-of-flight mass-spectrometry (Giesen et al., 2014).", "CyTOF analysis demonstrated that CD133 + AC-CSC are highly enriched with β-catenin and STAT3, two major targets actively explored in novel cancer-targeting therapies (Qu et al., 2016; Xiang et al., 2016).", "We and others previously implicated both STAT3 and Wnt/β-catenin signaling in ACC (Ivanov et al., 2013a; de Araujo et al., 2008; Daa et al., 2004; Frierson et al., 2002), and this study, for the first time, links both pathways to CSC suggesting their cooperation and providing targets for ACC-CSC eradication.", "An important step towards further ACC-CSC characterization is identification of CD markers which, in combination with CD133, may be used for FACS sorting and multiplexed CyTOF analysis of ACC-heterogeneity and their signaling states.", "Such studies may significantly advance understanding of ACC-CSC roles in tumor tissue and their relation to tumor growth, neural invasion, metastases, resistance to radiation, and recurrence.", "Association of ACC-CSC with the CD24 cell surface marker in this study provides a new insight into ACC cell of origin.", "Indeed, our hypothesis that ACC and basal-like breast cancers have similar CSC driven by SOX10 (Ivanov et al., 2013b) has been recently supported by a study that implicated SOX10 in stimulation of CD24hi fetal mammary stem cells (Dravis et al., 2015).", "Thus, CD24 appears to be a biologically and clinically relevant cell surface marker that can be used in combination with CD133 and CD44 in CyTOF studies and for more efficient ACC-CSC purification.", "As it was recently demonstrated, expression of CD24, CD44, and CD133 may be linked with activation of distinct pro-survival signaling pathways and have different effects on radiation resistance and patient survival (Sahlberg et al., 2014).", "It remains to see if similar associations take place in ACC and if differential expression of CD markers may predict prognosis or resistance to therapy.", "Another important goal of this study was to purify ACC-CSC from mixed mouse/human cell cultures and grow them as long-term cell cultures.", "In this regard, we found that Accx11 spheroids are made entirely of human CD133 + ACC-CSC, which made them a straightforward tool for ACC-CSC purification (Fig. 3.", "and Suppl. Fig. 3).", "Similarly, generation of monoclonal cell populations completely purified ACC-CSC from mouse cells (Fig. 5B).", "We then used MACS with antibodies to CD24 and CD133 as an alternative approach to ACC-CSC purification.", "Even though this approach turned out to be not as efficient to rid cultures of mouse cell contamination, these markers are human-specific and can be used with cell cultures that don't produce spheroids as well as in analytical FACS/CyTOF and preparative FACS studies on unpurified primary ACC cultures at early passages.", "Finally, we concluded that the Miltenyi kit for mouse cell elimination is an effective means for ACC-CSC purification since this approach produced cell populations without detectable mouse cells (Fig. 5C).", "Another important goal of this study was to purify ACC-CSC from mixed mouse/human cell cultures and grow them as long-term cell cultures.", "In this regard, we found that Accx11 spheroids are made entirely of human CD133 + ACC-CSC, which made them a straightforward tool for ACC-CSC purification (Fig. 3.", "and Suppl. Fig. 3).", "Similarly, generation of monoclonal cell populations completely purified ACC-CSC from mouse cells (Fig. 5B).", "We then used MACS with antibodies to CD24 and CD133 as an alternative approach to ACC-CSC purification.", "Even though this approach turned out to be not as efficient to rid cultures of mouse cell contamination, these markers are human-specific and can be used with cell cultures that don't produce spheroids as well as in analytical FACS/CyTOF and preparative FACS studies on unpurified primary ACC cultures at early passages.", "Finally, we concluded that the Miltenyi kit for mouse cell elimination is an effective means for ACC-CSC purification since this approach produced cell populations without detectable mouse cells (Fig. 5C).", "Overall, we report in this study on the assays, tools, and markers for authentication, purification, and research of a novel population of CSC that we recently identified in ACC.", "Marked by expression of SOX10, similar CSC populations appear to drive breast basal-like carcinoma, melanoma, and other cancers that appear to arise from neural crest (Ivanov et al., 2013b; Panaccione et al., 2017).", "We confirmed that ACC cultures maintained MYB fusions found in xenografts or primary tumors and identified a novel MYB fusion to a non-coding RNA.", "We demonstrated that ACC-CSC that we characterized earlier as SOX10 +/CD133 + (Panaccione et al., 2016; Yarbrough et al., 2016) also express CD24 and CD44 as well as signaling molecules commonly found in CSCs, STAT3 and β-catenin.", "To develop a new platform for drug screening with the goal to develop effective and specific therapies for ACC, we optimized ACC-CSC purification from mixed mouse/human cell cultures and created a novel ACC-CSC-initiated orthotopic model for pre-clinical studies.", "The following are the supplementary data related to this article.", "ACC-CSC purified via spheroid collection.", "Validation of human- and mouse-specific RT-PCR primers on bulk Accx11 cells, human breast cancer cell line MX-1, and mouse 3T3-J2 cells.", "RT-PCR analysis of CD133-sorted and spheroid-purified Accx11 cells for mouse contaminations.", "Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scr.2017.05.002.", "Authors declare no conflict of interests." ]	[ "ACC cancer stem cell cultures are authenticated using ACC-intrinsic MYB fusions.", "A novel MYB fusion, with a long non-coding RNA from 6q23.4, is identified.", "ACC-CSC is marked by expression of CD24 and CD44.", "ACC-CSC purification is optimized to establish cell lines for drug screening.", "CyTOF is used to explore STAT3 and β-catenin as novel targets for ACC-CSC eradication." ]
In vivo remodeling of a 3D-Bioprinted tissue engineered heart valve scaffold	Objective: To evaluate the recellularization potential of a bioprinted aortic heart valve scaffold printed with highly concentrated Type I collagen hydrogel (Lifeink® 200) and MSCs. Materials and methods: A suspension of rat mesenchymal stem cells (MSCs) was mixed with Lifeink® 200 and was 3D-printed into gelatin support gel to produce disk scaffolds which were subsequently implanted subcutaneously in Sprague-Dawley rats for 2, 4, 8, and 12 weeks. The biomechanical properties of the scaffolds were evaluated by uniaxial tensile testing and cell infiltration and inflammation assessed via immunohistochemistry (IHC) and histological staining. Results: There was an average decrease in both UTS and tensile modulus from 2 to 4 weeks followed by an increase between 4 to 8 weeks and a plateau from 8 to 12 weeks. IHC showed a continued expression of alpha smooth muscle actin and vimentin biomarkers throughout the study demonstrating continued presence of interstitial-like and fibroblast-like cells. Additionally, there was also an increase of elastin at each time point. Conclusion: The profile of the stress-strain curves of the bioprinted aortic heart valve scaffolds indicated that the scaffold transitioned through phases of resorption, synthesis, stabilization, and ultimately, remodeling. This is supported by IHC and histology which showed favorable remodeling capacity demonstrating potential feasibility for a 3D printed heart valve.	[ "BIOC", "COMP", "ENGI" ]	[ "Bioprinting", "Collage-based matrix", "Heart valve", "Mechanical behavior", "Tissue engineering" ]	[ "1305", "1706", "2204" ]	[ "According to the American Heart Association, there have been approximately 600,000 deaths due to valvular heart disease (VHD) since 1979 [1,2].", "Currently, the gold standard for aortic valve disease (AVD) is limited to either mechanical or bioprosthetic valve replacement.", "Mechanical valves require anticoagulation and are typically applied to younger patients, while bioprosthetic valves have limited durability and are prone to calcific degradation [3].", "Tissue engineered heart valves (TEHV) have become increasingly important in the development of valve replacement because of availability and customizability for individual patients.", "Although TEHV have limitations such as antigenicity and loss of extracellular matrix integrity to overcome, there is significant promise showing autologous host cell remodeling of these scaffolds [4].", "Three-dimensional (3D) bioprinting has become popular and increasingly used in medicine to produce biomaterials ready for implantation.", "The technology has applications that allow for living cells to be printed within a synthetic scaffold that presents in a variety of shapes, complexities, and mechanical properties [5].", "In recent years, the medical industry has increasingly used 3D-printing to create patient-specific prosthetics, surgical models, and drug delivery devices [6].", "Unlike biological tissue, bioprinting allows for the artificial creation of organs and tissues for on-demand manufacturing with patient cells [7].", "3D-bioprinting allows for host cell integration coupled with ECM remodeling to establish a viable TEHV that will grow and adapt with the patient.", "This technology can achieve homogenous porosity and allow deposition of different cell types within a construct [8].", "3D-bioprinted valves are customizable to patients though direct digital input from patient imaging utilizing computed tomography and ultrasound imaging.", "The precise spatial control has the capability to print complex native valvular structures with autologous cells.", "3D-bioprinting has been used to construct a heterogeneous aortic valve structure, using different materials for the root and the leaflets.", "More recently, the technique has been used to build a model of an aortic valve leaflet, giving each different layer specific mechanical properties [9,10].", "3D-bioprinting enables reproducibility that cannot be replicated with biological tissue scaffolds [11].", "The biomaterial selected to produce a scaffold must have the proper biocompatibility and degradation rate that will allow for structure maintenance, cellular maturity, and enable active cellular remodeling.", "Additionally, the material requires mechanical integrity and stability throughout the printing process in order to prevent layer collapse.", "Consideration of cell type and environmental conditioning is necessary to strengthen the valve to endure hemodynamic pressures.", "Potential mechanisms for crosslinking such as UV light or chemical crosslinking maybe considered to strengthen the scaffold and allow for the structure to maintain shape during environmental conditioning [12].", "Thus, a novel approach should be developed to constructing the 3D biological heart valve.", "The focus of the current manuscript is to evaluate the collagen-based bio-ink biocompatibility and its recellularization potential for use as a heart valve implant.", "This study involves a hybrid approach for 3D printing and in vivo biomaterial characterization of the collagen-based bio-ink via rat subcutaneous evaluation.", "A computer aided design model (CAD) for the implant disk scaffolds was created utilizing Solidworks (Dassault Systèmes, Waltham, MA) and Magics Materialise 3D rendering software (Materialise n.v. 2011, Magic EnvisionTec 32 bit V16.2.0.20).", "CAD model files were exported to Perfactory software (EnvisionTec v 3.2.2945), converted to stereolithography (.", "STL) files, and uploaded to the 3D-bioplotter control software Visual Machines (version 2.8.129r1).", "Freeform Reversible Embedding of Suspended Hydrogels (FRESH) slurry was made under aseptic conditions according to previously published methods [13].", "FRESH slurry was dispensed into 50 mL Falcon tubes.", "Centrifugation was performed and supernatant removed and replaced with DMEM.", "Tubes were shaken to homogenize the DMEM with resuspended gelatin particles.", "This was repeated several times to remove insoluble gelatin and to incorporate the cell culture medium into the FRESH slurry.", "The DMEM-FRESH slurry was dispensed into a 6-well plate.", "Allogenic rat mesenchymal stem cells (rMSCs) labelled with green fluorescent protein (GFP) (Cyagen Biosciences Inc., Santa Clara, CA) were cultured at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s Medium (DMEM, Gibco, Life Technologies).", "This was supplemented with 5% heat-inactivated fetal bovine serum (FBS, Atlas Biologicals) and 1% antibiotic-antimycotic (A/A, Gibco) until the number of necessary cells was reached.", "Media was changed every two days.", "Cultured cells, passaged at 80% confluence, were washed 3 times using phosphate buffer saline (PBS), detached from the culture vessel with Trypsin-EDTA (Gibco Life Technologies), suspended in DMEM, and centrifuged at 1800 RPM.", "The supernatant was removed and cells were counted and resuspended to achieve a concentration of 6 × 106 cells/mL of Bio-ink (5:2 ratio of Lifeink® 200: cell media) per manufacturer’s protocol.", "The cell suspension was loaded into a 5 mL syringe and attached to the tip of a 10 mL syringe containing Lifeink® 200® using a lumen connector.", "Lifeink® 200 and the cell suspension were mixed until a homogenous Bio-ink was achieved.", "The cell-laden Bio-ink was loaded into a syringe (EFD Nordson) compatible with the EnvisionTEC 3D-Bioplotter (EnvisionTEC GmbH, Gladbeck, Germany).", "The CAD model for the implant disk scaffolds measured 1 mm-thick and 28 mm-diameter was sliced into a 0.1 mm thickness, and uploaded to Visual Machines.", "This geometry was utilized as it could be easily implanted in the rat subcutaneous space and easily mounted in the support frame which prevented migration and folding.", "A more physiologically relevant shape could not be easily implanted in the subcutaneous space and likely would not have a large impact on the recellularization or remodeling as the biological and physical cues would not have been greatly altered.", "One disk scaffold was printed per well-plate.", "The inner diameter of the needle to extrude the Bio-Ink was 0.1 mm and 25 mm in length.", "The needle transfer height was 35 mm, needle offset was 0.1 mm, and printing speed and pressure were 14 mm/s and 1.6 bar, respectively.", "The temperature of the printing platform and the printing head were set at 4 °C and 23 °C, respectively.", "Disk scaffolds were printed in a layer-by-layer fashion, each layer printed in a continuous manner with a 0.3 mm distance between individual extruded strands and a 90° offset between the strand orientation in each layer.", "The description of the strands here is applicable during the printing process; during cross-linking, the individual strands fuse together.", "Pre-flow and post-flow delays were set at 200 ms and 100 ms, respectively.", "After the printing process, well-plates were placed in a 37 °C, 5% CO2 environment for 45–60 min.", "The liquefied gelatin was subsequently removed and the Bio-ink disk scaffolds were cultured for 5 days in DMEM with 1% A/A and 5% FBS.", "Under aseptic conditions, the cultured Bio-ink disk scaffolds were placed between polycaprolactone (PCL) support frames.", "Vetbond (3 M, Maplewood, MN) surgical glue was used to fix both PCL frames together with the disk scaffolds in the middle [14].", "In our previous study we investigated the effect of a PCL support frame on tissue remodeling in the rat subcutaneous space with decellularized pericardium.", "We found superior remodeling and fewer propensities for tissue folding or migration with the PCL support frames and so utilized this approach in this work [14].", "Twelve Sprague Dawley rats were obtained and raised in a pathogen-free environment.", "In vivo assessment of the scaffolds was conducted at 2, 4, 8, and 12 weeks (each rat had n = 4 implants, for a total of n = 48 and n = 12 for each time point).", "All experiments were performed in strict accordance with the guidelines in the Guide for Institutional Animal Care and Use Committee (IACUC protocol number: A00003286-17).", "The surgical procedure and the PCL support frames (Fig. 1A) used to house the Bio-ink disk scaffolds were 3D-printed following the same method described in previous studies [14].", "Explanted disk scaffolds were divided for 1) mechanical testing, and 2) fixed in 10%Formalin and preserved in 70% EtOH for immunohistochemistry.", "Bioprinted implant disk scaffolds were paraffin-embedded then sectioned at the Mayo Clinic Pathology Core (Scottsdale, AZ).", "Immunohistochemistry (IHC) methodology can be found in previous publications [4,14].", "Primary antibodies were used to express recellularization, extracellular matrix remodeling, and immune reaction of the bioprinted explant.", "Primary antibodies utilized were CD163 (Abcam, Cambridge, UK) and CD3 (Abcam) for immune reaction, Vimentin (Abcam) and Alpha smooth muscle Actin (Abcam) for interstitial-like cell phenotypes, CD31 (Abbiotec, San Diego, CA) for endothelialization, Elastin (Abcam) for elastic tissue, and Collagen I (Thermofisher) for Collagen Type 1.", "Images were quantitatively analyzed in terms of antibody signal surface area using ImageJ software and a digital slide scanner (MoticEasyScan, Motic).", "Immunofluorescence (IF) was performed to visualize the green fluorescent protein (GFP)-positive cells preseeded on the bioprinted acellular implant disk scaffolds.", "Sections were incubated with the primary antibody rabbit polyclonal to GFP (ThermoFisher, Waltham, MA) overnight at room temperature.", "The stain was labelled using a secondary antibody donkey anti-rabbit IgG H&L Alexa Fluor 488 (Thermofisher) mounted using Prolong Gold Anti-Fade Mountant with DAPI (Thermofisher), and visualized with a confocal microscope.", "Additionally, samples were stained with Hematoxylin-Eosin (H&E), Masson Trichromeand elastin stains to observe cellularity, collagen depositionand fibrosis; respectively.", "The mechanical properties of the explanted bioprinted scaffold were assessed by conducting uniaxial tensile testing per ASTM F2150-13 (n = 12 per time point 2, 4, 8, 12 weeks).", "The tensile testing was performed using a tensile tester (Instron, USA, Series 5900).", "Each sample was cut with a constant width of 4 mm and varying length.", "The thickness was measured with a drop gauge (Mitutoyo America Corp., Aurora, IL, USA).", "The samples were placed within the grips of the machine, and the specimen gauge length was measured and recorded.", "The samples were stretched until failure, which occurred midway between the grips, using a displacement rate of 0.16667 mm/s.", "The load as a function of the extension data was recorded and processed using MATLAB and Statistics Toolbox Release R2016a (The MathWorks Inc., Natick, Massachusetts).", "The ultimate tensile strength was computed as being the maximal value on the stress-strain curve, and the tensile modulus was computed as being the slope in the linear region on the stress-strain curve.", "Data distribution was analyzed utilizing MATLAB statistics toolbox using a one way ANOVA followed by a multi-comparison test.", "Data is presented as mean ± SD with statistical significance defined as P value < 0.05.", "Furthermore, to determine if there was a relationship between the tissue stiffness and the quantified concentrations of the IHC parameters linear regression analysis was performed across all time points with the R2 value reported to indicate goodness-of-fit.", "All animals (n = 12) survived the implantation procedure without any clinical evidence of wound infection or inflammation.", "Prior to implantation, the Bioink implants were observed to be fragile; however, after each time point, the physical handling of each explant had improved.", "After four weeks, the Bioink explants contained a fibrous capsule with neovascularization.", "Additionally, it was observed that layers of the host tissue had integrated with the Bioink explant, and after eight weeks, both layers had become indistinguishable (Fig. 1).", "Despite this integration, the structural integrity of the extracellular matrix remained intact with cellularization of host cells on the Bioink explant.", "Fibrosis was prevalent on the cutaneous side of the Bioink explant as seen with Masson’s trichrome (Fig. 1).", "For immunohistochemistry, immunofluorescence and histology, semi-quantification was done utilizing the surface area of the tissue and expression of specific biomarkers.", "Chronic inflammation and ECM remodeling were observed with the biomarkers CD3 and CD163 (Fig. 2).", "The linear regression analysis found that no IHC parameters had strong correlation with the tissue stiffness (Table 1).", "The highest R2 value was for vimentin (0.277) and the lowest for collagen (0.002).", "There was a decreased expression of CD3 as observed from the cross-sectional surface area of explanted Bioink tissue from week 2 (2.47 ± 0.5) to week 4 (1.16 ± 0.09) due to a reduced presence of chronic inflammatory cells (p = 0.002).", "There was a decrease in CD163 expression from week 2 (2.55 ± 0.24) to week 4 (2.15 ± 0.21).", "From week 2 (1.36 ± 0.17) to week 4 (3.43 ± 0.58), there was a statistically significant increase in the expression of the CD31 biomarker for endothelialization and angiogenesis between the host tissue and Bioink explant (p < 0.05) (Fig. 3).", "Alpha smooth muscle actin and vimentin biomarkers revealed an increase of interstitial-like cells infiltrating the Bioink explants over time (Fig. 4).", "At weeks 2 and 4, there was an increased concentration of vimentin in the surrounding tissue (Week 2: 3.84 ± 1.12; Week 4: 5.49 ± 0.31).", "There was also an increase in presence of elastin during this time period.", "From a mechanical property standpoint; stress versus strain plots from tensile testing are shown in Fig. 5.", "Between 2 and 4 weeks, the strength of the mean sample decreases.", "The UTS and tensile modulus are given in Fig. 6 as mean ± standard deviation.", "One-way analysis of variance (ANOVA) was run.", "The values for UTS were 0.344 ± 0.120 and 0.169 ± 0.077 MPa for 2 and 4 weeks, respectively, showing a decrease over time.", "The same statistical analysis was run for the tensile modulus, which was taken to be the slope of the stress-strain curve.", "There was a decrease in the tensile modulus from 2 to 4 weeks (1.186 ± 0.872 and 0.548 ± 0.341 MPa, respectively).", "There was a decrease in CD163 expression from week 4 (2.15 ± 0.21) to week 8 (1.74 ± 0.14) (Fig. 2).", "Overall, there was a steady decrease of CD163 expression from week 1 (2.55 ± 0.24) to week 8 (1.74 ± 0.14; p < 0.05) reflecting a decrease in inflammation.", "At week 4, the expression of CD31 biomarker started to taper (Week 4: 3.43 ± 0.58; Week 8: 2.56 ± 0.25).", "During this time period, the elastin presence continued to increase, and the largest increase of elastin occurred between week 2 (3.74 ± 0.62 and week 8 (7.12 ± 1.13) (p = 0.03).", "The stress-strain plots show a change in shape in the general curve and a visible increase in strength from 4 to 8 weeks.", "There was an increase in the UTS from 4 to 8 weeks (0.275 ± 0.166 MPa).", "There was also a statistically significant increase in the tensile modulus from 0.548 ± 0.341 to 1.425 ± 0.620 MPa (p < 0.05).", "There was an increase in CD163 expression from week 8 (1.74 ± 0.14) to week 12 (2.28 ± 0.32) due to subsequent infiltration of M2 macrophages (Fig. 2).", "The expression of CD31 biomarker continued to decrease (Week 8: 2.56 ± 0.25, Week 12: 2.20 ± 0.25) and fibrotic tissue started to encapsulate the Bioink explant by week 12.", "After week 8, vimentin concentration within the Bioink explant increased (Week 8; 6.14 ± 0.85) (p = 0.05) (Fig. 4).", "From 8 to 12 weeks, there is a decrease in strength again, along with a change in the shape of the plot curve, which returns to more of a shape similar to 2 and 4 week samples but with lower strain at fracture.", "The UTS was 0.306 ± 0.176 MPa at 12 weeks, showing an increase between 8 weeks and 12 weeks.", "There was a very slight decrease in the tensile modulus from 1.425 ± 0.620 to 1.376 ± 0.616 MPa.", "The increase of the tensile modulus from 4 to 12 weeks was computed to be of statistical significance (p < 0.05).", "The current study demonstrated that 12 week rat subcutaneous implantations revealed the pathway to remodeling for this scaffold that included resorption, synthesis, stabilization, and remodeling phases.", "The in vivo studies showed increased host cellularization potential, biocompatibility, and biomechanical behavior results.", "The bio-ink was successfully printed with MSCs and showed remodeling.", "Thus, the current study supports the potential use of a collagen-based bio-ink as an alternative for a tissue engineered heart valve implant.", "Throughout the in vivo study, the bioprinted implant scaffolds went through a remodeling pathway characterized by 1) resorption, 2) synthesis, 3) stabilization, and 4) remodeling.", "These stages were critical for the tissue engineered scaffold to be strengthened over time.", "The resorption stage, where the host environment integrates the scaffold and partially degrades it, is seen by the initial drop in the tensile modulus and UTS for the scaffold from week 2 to week 4.", "During this time, there was an initial increase in CD3 biomarker from acute inflammation.", "In similar studies, degradation and resorption responses in tissue engineered scaffolds made from porcine pericardial tissue and small intestinal submucosa were also observed [13–16].", "The synthesis stage, where the host tissue begins infiltrating the scaffold, found an increase in UTS and tensile modulus from week 4 to week 8.", "When evaluating CD31a biomarker for angiogenesis and endothelialization, there was a significant increase from week 2 to week 4.", "However, upon explantation the scaffold had gross encapsulation from the host tissue and signs of neovascularization.", "The stabilizing stage occurred between weeks 8 and 12.", "The inflammation leveled out as shown by the decreased CD3 biomarker expression.", "At this point; elastin, vimentin, and alpha SMA production were at its peak as observed by immunohistochemistry staining, which indicated that the infiltrated cells were actively depositing collagen and strengthening the extracellular matrix in the bioprinted scaffold.", "Both the UTS and the tensile modulus peaked and plateaued, which indicated a biomechanical equilibrium for the scaffold.", "The final stage and perhaps the most important was classified as a period of remodeling and occurred starting at week 12.", "This stage was detected microscopically as well as through the IHC straining results where the CD31 biomarker for angiogenesis was at its maximum at week 12.", "The CD163 biomarker for M2 macrophages showed a steady increasing trend, and was at its greatest at week 12 suggesting good regenerative capacity over time.", "Biomechanical evaluation showed that both tensile modulus and UTS increased significantly from week 4 to week 12.", "The UTS and tensile modulus are comparable to findings previously published with fibrin-based gel to engineer a pediatric heart valve [17].", "In addition to the results from the rat subcutaneous implantation, we were able to successfully print a heart valve utilizing the same bio-ink from the in vivo study with MSCs.", "Obtaining valve structure is a complex balance of processes and materials.", "The bio-ink material must allow cells to infiltrate, support itself during the printing process, and achieve cell survival throughout the extrusion process.", "Researchers are exploring new material combinations to obtain cell viability.", "Some materials investigated include, but are not limited to alginate, agarose, collagen, gelatin, hyaluronic acid (HA), decellularized tissue and polycaprolactone, polyethylene glycol (PEG) [9,18–23].", "Duan et al. 3D-bioprinted a living alginate/gelatin hydrogel valve using smooth muscle cells and valvular interstitial cells, which showed viability in vitro for 7 days [9].", "Similar mechanical behavior to the remodeling phase seen in this work can be identified in reverse for tissues with elastin removed by enzymatic digestion.", "The change in mechanical response between 12 and 8 weeks is similar to arterial tissues where elastin has been removed [24–26].", "Collagen, held in a crimped configuration, is released and so is immediately stretched giving an immediate stiff response rather than there being an initial toe region where the elastin is stretched and the collagen unfurled.", "This coincides with our theory of tissue remodeling where we speculate that the synthesized components are being reorganized such that collagen is being crimped to give the two stage mechanical response common for soft tissues.", "The bioprinted heart valve scaffolds have much lower strength compared to native aortic valve cusps, where native valve cusps have an average UTS of 2.6 and 0.4 MPa in the circumferential and radial directions, respectively [27].", "Therefore, preconditioning will be of utmost importance, slowly increasing the pressures applied to the scaffold until physiological pressures are met and the scaffold can strengthen [6–8,11].", "Potentially after preconditioning, if the scaffold is strong enough before implantation the resorption phase will be smaller or may not occur at all.", "There have also been various cross-linking methods used to increase mechanical strength of the bioprinted structures post-printing, but most of them involve a process that is cytotoxic [5].", "Future steps will involve using visible-light crosslinking with ruthenium and sodium persulfate prior to valve preconditioning to achieve greater mechanical stability [28–30].", "Outside of valvular applications, current technologies that have been explored include the work by Yu et al. where spheroid technology was utilized to create tissue strands by printing alginate micro-conduits.", "They synthesized cell tissue strands that were formed up to 4 days post-fabrication with viability close to 90% [31].", "For application in the nervous system, Gu et al. constructed neural tissue by printing neural stem cells using alginate, carboxylmethyl-chitosan, and agarose bio-ink [18].", "While, Lee et al. 3D-bioprinted collagen bio-ink, reinforced with polycaprolactone, with 3 types of cells to construct artificial liver tissue where a co-culture of the cells was maintained over time [19].", "Skardal et al. also 3D-bioprinted primary liver spheroids with a modular HA and gelatin-based hydrogel and showed high cell viability [20].", "Lee et al. used PCL and a cell-laden hydrogel to 3D-bioprint an ear and showed chondrogenesis and adipogenesis through in vitro assays [23].", "Finally for printing of generic scaffolds, Ouyang et al. studied a dual-cross-linking hyaluronic acid system that enabled them to achieve structures that were stable for over a month [32].", "While Pati et al. were able to 3D-bioprint with decellularized extracellular matrix material showing high cell viability and functionality [33].", "These studies provide insight to the challenges of obtaining shape, geometry, and function of the printed construct while maintaining cell viability.", "One limitation is that the cells used for the bioprinted scaffolds were commercially bought, and not from the actual implanted rats.", "This could be the cause of some of the inflammatory reaction and would need to be further assessed.", "Additionally, this study did not evaluate the influence of the presence of the PCL scaffolds on the remodeling process.", "PCL has been shown to be biocompatible and to induce minimal inflammatory responses [34].", "Additionally, the implantation time was too short to expect any type of degradation of the PCL, as PCL has been shown to have long degradation times, exceeding the timeframe of this study [35,36].", "An aspect that must be studied further is the effect of the remaining FRESH gelatin in the Bio-Ink lattice, after cross-linking.", "However, we do not expect this to have a negative effect.", "As mentioned in Ref. [37], gelatin is biocompatible, and may actually enhance cell adhesion and integration with the bioprinted scaffold [38].", "The rat subcutaneous space does not provide the same mechanical cues as the physiological valve environment.", "However, there are obvious difficulties in performing large animal studies in sufficient numbers to provide statistical power to the results.", "The goal of this study was to evaluate the collagen-based bio-ink biocompatibility and its recellularization potential for use as a heart valve implant.", "From this, the bioprinting approach can be refined prior to more costly and exhaustive larger animal studies wherein the final optimized approach can be ascertained from a reduced number of possible designs.", "Our previous study utilized a similar approach with decellularized-sterilized porcine pericardium to similarly evaluate the recellularization and remodeling potential and performed subsequent finite element analyses to evaluate the performance as a TEHV prior to large animal studies [39].", "For this work, the good association between the mechanical performance, histology, and IHC of the 12 week explants and the native tissues lends confidence to the current scaffold structure for this purpose and going forward.", "Finally, the fibrous capsule was not investigated in this study.", "It is known that this is a key indicator to the host response to the implanted tissue including internal cellularization and differentiation [40–42].", "However, we lacked the sample size to analyze this independently and thus this must be considered a limitation of this work.", "We evaluated a 3D printed collagen based bio-ink tissue engineered heart valve scaffold.", "In vivo studies proved the scaffold had favorable remodeling capacity with increasing elastin, vimentin, alpha SMA, and CD31 throughout the 12 weeks.", "The pathway for remodeling stages included resorption, synthesis, stabilization, and remodeling.", "Each phase played a key role to improve strength and mechanical stability over time.", "Additionally, the current study introduced a 3D bioprinted scaffold that contains rMSCs that would assist in recellularization.", "The addition of rMSCs to the 3D scaffold attracted host cells to repopulate the scaffold, which further strengthened the synthetic ECM architecture.", "We hypothesized that seeded rMSCs encouraged host cell recellularization behavior.", "An aortic heart valve was 3D printed with MSCs and sustained a valve shape.", "The results show great promise in the use of a collagen-based bioink for heart valve construction using 3D-bioprinting technology.", "This work is supported by the HH Sheikh Hamed bin Zayed Al Nahyan Program in Biological Valve Engineering.", "In addition, this research was supported in part by a National Institutes of Health T32 (HL007111) training grant in Cardiovasology.", "The author(s) declare no competing interests." ]	[]
Is fMRI "noise" really noise? Resting state nuisance regressors remove variance with network structure	Noise correction is a critical step towards accurate mapping of resting state BOLD fMRI connectivity. Noise sources related to head motion or physiology are typically modelled by nuisance regressors, and a generalised linear model is applied to regress out the associated signal variance. In this study, we use independent component analysis (ICA) to characterise the data variance typically discarded in this pre-processing stage in a cohort of 12 healthy volunteers. The signal variance removed by 24, 12, 6, or only 3 head motion parameters demonstrated network structure typically associated with functional connectivity, and certain networks were discernable in the variance extracted by as few as 2 physiologic regressors. Simulated nuisance regressors, unrelated to the true data noise, also removed variance with network structure, indicating that any group of regressors that randomly sample variance may remove highly structured "signal" as well as "noise." Furthermore, to support this we demonstrate that random sampling of the original data variance continues to exhibit robust network structure, even when as few as 10% of the original volumes are considered. Finally, we examine the diminishing returns of increasing the number of nuisance regressors used in pre-processing, showing that excessive use of motion regressors may do little better than chance in removing variance within a functional network. It remains an open challenge to understand the balance between the benefits and confounds of noise correction using nuisance regressors.	[ "NEUR" ]	[ "Connectivity", "FMRI", "Motion", "Noise correction", "Regression", "Resting state" ]	[ "2805", "2808" ]	[ "Blood oxygenation-level dependent functional magnetic resonance imaging (BOLD fMRI) data can be analysed using a generalised linear model (GLM) to identify brain regions exhibiting signal changes time-locked to known stimuli.", "In addition to the activations of interest, BOLD data contain numerous noise signals reflecting thermal noise, scanner drift, magnetic field inhomogeneities, head motion, and human physiology.", "Some noise sources can be well modelled (e.g., using the results of rigid body registration algorithms or measuring respiration) and these nuisance regressors can be added to the GLM to improve sensitivity, specificity, and validity of activation maps (Bullmore et al., 1999; Lund et al., 2006).", "Noise correction becomes increasingly important when mapping resting state signal fluctuations, which cannot be modelled by a known stimulus.", "Nuisance regressors derived from external measurements or from within the resting state dataset itself are removed prior to connectivity analysis (Murphy et al., 2013).", "Initial attempts at this noise correction approach have proved to be insufficient: numerous studies have demonstrated that head motion remains a critical confound in mapping functional connectivity despite removal of up to 12 motion-related nuisance regressors (Power et al., 2012; Satterthwaite et al., 2012; Van Dijk et al., 2012).", "Recent evidence supports the use of up to 24 motion-related nuisance regressors in order to best remove this confound (Satterthwaite et al., 2013), while physiological noise is commonly removed using a combination of 12 or more cardiac and respiratory regressors (RETROICOR (Glover et al., 2000), blood gas levels (Bright and Murphy, 2013a; Wise et al., 2004), and respiratory and cardiac rates (Birn et al., 2008; Chang et al., 2009; Shmueli et al., 2007)).", "This expanding number of nuisance regressors raises concerns.", "Measures of resting-state correlations do not typically account for the reduction in degrees of freedom associated with these noise correction approaches (Satterthwaite et al., 2013).", "Increasing the number of nuisance regressors also increases the risk of removing true signal of interest.", "In this paper, we use independent component analysis (ICA) to characterise the portion of resting-state data typically removed and discarded during noise correction.", "First, we demonstrate that the signal variance removed by the regression of 24 head motion parameters can be decomposed into networks typically associated with functional connectivity.", "Several of these network structures can be observed in the variance extracted by as few as 3 head motion regressors or 2 physiologic regressors.", "Next, we consider simulated nuisance regressors, unrelated to the fMRI data: these simulated regressors also remove data with network structure, suggesting that any regressors may remove highly structured “signal” as well as “noise.”", "To directly address this, we show that sampling a small percentage of volumes at random from the original resting state data continues to produce robust network maps using ICA.", "Finally, we compare the variance explained by different numbers and combinations of true and simulated nuisance regressors within functional networks.", "The implications of these observations on our analysis and understanding of resting state fMRI are discussed.", "We have shown that the data variance typically removed and discarded during noise correction of resting state data can be decomposed into network structure highly correlated to networks documented in the functional connectivity literature.", "Simulated regressors, unrelated to the true noise, also remove data with network structure.", "This suggests that any set of regressors may remove highly structured “signal” as well as “noise” and that this may become increasingly apparent as the number of regressors increases.", "We have shown that reduced subsets of the original resting state data can also be decomposed into robust network maps using ICA.", "With as few as 20% of the original volumes considered, we observe spatial network maps similar to those identified in the entire cleaned dataset.", "These results show that the spatial distribution of intrinsic connectivity networks can be robustly mapped using very sparse temporal sampling of fMRI data.", "Finally, we have demonstrated that using excessive true noise regressors related to head motion may be detrimental, removing amounts of variance similar to that of simulated noise regressors while affecting degrees of freedom such that interesting “signal” is removed from the resting state data.", "This balance between de-noising and signal loss can be adjusted by considering datasets with greater numbers of time-points.", "It remains an open challenge to identify when nuisance regressors are no longer beneficial and become an added confound themselves in resting state fMRI.", "The following are the supplementary data related to this article.", "True and simulated noise regressors for an example subject, demonstrating the results of the phase-randomisation technique.", "Correlation across the regressors considered in the true noise, simulated noise, and “incorrect subject” noise analyses in this manuscript.", "For simplicity, only the first iteration of the phase-randomised simulated regressors is included in this figure.", "For subject N, the true noise regressors from subject N + 1 are used as the “incorrect subject” noise data.", "Correlation coefficients were transformed using the Fisher r-to-z transform prior to averaging across subjects, and the inverse transformation of the group mean is presented.", "As intended, minimal correlation structure is observed between the true and simulated noise regressors of a given subject.", "By design, the true noise regressors and the “incorrect subject” regressors represent the same data and demonstrate identical internal correlation structure; there is also consistent correlation in the regressors across the pairs of subjects considered.", "Networks identified in resting state functional connectivity studies (Smith et al., 2009, PNAS 106 (31):13040–5) are observed in the unfiltered cleaned data (24 head motion nuisance regressors removed).", "Spatial correlation values (r, left) and Dice's overlap coefficients (d, right) quantify the similarity between the unfiltered cleaned data network maps and networks identified in true noise data.", "Examples of network structure in the noise data created using nuisance regressors from a different (incorrect) subject, and thus unrelated to the BOLD data.", "Spatial correlation values (r, left) and Dice's overlap coefficient (d, right) quantify the similarity of the networks observed with those present in the correctly cleaned data.", "Correlation matrices representing the temporal relationships between the four nodes of Default Mode Network.", "Each subject's data were transformed into %BOLD units, and the mean time-series within each of the four nodes was extracted.", "Correlation was calculated between each node pair, and values were transformed using the Fisher r–z transform prior to averaging across subjects.", "The inverse transform was performed and results are provided.", "No significant changes were observed between the correlation values of the cleaned and original data for any number of regressors considered (3, 6, 12, or 24 motion regressors).", "Only one significant difference was observed between the cleaned and true noise data correlations after correcting for multiple tests (p < 0.05, indicated by asterisk).", "Supplementary Data.", "ICA results for all true noise datasets (2phys, 3motion, 6motion, 12motion, and 24motion) and cleaned datasets (24 motion regressors removed, the resulting data either unfiltered or band-pass filtered to include only frequencies between 0.02 and 0.1 Hz).", "The data from 12 subjects were normalised to the MNI template space, temporally concatenated and decomposed into 20 spatially independent components using MELODIC (FSL).", "All files are in NIFTI format and contain the resulting spatial maps for each component.", "Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.neuroimage.2015.03.070.", "Twelve healthy subjects (aged 32 ± 6 years, 5 female) were scanned using a 3T GE HDx scanner (Milwaukee, WI, USA) equipped with an 8-channel receive head coil.", "An eyes-open resting state scan lasting 5.5 min was acquired using a BOLD-weighted gradient-echo echo-planar imaging sequence (TR/TE = 2000/35 ms; FOV = 22.4 cm; 35 slices, slice thickness = 4 mm; resolution = 3.5 × 3.5 × 4.0 mm3, 165 volume acquisitions).", "These data were collected as part of a larger study (Bright and Murphy, 2013b).", "A whole-brain high-resolution T1-weighted structural image was acquired (resolution = 1.0 × 1.0 × 1.0 mm3), for the purpose of image registration.", "Cardiac pulsations were recorded using the scanner finger plethysmograph.", "Expired gas content was continuously monitored via a nasal cannula, and O2 and CO2 data were recorded (AEI Technologies, PA, USA).", "This study was approved by the Cardiff University School of Psychology Ethics Committee, and all volunteers gave written informed consent.", "The resting-state functional data were volume registered, motion corrected, time-shifted to a common temporal origin, and brain extracted (AFNI, http://afni.nimh.nih.gov/afni (Cox, 1996)).", "The first five volumes, during which steady-state magnetisation was not yet achieved, were removed from the data.", "End-tidal CO2 and heart rate values were extracted from the physiological data (MATLAB, MathWorks, Natick, MA, USA) and convolved with an HRF and CRF (Chang et al., 2009), respectively.", "Derivatives of the 6 head motion regressors determined during motion correction were calculated, and the quadratic terms of these 12 regressors were derived.", "The mean functional volume for each subject was registered to the corresponding high-resolution T1-weighted image, which was then normalised to the MNI-152 brain template (MNI152, nonlinearly derived, McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada).", "The combined transformation matrices were saved for later use.", "Three types of “noise” datasets were created using the original BOLD fMRI data and processed as shown schematically in Fig. 1.", "The head motion and physiologic noise for each resting state scan were used as regressors in a general linear model (GLM) using AFNI.", "The 4-dimensional dataset comprising the fit of the original data to these nuisance regressors was extracted, spatially smoothed (FWHM = 5 mm), and registered to MNI space using the transformations obtained earlier.", "This process was performed for motion-related noise, using 3 (x-, y-, z-translations), 6 (and three rotations), 12 (and their derivatives), or 24 (and their quadratic terms) head motion regressors (denoted Motion3, Motion6, Motion12, and Motion24 respectively), and for 2 physiologic regressors (end-tidal CO2 and heart rate).", "The use of 24 (or more) head motion regressors has been implemented in different ways throughout the literature (Friston et al., 1996; Satterthwaite et al., 2013; Yan et al., 2013a); in this paper, we adopt the method of Satterthwaite and colleagues (Satterthwaite et al., 2013).", "In all cases, linear and quadratic trends were included in the model to optimise fitting.", "The resulting datasets, which reflect variance typically removed during fMRI pre-processing steps (including linear and quadratic trends), are referred to as the “true noise” data.", "Physiologic regressors were always considered independently from the motion regressors in order to assess these distinct noise sources in isolation.", "For example, in the 3 regressor case, the true noise data reflects the 3 translation motion regressors and the linear/quadratic trends, but not the CO2 or heart rate variance.", "The true noise regressors were phase-randomised to create simulated noise regressors with similar frequency distributions to the true noise regressors, but with no relation to the measured head motion or physiology.", "To achieve this, the frequency spectra of the true noise regressors were obtained using a Fourier transform, and the phase of each frequency in half of the spectrum was randomised and mirrored before performing the inverse Fourier transform (this phase randomization was repeated until the temporal correlation with the true regressor was r < 0.1).", "The entire set of resulting time-series was then orthogonalised to the complete set of original regressors to make them independent from the true noise.", "These simulated regressors were input into a GLM in the same manner as the true noise regressors, and the resulting fits were also spatially smoothed (FWHM = 5 mm) and registered to MNI space and are referred to as “simulated noise” data.", "This process was repeated 10 times to allow us to test whether the true noise regressors behaved differently relative to simulated and unrelated ones.", "Examples of true and phase-randomised simulated noise regressors for one subject are provided in Supplementary Fig. S1.", "The true noise regressors were phase-randomised to create simulated noise regressors with similar frequency distributions to the true noise regressors, but with no relation to the measured head motion or physiology.", "To achieve this, the frequency spectra of the true noise regressors were obtained using a Fourier transform, and the phase of each frequency in half of the spectrum was randomised and mirrored before performing the inverse Fourier transform (this phase randomization was repeated until the temporal correlation with the true regressor was r < 0.1).", "The entire set of resulting time-series was then orthogonalised to the complete set of original regressors to make them independent from the true noise.", "These simulated regressors were input into a GLM in the same manner as the true noise regressors, and the resulting fits were also spatially smoothed (FWHM = 5 mm) and registered to MNI space and are referred to as “simulated noise” data.", "This process was repeated 10 times to allow us to test whether the true noise regressors behaved differently relative to simulated and unrelated ones.", "Examples of true and phase-randomised simulated noise regressors for one subject are provided in Supplementary Fig. S1.", "As an additional model of unrelated nuisance regressors, we also considered the noise datasets created by using the true noise regressors from another subject's data.", "Specifically, we used the noise regressors from Subject “N + 1” to remove variance from the fMRI data of Subject “N” (and using the regressors of Subject 1 for Subject 12).", "Correlations across true regressors, simulated regressors, and true regressors from an incorrect subject are presented, averaged across the cohort, in Supplementary Fig. S2.", "As an additional model of unrelated nuisance regressors, we also considered the noise datasets created by using the true noise regressors from another subject's data.", "Specifically, we used the noise regressors from Subject “N + 1” to remove variance from the fMRI data of Subject “N” (and using the regressors of Subject 1 for Subject 12).", "Correlations across true regressors, simulated regressors, and true regressors from an incorrect subject are presented, averaged across the cohort, in Supplementary Fig. S2.", "Finally, we created reduced versions of the original data, retaining only a fraction of the signal variance.", "This provides a toy example imitating how a given set of nuisance regressors may randomly sample variance from the original fMRI data.", "A given percentage of the original data volumes (unfiltered, spatially smoothed and aligned to MNI space as described above) were randomly selected and replaced with the mean volume; this procedure retained 10, 20, 30, 40, 50, or 60% of the volumes, and was iterated 100 times.", "These datasets are referred to as “reduced” data (reduced10 through reduced60 as appropriate).", "We also considered reduced data consisting of the initial consecutive volumes only; these are referred to as “shortened” data (short10 through short60 as appropriate).", "The residuals from the fitting of the nuisance regressors to the resting state data, as indicated in Fig. 1, represent the de-noised or “cleaned” data.", "We constructed the cleaned datasets from the residuals of the fit to 24 true motion regressors.", "This dataset was then bandpass filtered (AFNI 3dBandpass, FFT-based filter, 0.02–0.1 Hz) to reflect current practises in the field of resting state pre-processing (Di Martino et al., 2013; Power et al., 2012, 2014), spatially smoothed, and registered to MNI space as described above.", "We also considered the unfiltered cleaned data, to test the influence of this processing step on network similarity.", "The cleaned data, true noise data, simulated noise data, reduced, and shortened data were analysed using spatial ICA as implemented in MELODIC as part of FSL (Beckmann et al., 2005; Jenkinson et al., 2012).", "The datasets of the 12 subjects were temporally concatenated, and dimensionality was fixed to output 20 components (except in the case of the short10 dataset, which had too few degrees of freedom to be decomposed into 20 components, where dimensionality was fixed to 10).", "The resulting networks were then compared as follows.", "In the first instance identifying networks in the noise variance, components were manually matched following visual inspection.", "Spatial correlation was used to quantify the similarity of network patterns in the noise variance and the cleaned data, and in the cleaned data relative to network structures provided in the literature (Smith et al., 2009).", "An alternative metric in the literature (Moodie et al., 2014; Tie et al., 2014; Wisner et al., 2013; Zhu et al., 2013), Dice's coefficient, was also calculated for these network pairs using thresholded maps (thresholded p < 0.05, using the alternative hypothesis testing and mixture model approach within the MELODIC framework).", "In the simulated noise data and reduced data, the maximum spatial correlation was used to identify matching components in each iteration.", "The iteration reflecting the median spatial correlation across all iterations was identified, and the network map presented (thresholded Z > 3.0) in the figures.", "The variance explained (and removed) by true and simulated noise regressors was calculated within the 9 functional networks of interest observed in the cleaned data.", "Network maps from the cleaned data were thresholded (Z > 3.0) to form region of interest masks.", "The AFNI software used to perform GLM fitting in the Construction of “noise” datasets section automatically output maps of voxelwise R2 values, representing the variance explained by the full noise model.", "The median voxelwise R2 value was calculated within the network-based ROI masks for each combination of true and simulated noise regressors and for each subject, using the first iteration of phase-randomised simulated noise regressors for simplicity.", "Additional GLM analyses were performed to test combinations of true and simulated noise regressors: 3 true (translations) and 9 simulated, 6 true (translations and rotations) and 6 simulated, 3 true (translations) and 21 simulated, and 6 true (translations and rotations) and 18 simulated head motion regressors.", "In the data variance extracted by the true noise regressors (typically removed from fMRI data during pre-processing), we observed numerous network structures similar to those identified in the functional connectivity literature and in the cleaned data.", "Fig. 2 demonstrates the breadth of network structure identified in the variance removed by different combinations of nuisance regressors, including visual, sensorimotor, executive, auditory, fronto-parietal and default mode networks.", "Spatial correlation values and Dice coefficients show that networks in the noise variance are in good agreement with the networks identified in the cleaned data, which in turn can be matched to established functional networks from the resting state literature (Smith et al., 2009).", "In the noise variance reflecting 24 motion regressors, spatial correlation values ranged from 0.44 to 0.82 across the 9 networks examined; values above 0.25 are typically considered significant in the literature (Forman et al., 1995; Smith et al., 2009; Taylor et al., 2012).", "A similar figure using the unfiltered cleaned data as the reference is presented in the supplementary material (Fig. S3).", "In the data variance extracted by the true noise regressors (typically removed from fMRI data during pre-processing), we observed numerous network structures similar to those identified in the functional connectivity literature and in the cleaned data.", "Fig. 2 demonstrates the breadth of network structure identified in the variance removed by different combinations of nuisance regressors, including visual, sensorimotor, executive, auditory, fronto-parietal and default mode networks.", "Spatial correlation values and Dice coefficients show that networks in the noise variance are in good agreement with the networks identified in the cleaned data, which in turn can be matched to established functional networks from the resting state literature (Smith et al., 2009).", "In the noise variance reflecting 24 motion regressors, spatial correlation values ranged from 0.44 to 0.82 across the 9 networks examined; values above 0.25 are typically considered significant in the literature (Forman et al., 1995; Smith et al., 2009; Taylor et al., 2012).", "A similar figure using the unfiltered cleaned data as the reference is presented in the supplementary material (Fig. S3).", "As apparent in Fig. 2, network structure continues to be present in the true noise data as the number of contributing nuisance regressors is reduced from 24 to 2, although not every network is discernable in every noise dataset examined and the splitting of the motor, default mode, and fronto-parietal networks varies.", "Both similarity metrics as well as qualitative agreement reduce with the number of contributing regressors; spatial correlations generally reduce to non-significant levels (r < 0.25) in the case of 2 nuisance regressors.", "ICA results from the true noise data and cleaned data presented in Figs. 2 and S3 are available as NIFTI files in the supplementary material.", "As apparent in Fig. 2, network structure continues to be present in the true noise data as the number of contributing nuisance regressors is reduced from 24 to 2, although not every network is discernable in every noise dataset examined and the splitting of the motor, default mode, and fronto-parietal networks varies.", "Both similarity metrics as well as qualitative agreement reduce with the number of contributing regressors; spatial correlations generally reduce to non-significant levels (r < 0.25) in the case of 2 nuisance regressors.", "ICA results from the true noise data and cleaned data presented in Figs. 2 and S3 are available as NIFTI files in the supplementary material.", "Maximum spatial correlation with the cleaned data networks was used to identify nine components in the simulated noise datasets (for each of the 10 iterations).", "Fig. 3 summarises the similarity between the true/simulated data networks and the cleaned data networks.", "The simulated noise data contain network structures with similar spatial correlation to the cleaned data maps as the true noise data, suggesting that the network structure in the noise variance is not inherently linked to either true head motion or true physiological noise and can be achieved via unrelated (orthogonalised) nuisance regressors.", "Due to the way simulated noise regressors were derived (i.e., forced orthogonality), statistical tests for significant differences between these results are not valid.", "However, for most networks the simulated noise data show a monotonically increasing relationship between the spatial similarity relative to the cleaned data map and the number of nuisance regressors contributing to the simulated noise dataset.", "This demonstrates the potential impact of the “degrees of freedom” associated with nuisance regressors on the network-related variance extracted by the regression.", "This concept is revisited in the Network structure in random sampling of fMRI variance section and Variance explained by true and simulated noise regressors section.", "Finally, the results from the noise data using true regressors from a different subject's data are presented in Supplementary Fig. S4, providing additional evidence that nuisance regressors unrelated to the data can remove variance of interest, even with minimal numbers of regressors (degrees of freedom) used in the fitting procedure.", "Finally, the results from the noise data using true regressors from a different subject's data are presented in Supplementary Fig. S4, providing additional evidence that nuisance regressors unrelated to the data can remove variance of interest, even with minimal numbers of regressors (degrees of freedom) used in the fitting procedure.", "Because we observe network structure in the variance associated with simulated nuisance regressors, it is likely that random sampling of the fMRI signal variance is sufficient to observe the same phenomenon.", "Using the reduced data to test this hypothesis, we observed network structure in subsets of the data with as few as 10% of volumes (16 volumes per subject) randomly “sampled.”", "Fig. 4 shows the spatial correlation between the reduced data network maps and the cleaned data maps for 4 networks.", "The boxplots represent the median and quartile distributions of spatial correlation coefficients (r) for the 100 iterations of randomly reduced data.", "The network maps associated with the median correlation values are provided for reference.", "The mean time-series within the default mode network for one subject is shown for the 6 reduced datasets (map thresholded Z > 3 before signal averaging within this mask).", "There is little decrease in spatial correlation as the data is reduced from 60% to 10% of the original volumes.", "This raises the important question of whether substantially shortened scans could achieve spatial maps of functional networks with fidelity.", "Fig. 5 demonstrates that the 9 networks observed in the total cleaned dataset are also observed in the shortened data, with as few as the initial 20% of volumes (32 volumes, approximately 1 min of scan time) considered for each subject.", "The default mode network, visual network, motor network, and auditory network are also visible in the short10 data, which represents 32 s of fMRI acquisition for each subject.", "The remaining networks are not visible, potentially as a result of the limited dimensionality in the ICA decomposition.", "The results of these toy examples indicate that network structure is extremely robust, and can be extracted from small subsets of the original data variance.", "In Fig. 6a we demonstrate that true noise regressors remove consistently more variance from the original BOLD fMRI data than the simulated noise regressors, as may be expected given that true noise regressors reflect theoretical and empirical noise models designed to remove known signal confounds from resting state data.", "In addition, the amount of variance explained increases as the number of nuisance regressors increases, whether true or simulated, as expected when increasing the degrees of freedom in the GLM fitting procedure.", "We also observe a change in the relationship of additional true/simulated noise regressors and the amount of BOLD variance explained when considering few (2–6) compared to many (12–24) nuisance regressors.", "Specifically, the addition of up to 6 nuisance regressors results in sharp deviation between the amount of variance explained by true versus simulated noise regressors, while beyond 6 regressors there is a more similar increase in the variance explained.", "This suggests that beyond 6 true noise regressors (translations and rotations), the addition of more true noise regressors to the GLM is akin to adding unrelated simulated regressors with respect to the amount of variance removed within a functional network.", "To test this, Fig. 6b presents the correlation between variance explained by combinations of true and simulated noise regressors compared with the variance explained by all true noise regressors, while maintaining the total number of nuisance regressors (12 or 24) to control for degrees of freedom in fitting.", "There is no correlation across subjects in any network examined between the variance explained by all true and all simulated noise regressors (black dots).", "Replacing 3 simulated regressors with the true 3 head motion regressors (x-, y-, and z-translations) does not improve this.", "However, replacing 6 simulated regressors with the true 6 head motion regressors results in significant correlation (p < 0.05, corrected for 18 comparisons) with the variance explained by all true noise regressors.", "(Note, one non-significant finding was observed in the case of 24 nuisance regressors in the Fronto-parietal2 network).", "These results quantitatively support the qualitative observation in Fig. 6a: in our data, beyond 6 true head motion regressors, additional regressors may act similarly to unrelated simulated nuisance regressors, which function to randomly sample (and remove) variance.", "Given that we observe network structure in the true noise datasets (Fig. 2), it is important to consider whether there may be a real relationship between the neural activity in these functional networks and the noise sources modelled by our nuisance regressors.", "In this study, the network structure is most robust in the noise variance associated with 12 or 24 head motion regressors.", "Yan and colleagues (Yan et al., 2013a,b) proposed that the relationship between motion regressors and the BOLD signal partly reflect a neural signature of head motion rather than strictly artefact.", "In support of this, recent studies have shown that head motion is linked to signal changes in certain brain regions, including sensory, motor, visual and default mode network areas (Pujol et al., 2014), or may be associated with an inherent subject-specific trait linking connectivity and tendency for head motion (Zeng et al., 2014).", "Regarding physiological noise regressors, voluntary breathing has been linked to activation of bilateral sensory and motor cortices (McKay et al., 2003).", "Cerebrovascular reactivity to arterial CO2 changes, independent of neural activation, may also result in BOLD signal changes coupled to our physiologic regressors.", "There are consistent regional differences in the vascular response to this CO2, with areas such as sensorimotor and visual cortices distinguished by early or delayed response times (Bright et al., 2009).", "Although these factors do allow for the possibility of an inherent link between true noise and network structure, it seems unlikely that this is the driving source of our observations.", "Simulated noise regressors that are orthogonalised (and thus unrelated) to the true noise regressors result in similar network decomposition, and often these networks are as similar or more similar to the cleaned data networks than are the true noise equivalents (Fig. 3a).", "This suggests that any set of nuisance regressors may extract variance that can be decomposed into network structure.", "The reduced data analysis (Fig. 4) supports this finding, demonstrating that even very small (10%) sampling of the original data, which is akin to randomly sampling data variance with unrelated nuisance regressors (as in Fig. 6a), will result in similar network structure as the cleaned data.", "The literature demonstrates that true noise regressors remove signal variance associated specifically with the intended noise confound (see, for example, (Power et al., 2014)); however, we assert that these regressors will also inherently remove some additional variance at random, and that this random sampling of the data drives the observation of interesting network structure in the associated noise datasets.", "We also assert that this randomly sampled variance must be considered “signal” because it contains multiple, distinct “intrinsic connectivity network” structures: our model of nuisance regressors fits different variance in, say, the Default Mode Network compared to the Visual Network compared to the Sensorimotor Network.", "Under the assumption that intrinsic connectivity networks are neural networks that express synchronised neural activity, rather than areas where motion confounds are expressed differently due to differences in gyri structure and other brain boundaries (for example), we must also conclude that the variance we are extracting to differentiate these networks represents neural signals as well.", "Finally, we observe a wide range of network structures, including short and long-distance nodes across many cortical areas, both bilateral and lateralised, extending beyond the regions indicated in the recent literature as having potential for neural linkage between motion “noise” models and neural activity (Pujol et al., 2014; Zeng et al., 2014).", "The complete picture is potentially complicated: the relationship between intrinsic connectivity networks and true noise sources is likely to be variable across different brain regions.", "However, we observe network structure in nearly all cases examined in this paper.", "It may be important to characterise the variance that is removed from resting state analysis by nuisance regressors, to assess the impact of how many and what type are used, and to determine whether this is problematic for a given subject or cohort.", "In the cleaned data and the true and simulated noise data, we consistently identified network structure (Figs. 2 and 3).", "In the true noise variance, 12–24 head motion regressors removed variance that exhibited all functional network structure identified in the cleaned data.", "Considering fewer nuisance regressors (or fewer volumes, as in the short10 data of Fig. 5), the ICA decomposition identified some, although not all, network structures.", "This potentially reflects sub-optimal decomposition due to fixed ICA dimensionality in situations with differing degrees of freedom in the data, or closer coupling between those true noise regressors and true signal confounds that are spatially unrelated to network structures.", "Fig. 4 demonstrates that random sampling of only 16 volumes per subject was sufficient to identify most of these same functional networks in our data.", "Randomly reducing the data to 10% of volumes is similar to sampling 10% of the total data variance, demonstrating that fMRI “signal” with interesting network structure is likely fit and isolated even when nuisance regressors are removing only very small amounts of variance at random.", "We also observe that 2 true physiologic or 3 true head motion regressors sample slightly less than 10% of the variance in the data of most subjects and networks examined in this study (Fig. 6a), representing both true noise variance as well as randomly extracted variance, and not all functional brain networks are observable in these true noise datasets.", "Combined, these results indicate that network structure is sometimes observable in less than 10% of our data variance, but only becomes robust and consistent when 10% or greater random variance is considered.", "This suggests that the structure of functional networks—the identification of nodes that exhibit temporally correlated BOLD signal fluctuations in the resting state—is extremely robust beyond this threshold of 10% of our data (16 volumes, 32 s, per subject).", "By contrast, in the shortened data that considers only the initial consecutive volumes, we observe that 20% of volumes (32 volumes, 64 s, per subject) are necessary to discern all networks considered in this study.", "The “shortened” data analysis examines how little data is needed to observe networks, whereas the “reduced” data analysis examines how little variance is needed to observe these networks in a full length dataset.", "Using consecutive volumes to differentiate intrinsic signal fluctuations may also be less efficient at extracting robust network structures than using randomly sampled volumes, particularly if these fluctuations are at low frequencies of approximately 0.01 Hz.", "Noise confounds, truly unrelated to the intrinsic signal fluctuations of interest, are also likely to be of more concern in consecutive volumes, potentially hindering our ability to discern the network structures.", "We conclude that ~ 1 min of the initial data is necessary to observe robust network structure in this study, although randomly sampling from a longer dataset may require less data.", "This initially appears at odds with the literature, where it has been suggested that between 5 and 12 min of data (Birn et al., 2013; Van Dijk et al., 2010), or 3 min of “scrubbed” data (Yan et al., 2013a), is desirable for reliable connectivity estimates.", "These previous studies, however, have assessed connectivity measures such as temporal correlation, whereas here we examine spatial structure.", "Note also that we include data from 12 subjects in the ICA decomposition, and thus approximately 12 min of data are used in the automatic extraction of network structure in this paper, despite needing very little data from an individual subject.", "Within the resting state literature, temporal correlations are commonly used to map network structure, and correlation strengths are then calculated within this structure—a potentially circular procedure.", "Knowing that a small number of fMRI volumes (~ 1 min with a TR of 2 s) are sufficient to map network structure in a population, we could use short scans to spatially define networks independently of the temporal correlations in a longer functional scan and prevent the possibility of “double dipping” in our analysis.", "Alternatively, a random sampling of an even smaller number of volumes could be extracted from a longer resting-state dataset for this purpose.", "Both approaches could be applied to existing data with minimal cost.", "In this study, we highlight the potentially detrimental aspects of using nuisance regressors to remove noise from resting-state data; particularly, that increasing the number of nuisance regressors is more likely to result in the removal of variance containing “interesting” structure.", "Here we examine the variance removed by the different noise models in the context of the field, and how nuisance regressors could be limited to reduce over-fitting of interesting signal variance.", "In the original paper proposing the cardiac rate noise correction technique, multiple lagged heart rate regressors were observed to explain 1.5% of variance beyond that explained by polynomial and RETROICOR regressors (Shmueli et al., 2007); this underestimates the contribution of heart rate in the current study, where we consider only 1st and 2nd order polynomial terms and do not consider RETROICOR.", "In the original paper proposing end-tidal CO2 as a nuisance regressor, it was observed to explain 4.7% of variance (Wise et al., 2004) within grey matter, although this study only considered voxels significantly correlated to the end-tidal CO2 regressor and thus overestimates this effect.", "In our study, we observe that combined, these physiological regressors, in conjunction with linear and quadratic detrending terms, explain approximately 5–10% of signal variance across subjects, which is in agreement with the literature reports albeit difficult to make a fair comparison.", "We observe that 6 motion regressors explains slightly more variance (approximately 10–30%) than reported elsewhere (~ 5–20%, (Jo et al., 2010)), however, differences in calculating the mean/median across voxels, using voxelwise values or considering mean time-series, examining all grey matter versus smaller regions of interest, and the use of bandpass filtering may affect specific quantification.", "To clarify for future comparisons, the values presented here represent the median voxelwise R2 value for the entire nuisance regressor model fit, including linear and quadratic detrending terms, across network regions of interest defined elsewhere, with no additional grey/white-matter masking, and no bandpass filtering.", "The concern over the expanding number of nuisance regressors in resting state fMRI pre-processing and the impact on degrees of freedom has been raised in the literature.", "Satterthwaite and colleagues tempered their proposed 24-regressor model with the caveat that connectivity values do not yet take into account the degrees of freedom contributing to that value (Satterthwaite et al., 2013).", "Others have applied Principal Component Analysis to reduce 6 motion regressors to 2, still containing up to 85% of the original motion-related variance but with a smaller impact on degrees of freedom (Churchill et al., 2012).", "Similarly, Jo and colleagues state that the number of physiological nuisance regressors (e.g., RETROICOR (Glover et al., 2000)) should be reduced because they do not explain much variance compared to their effect on degrees of freedom (Jo et al., 2010).", "Precisely where to draw the line between beneficial and confounding nuisance regression is inherently ambiguous.", "In our data, we find that more than 6 motion regressors remove a similar amount of variance to unrelated simulated noise regressors.", "If we make the assumption that simulated or random regressors remove variance at random while true noise regressors remove both the intended noise variance as well as some degree of random variance, the fact that the amount of variance removed is similar in these two scenarios demonstrates the inherent difficulty in quantitatively proving that additional true noise regressors perform “better” than random equivalents.", "In addition, the variance explained by just 6 or fewer regressors did not demonstrate as robust network structure as 12 or 24 regressors (Fig. 2), indicating that analysis of our data may be best served by limiting the number of motion-related nuisance regressors to 6.", "Besides reducing the size of the nuisance regressor model, there is an alternative means of addressing the balance between beneficial de-noising and random removal of signal.", "As the underlying concern is one of degrees of freedom, the problems associated with excessive regression can be attenuated by extending the number of time-points in the data.", "This study considers data with 160 time-points, and observes that approximately 6 motion regressors may be optimal; however, longer datasets with many hundreds of fMRI volumes are likely to be well-suited for increased numbers of nuisance regressors.", "To test this, we simulated fMRI “signal” as a low frequency sinusoid (0.01 Hz), and examined the variance removed by random nuisance regressors generated using evenly distributed random numbers.", "The results of 100 iterations (mean and standard deviation) are presented in Fig. 7, demonstrating how the random sampling of signal variance is attenuated as the duration of the fMRI dataset is extended (up to 640 volumes, or 4 times longer than data considered in this study).", "We observe that the attenuation exhibits a transition or \"bend\" before asymptotically approaching negligible R2 values.", "At 160 time-points, the variance randomly sampled by 3 or 6 regressors is small and we are operating beyond the \"bend\" in these plots, whereas 12 or 24 regressors place us within this transition region and the variance being sampled is much higher (approximately 10–20%).", "Extending the data to 600 or more volumes would ameliorate this concern, bringing us into the asymptotic regime in all plots in Fig. 7.", "However, note that we have used a very simplistic simulation in which regressors are truly unrelated to the data; in practise, regressor accuracy and total noise levels are likely to influence these attenuation plots.", "Together, these findings suggest that limited amounts of physiologic or motion regressors may be the best way forward.", "We recommend that simulations, similar to those presented here, are applied to determine the most appropriate number of nuisance regressors for a given analysis.", "For example, regression could be performed on subsets of data with increasing numbers of time-points, to determine if or when the \"plateau\" is reached.", "This approach would assist future studies aiming to maximise removal of noise confounds while preserving the existing signals of interest in the data.", "We also recommend that future studies characterise the variance removed by any pre-processing nuisance regression, to determine whether this balance is altered in different datasets.", "For example, some cohorts may display greater head motion artefacts than other cohorts (Van Dijk et al., 2012); in subjects with large amounts of head motion, the addition of extra motion-related nuisance regressors may make the cleaned data more accurate, while in subjects with much less head motion these additional regressors may serve more to randomly sample and remove signals within functional networks.", "Thus nuisance regressors could create false positives in addition to false negatives in resting state studies.", "The main aim of this paper is to demonstrate that nuisance regressors extract and discard signal variance with network structure.", "Here we also assess the impact of nuisance regression on temporal correlations between network nodes, using the Default Mode Network as an example.", "The four largest clusters were extracted from a map of the Default Mode Network taken from the literature (Smith et al., 2009), and the mean %BOLD time-series within these four nodes were extracted in each subject's original, true noise, and cleaned (unfiltered) data.", "Correlation between each node pair was calculated and converted using the Fisher r–z transform before averaging across subjects.", "We have presented the resulting group average correlation matrices in supplementary material (Fig. S5), for the Motion24, Motion12, Motion6, and Motion3 analyses.", "The main aim of this paper is to demonstrate that nuisance regressors extract and discard signal variance with network structure.", "Here we also assess the impact of nuisance regression on temporal correlations between network nodes, using the Default Mode Network as an example.", "The four largest clusters were extracted from a map of the Default Mode Network taken from the literature (Smith et al., 2009), and the mean %BOLD time-series within these four nodes were extracted in each subject's original, true noise, and cleaned (unfiltered) data.", "Correlation between each node pair was calculated and converted using the Fisher r–z transform before averaging across subjects.", "We have presented the resulting group average correlation matrices in supplementary material (Fig. S5), for the Motion24, Motion12, Motion6, and Motion3 analyses.", "We observe no significant changes in the node-pair correlation values between the original data and the cleaned data using 24, 12, 6, or 3 motion regressors to de-noise, and only one instance of significant difference in the correlations in the cleaned data relative to the corresponding noise data.", "The lack of significant changes in the temporal correlation values following nuisance regression may mean that, to many users, our observations of network structure in the noise variance are not particularly concerning.", "However, temporal correlation values are similar no matter what portion of the variance is examined.", "Given that we can divide our data variance in multiple ways depending on how many nuisance regressors are used, attributing one portion to signal and the other to noise, the consistency of temporal correlations in the “signal” and “noise” variance suggests it is ambiguous to use temporal correlations alone as a marker for optimising pre-processing methods.", "Finally, we have provided a new framework for examining the benefits of nuisance regressors by comparing (across subjects) the amount of variance removed by the proposed nuisance regressor relative to the variance removed by a phase-randomised regressor.", "Simulations could also be used to quantify the amount of data variance expected to be removed purely at random with each additional nuisance regressor added to the noise model, as described in Fig. 7.", "This approach may contribute towards a test for determining whether a new nuisance regressor removes noise rather than randomly sampled variance, particularly when the intent is to compare the results across subjects with varying noise contributions." ]	[ "Data variance removed by nuisance regressors contains network structure.", "Simulated regressors unrelated to noise also extract data with network structure.", "Random sampling of original data (as few as 10% of volumes) reveals robust networks.", "After optimal number, motion regressors remove similar variance as simulated ones.", "Excessive nuisance regressors extract random signal variance with network structure." ]
Mapping combined wildfire and heat stress hazards to improve evidence-based decision making	Heat stress and forest fires are often considered highly correlated hazards as extreme temperatures play a key role in both occurrences. This commonality can influence how civil protection and local responders deploy resources on the ground and could lead to an underestimation of potential impacts, as people could be less resilient when exposed to multiple hazards. In this work, we provide a simple methodology to identify areas prone to concurrent hazards, exemplified with, but not limited to, heat stress and fire danger. We use the combined heat and forest fire event that affected Europe in June 2017 to demonstrate that the methodology can be used for analysing past events as well as making predictions, by using reanalysis and medium-range weather forecasts, respectively. We present new spatial layers that map the combined danger and make suggestions on how these could be used in the context of a Multi-Hazard Early Warning System. These products could be particularly valuable in disaster risk reduction and emergency response management, particularly for civil protection, humanitarian agencies and other first responders whose role is to identify priorities during pre-interventions and emergencies.	[ "ENVI" ]	[ "Decision making", "Fire weather index (FWI)", "Geospatial analysis", "Multi hazard", "Universal thermal climate index (UTCI)", "Wildfire" ]	[ "2300" ]	[ "The frequency and length of high-temperature driven hazards such as wildfires and heat stress is likely to worsen under a changing climate (Running, 2006; Westerling et al., 2006; Diffenbaugh et al., 2007; Forzieri et al., 2016; Kurnik et al., 2017).", "According to the EM-DAT International Disaster Database (https://www.emdat.be/), between 1966 and 2017 approximately 167 major fires and heat events occurred in Europe, causing about 140,000 deaths.", "In 2017 alone, the 6 largest wildfire events spanning Spain, Portugal, Croatia and Montenegro resulted in 732 Million USD in total damages (data from EM-DAT).", "The summer period of 2017 also witnessed two major heat-related events, one in June and one in August, which led to unprecedented high temperatures across Europe (Di Giuseppe et al., 2017; Sánchez-Benítez et al., 2018).", "An increase in mortality was correspondingly observed by national health services in Belgium, France, Italy and Spain (Gil Bellosta et al., 2017; Ministero della Salute, 2017; Bustos Sierra and Asikainen, 2018; Beaudeau et al., 2018).", "Extremely high temperatures are the common denominator for both heat stress and wildfires.", "According to Gill and Malamud (2014), the occurrence of a heat event, i.e. a heatwave, can trigger dangerous fire weather conditions.", "In turn, the emissions from wildfires can exacerbate the effects of heat stress on human body, especially on the cardiovascular and respiratory systems (Finlay et al., 2012).", "Although there is a wealth of research on mapping and analysing wildfires and heat events individually, the analysis of their simultaneous occurrences is relatively less explored.", "This is because the analysis and visualisation of multiple-hazards is considered a rather challenging task (Kappes et al., 2012).", "Relevant contributions include: monitoring wildfires during major heatwaves (Bondur, 2011; Gouveia et al., 2016), combined assessment of multi-hazard indicators under climate change scenarios (Lung et al., 2013; Forzieri et al., 2016), quantification of emissions and the combined long-term effects of wildfires and heatwaves on mortality (Tressol et al., 2008; Shaposhnikov et al., 2015), and assessment of weather driven disasters in the context of protection for the environment and citizens (Lavalle et al., 2006).", "In this work, as we contribute to the goal to develop a European Multi Hazard - Early Warning System (MH-EWS) platform, we focus on the last aspect, specifically on enhancing emergency management and response to extreme weather and climate events.", "Marzocchi et al. (2009) state that the “assumption of independence of the risk sources leads to neglect possible interactions […] a potential ‘multi-risk’ index could be higher than the simple aggregation of single risk indexes”.", "In this context, analysing the historical occurrence of multiple hazards and applying that knowledge to future scenarios is paramount because the simultaneous occurrence of multiple hazards can potentially lead to substantial impacts, as the impacts of one hazardous event are often exacerbated by interaction with another (Marzocchi et al., 2009).", "Here we propose a general framework to locate and predict the simultaneous occurrence of two high-temperature driven hazards: wildfires and heat stress.", "This type of information could be used within an operational MH-EWS as a reference layer to provide timely information with which decision makers could allocate resources and prioritise interventions.", "The framework is general and can be expanded to include other natural hazards.", "First responders, emergency managers, decision makers and other stakeholders could make use of a MH-EWS by simply browsing individual hazard layers and extrapolating the consequences of concurring hazards on the basis of their experience and expertise.", "However, forecasters and forecast users are keen for innovation in the visualisation of information that can concisely and quickly highlight the possibility of upcoming natural hazards (Pappenberger et al., 2018), and this is particularly important for concurrent hazards.", "Here we propose four information layers for MH-EWS users: (1) maps of climatological thresholds for single hazards, (2) maps of hotspots for concurrent hazards, (3) maps of related probability of occurrence of future events and (4) a monthly summary of forecasted combined danger.", "In order to produce these information layers, we make use of a pan-European reanalysis database and medium-range (3–15 days) forecasts of heat stress and wildfire danger indices available from the European Centre for Medium-range Weather Forecasts (ECMWF).", "Below is a description of the hazard indices and datasets considered in the study, as well as a brief explanation of the three steps followed in the modelling workflow: (i) the calculation of daily climatology to define warning levels; (ii) the mapping of multi-hazard hotspots for past and future dates using reanalysis and high resolution medium range forecasts, respectively; and (iii) the mapping of the probability of occurrence of future simultaneous hazards using ensemble medium-range forecasts.", "The workflow is also illustrated in the infographics in Fig. 1.", "The models calculating fire danger and heat stress are driven by atmospheric forcings.", "Two types of forcings are used: a historical database, the ECMWF Re-Analysis (ERA-Interim, Dee et al., 2011) and the medium-range forecasts both generated by the Integrated Forecasting System (IFS) and produced by ECMWF.", "A reanalysis dataset is generated using a data assimilation scheme and a physical model that ingests quality controlled observations and produces a dynamically consistent estimate of the state of the atmosphere.", "ERA-Interim has a spatial resolution of approximately 80 km and a temporal extent from 1979 to the present day.", "In addition to this dataset, ECMWF provides weather forecasts using different configurations of its dynamical model, amongst which are: the high resolution configuration (also called HRES or deterministic forecast), a single run with a horizontal resolution of ~9 km and 10 days time horizon; and the ensemble configuration (called ENS or probabilistic forecast) comprising 51 realizations of the same forecast from slightly perturbed initial conditions and different model configurations.", "The spread in the ENS forecasts provides information about the accuracy and confidence of the forecast as similar outcomes are regarded as a proof of enhanced predictability for a given field.", "ECMWF ENS forecast has a spatial resolution of ~18 km and 15 days horizon.", "Wildfires are natural phenomena and are controlled by fuel availability, weather and ignition agents.", "As ignition is a highly unpredictable factor, fire Early Warning Systems are based on the estimation of fire danger rather than on potential ignition.", "Most fire authorities in Europe rely on weather measurements from meteorological observations at point locations, which are then extrapolated to the surrounding areas.", "Examples of fire danger rating systems are the US Forest Service National Fire Danger Rating System (Deeming et al., 1977), the Canadian forest service Fire Weather Index (Van Wagner, 1974, 1987; De Groot, 1998) and the Australian McArthur rating systems (McArthur, 1966).", "All these systems provide estimates of fire danger in terms of indices that measure fire behavior, energy release and rate of spread if a fire were to start (San-Miguel-Ayanz et al., 2003a).", "The Fire Weather Index (FWI), is a simple model that provides good performance worldwide and has become the backbone model of the European Forest Fire Information System (EFFIS, http://effis.jrc.ec.europa.eu/) and the Global Wildfire Information System (GWIS, http://gwis.jrc.ec.europa.eu/), respectively the European and Global platforms for fire danger information in the framework of the Copernicus program (Di Giuseppe et al., 2016).", "The FWI is used here as a metric to quantify fire danger at the European scale.", "The system contains three soil moisture codes which represent fuel beds of different consistency and depths.", "They are therefore characterised by different time lag responses to the atmospheric forcings.", "From these codes, the model calculates the expected rate of spread immediately after ignition and a build-up component related to the fuel availability for combustion.", "The FWI combines the rate of spread and fuel availability to provide a generic numeric rating of potential fire intensity.", "The higher the numerical value, the more uncontrollable the fire is expected to be.", "Forestry agencies usually estimate daily FWI from observations, therefore with uneven spatial coverage.", "Under the umbrella of Copernicus Emergency Management Services, ECMWF produces a historical dataset of FWI values using the atmospheric reanalysis dataset as forcings (Vitolo et al., 2019) with an even spatial coverage at global scale.", "In this dataset, FWI varies in the range [0, +inf[.", "However, in practical applications such as EFFIS and GWIS, values are usually binned into danger classes (e.g. very low, low, moderate, high, very high and extreme).", "These classes can be estimated on the basis of the local distribution of historical quantiles (Vitolo et al., 2017, 2018).", "ECMWF also provides daily forecasts of FWI using both the high resolution (HRES) and ensemble (ENS) configurations of the Integrated Forecasting System (Di Giuseppe et al., 2016, under review).", "The reader should note that the current FWI ensembles have shorter time horizon compared to the IFS ENS: 10 rather than 15 days.", "Heat stress is the physiological heat load enforced by the outdoor environment on the human body which correspondingly reacts via different physiological responses, such as sweating, to maintain its core temperature within certain boundaries, even when the surrounding temperature is very different (McGregor and Vanos, 2018).", "A measure of heat stress is provided by the Universal Thermal Climate Index (UTCI), a bioclimate index that well reflects health hazards attributable to extreme high temperatures both at the European and global level (Pappenberger et al., 2015; Di Napoli et al., 2018; under review).", "The UTCI is defined as the isothermal temperature, expressed in degrees Celsius, of a reference condition that, following the human energy balance model, would elicit the same dynamic physiological response of the real condition (Jendritzky et al., 2012; Błażejczyk et al., 2013).", "Whereas other indices are based exclusively on meteorological parameters such as air temperature and humidity (e.g., humidex; Masterton and Richardson, 1979), the UTCI is based on a human energy balance model that considers heat and mass transfer within the body, thermoregulatory reactions of the central nervous system, perceptual responses and a temperature-adaptive clothing insulation for outdoor climates (Fiala et al., 2012).", "The UTCI is computed via a six-order polynomial equation in four environmental parameters (Bröde et al., 2012), namely 2 m air temperature, the mean radiant temperature (i.e., solar and thermal radiation), wind speed and humidity.", "While the UTCI is a continuous variable it is, like the FWI, categorised using the corresponding reanalysis database.", "ECMWF ENS and HRES forecasts of 2 m air temperature, humidity, radiation and wind speed are then used to calculate UTCI forecasts.", "In this section we demonstrate how to define warning levels knowing the range of possible values for both the FWI and UTCI indices.", "There are two methodologies that are usually adopted to derive warning levels for the FWI: one is based on the number of events that occurred during fire seasons, and the other is based on the analysis of the probability distribution (i.e. using representative percentiles) of the historical record of the FWI values.", "In this work, we employ the latter method.", "Warning levels (or danger thresholds) are usually calculated as an average over large regions and/or at country level (Di Giuseppe et al., 2019).", "However, Vitolo et al. (2018) tested the same approach on European Nomenclature of Territorial Units for Statistics (NUTS) 1–2 levels and found that averaging fire danger over an area reduces the probability of detection of an event as the information on high danger levels is lost.", "Therefore, a better approach is to rely on statistics calculated cell-by-cell, even though this is more computationally demanding.", "With regards to heat thresholds, Błażejczyk et al. (2013) suggested that the UTCI should be categorised in a ten thermal stress levels scale: extreme cold stress (UTCI < −40 °C); very strong cold stress (−40 °C ≤ UTCI < −27 °C); strong cold stress (−27 °C ≤ UTCI < −13 °C); moderate cold stress (−13 °C ≤ UTCI < 0 °C); slight cold stress (0 °C ≤ UTCI < 9 °C); no thermal stress (9 °C ≤ UTCI < 26 °C); moderate heat stress (26 °C ≤ UTCI < 32 °C); strong heat stress (32 °C ≤ UTCI < 38 °C); very strong heat stress (38 °C ≤ UTCI < 46 °C); and extreme heat stress (UTCI ≥ 46 °C).", "Each level corresponds to specific physiological responses from the human body.", "Although physiological responses are common across humans, populations across Europe have adapted to different heat stress levels, resulting in a variability in heat acclimatisation in both time and space.", "Southern Europe generally experiences higher heat stress than northern Europe, and summer thermal stress is usually higher in July/August than in June (Di Napoli et al., 2018).", "To take into consideration these aspects, in this work, the UTCI warning levels are not fixed levels but are defined, consistently with the FWI, using the probability distribution of reanalysis records.", "For both hazards, a collection of maps of warning levels is generated, one for every day of the year and danger level.", "Every map corresponds to a predefined percentile and summarises the distribution of the values across the years.", "Although this procedure is more laborious and resource consuming than those adopted in previous studies, it provides a number of important advantages: it does not depend on the definition of the fire/heat stress season (often subjective and generally changing year on year) and the result is not smoothed because there is no spatial averaging.", "We generate the climatology of a given hazard index from historical reanalysis records over the period 1980–2016 extracting the following percentiles: 50th, 75th, 85th, 90th, 95th, 98th (corresponding to very low, low, moderate, high, very high and extreme danger, respectively).", "These percentiles are widely used to assess danger due to wildfire and heat stress in Europe (San-Miguel-Ayanz et al., 2003b; Di Napoli et al., 2019).", "We calculate these percentiles taking into account 333 days: the period that spans 4 days before and after a given date (total of 9 days) for the full record of 37 years.", "Values are evaluated cell-by-cell and provide thresholds of increasing danger.", "These thresholds are used as cut-off points to assess daily conditions from 2017 onwards.", "The procedure can be applied to other hazard indices for which a long historical record is available.", "By historical record we mean either a reanalysis or a long record of in-situ observations, generating a gridded or point-based map respectively.", "In order to limit processing time, we only generated thresholds for the months that are most likely to be affected by fire and heat stress hazards in Europe: June, July and August (referred to as JJA season hereafter).", "Once a database of threshold maps is generated, we have a means of comparison and can assess on any given day whether a hazard index falls in a concerning range (e.g. above very high danger threshold).", "Then, the spatial overlap of multiple hazard maps can reveal areas of particular concern.", "First responders, forecasters and other stakeholders might find it particularly useful to have access to an operational product that delivers a daily overview of where, in Europe, there is a chance of occurrence of one or multiple hazards.", "We call this daily overview a map of hotspots, to build upon work on mapping the observed occurrence of multiple weather driven disasters in the same location (Dilley et al., 2005).", "A map of hotspots is generated by analysing the spatial overlap of the high resolution forecasts of multiple hazard indices and the relevant climatology on a given day.", "The methodology follows three steps:", "Warning level maps, defined in the previous section, are resampled to match the resolution of the forecasts using the nearest neighbour technique.", "Binary maps are generated, with 1 (resp.", "2) in the cells where the fire (resp.", "heat) forecasted value is above a given threshold, and 0 otherwise.", "There will be a map for every hazard, danger threshold and day in the forecast horizon.", "The binary maps of different hazards are summed pairwise.", "The result is a four-value encoded map (per danger threshold and day) in which cells are encoded as follows:", "0 = no hazard", "1 = only fire hazard", "2 = only heat stress hazard", "3 = fire and heat stress hazard", "The same procedure can be employed to map hotspots during past dates, using daily reanalysis as input rather than high resolution forecasts.", "In this case, the computation is much faster as only 1 map is produced, per date of interest, and there is no need of resampling the layers as there is no change in the spatial resolution.", "In this work we focus on predicting hotspots and leave to future work the analysis of patterns of past co-occurrences.", "If a combined fire and heat stress hazard is detected in the previously described map of hotspots, it is important to inform decision makers of the confidence/uncertainty that accompanies this prediction, especially if this information is to be used for issuing alerts and warnings.", "The procedure described for the high resolution forecasts can be repeated for every member of the ensemble forecasts, generating a four-value encoded map per ensemble member, danger threshold and day in the forecast horizon.", "At each cell, the percentage of the ensemble members for which the fire and heat thresholds are both exceeded will return the probability of simultaneous occurrence of the two hazards.", "As ensemble members in the ECMWF forecasts can be treated as being equally likely, we can estimate the average statistics by comparing fire danger and heat stress forecasts deriving from the same weather realisation (fire danger forecast ensemble member 1 with heat stress forecast ensemble member 1, and so forth).", "Local authorities may also benefit from visualisation tools to track the progress of forecasts over time and be able to compare today's information with what was forecasted over the past few days.", "In light of these needs, we have designed a 1-month forecast summary, in which successive forecasts are plotted one on top of the other to help contextualize the current forecast and gain an appreciation of how these complex dynamics evolve over time.", "The plot can be generated for different types of information and be used to track the persistency and/or the fluctuation of a signal over time.", "For instance, when visualising a single danger index, such as fire danger, the plot can be used to identify the start/end of the fire season.", "In the results section we will show how to use this plot to track the expansion of the area characterised by very high combined danger, according to the forecasted maps of hotspots.", "In order to illustrate the results of the above methodology, we analyse the summer 2017 in relation to its climatological values at the European scale.", "As points of reference, we will use the locations mentioned by media articles reporting outbreaks of extreme heat and wildfires during June (see Table 1 and Fig. 2).", "We use media reports to distinguish major fire outbreaks (blue and yellow circles in Fig. 2) from the numerous sources of Fire Radiative Power (FRP, generated using CAMS-GFAS (2019) information, red dots in Fig. 2) detected by satellites.", "The two major fires in Portugal were also checked against the Copernicus Emergency Management Service activation points (Copernicus EMS, 2017a, 2017b).", "Fig. 3 shows the average FWI (top row) and UTCI (bottom row) very high danger values (95th percentile) for the months of June (left column), July (centre column) and August (right column).", "The climatology of wildfire and heat stress danger have different spatial distributions.", "The UTCI thresholds span the range [9, 44]°C and are more homogeneously distributed over Europe than FWI thresholds.", "FWI's spatial distribution presents a more pronounced difference between Mediterranean countries, central Europe and Nordic countries with values spanning a much larger range [2, 96].", "This shows that, while moderate or stronger heat stress (UTCI > 26 °C) is above human acclimatisation capability for most of the European population, the interpretation of fire ratings varies greatly in space.", "For example, an FWI of 30 can signal very unusual fire weather in Scandinavia, while representing the norm in Mediterranean countries.", "Climatological maps are very powerful tools that allow the re-classification of danger indices based on past decades of weather observations, on a cell-by-cell basis.", "Binning the data into categories defined by the historical percentiles allows expected biases in the reanalysis and forecasts to be accounted for.", "It is, in fact, when danger indices are reclassified that clearer patterns are revealed and multiple hazards can be compared.", "Fig. 4 shows the reclassified reanalysis layers corresponding to 17th June 2017, the day in which the major fire event in Pedrógão Grande, Portugal was ignited.", "On that day, high to extreme fire weather and heat stress were observed in various locations in Europe, e.g. Spain (El Mundo, 2017; The Guardian (2017b)), Portugal (BBC, 2017), France (Le Dauphine, 2017), Italy (CN24, 2017).", "A heatwave combined with numerous wildfires spreading across Europe caused evacuation of thousands of people (Copernicus EMS, 2017a, 2017b).", "The reanalysis of UTCI shows that the entire Portugal was under extreme heat stress, without clear spatial variation of danger.", "Fire danger, instead, was higher in the north and south of the country.", "In this particular case, combining the two danger maps means borrowing the spatial gradient of fire danger and applying it to both hazards, which would allow more targeted preparedness actions and prioritisation of interventions.", "Fig. 5 shows what the map of forecasted hotspots would have looked like for 17th June 2017, taking ‘very high danger’ as the hazard level of reference for both indices.", "Numerous areas appear under combined danger: Spain, Portugal and France (1–3 days horizon), then moving also to Italy, Greece and the Balkans.", "The spatial distribution of the events is confirmed by the FRP detected from satellites as well as by news articles (Table 1).", "Some of the information in Fig. 5 could be considered a little surprising because it shows that wildfires and heat stress are not very spatially and temporally correlated for this case.", "Unusually high temperatures are certainly the common denominator but, the (high) relative humidity is likely to also have an important role in mitigating wildfire danger and exacerbating heat stress.", "Conversely, low relative humidity, drying out vegetation, can increase wildfire risk but mitigate heat stress.", "The key to predict combined danger is, therefore, being able to detect dangerous trade-offs in time and space.", "The probability of occurrence of both hazards is explored by reclassifying and combining all the ensemble members of the probabilistic forecasts, as we have done for the single member of the deterministic forecasts.", "The percentage of the members reporting combined hazard is a measure of the confidence in the multi-hazard forecasts.", "Fig. 6 shows the 10-day ensemble forecasts for the combined danger that could have been issued on 17th June 2017.", "The highest probability of occurrence of both hazards was expected to move from West Europe (Spain, Portugal) to Central-East Europe (France, Italy, Greece, Croatia) which accurately reflects what was subsequently reported by the Copernicus Emergency Management Service (2017a, 2017b) and media (Table 1).", "With regard to the Iberian peninsula, where numerous media reported extreme heat (BBC, 2017; El País, 2017; Phys.org, 2017), Fig. 7 shows the areas affected by the Pedrógão Grande/Castanheira de Pera (Portugal) fires had a forecasted probability of occurrence between 6% and 40%.", "In Spain, instead, the probability raises to approximately 50% in Huelva and 74% in Serra Calderona.", "Lastly, we use the monthly summary plot, in Fig. 8, to track the expansion of areas characterised by very high combined danger during June 2017 in Portugal.", "On the x-axis is the forecasted day and on the y-axis the issuing date of each forecast.", "Cells are color-coded based on the percentage of total area classified as hotspot.", "The bottom-left cell corresponds to the first day of the forecast issued on 1st June 2017.", "The forecasts for day 2 to day 10 are on the same row.", "The forecasts issued on the following day are one row above and so forth.", "The purpose of this plot is two-fold: on the one hand it provides an immediate estimate of the area under very high danger conditions and, on the other hand, it shows how the extent of this area evolves over time.", "The area of interest can be as small as a single pixel in the high resolution forecast or as large as a province/region/country.", "However, as the total area increases, the plot on its own becomes less informative because it does not provide the spatial distribution of the area at risk.", "When assessing large areas, therefore, the plot should be used in conjunction with maps of hotspots.", "Forecasts issued on 7th June started showing that >50% of the country would have been affected by very high combined danger starting from 16th June.", "This information would have warned authorities 10 days before the major fire event in Pedrógão Grande, and give them time for pre-emergency planning and allocation of resources.", "In the future, collaborations with first responders and decision makers could clarify how these new layers could be best used operationally.", "Ideally, such a collaboration could lead to the co-production of forecasting and early warning products.", "This can be achieved by creating a collaborative eco-system in which all parties interested in designing new data products will build, test and deploy them through sequential iterations and regular feedbacks.", "Assessing the uncertainty and robustness of the newly generated combined danger maps is particularly complex because they are forecasted maps of hazard.", "The quantification of the risk to the population and environment would require information on exposure and vulnerability.", "This means any actual occurrence of fire requires the appropriate conditions (expressed by the fire danger index), an ignition source as well as populated areas, presence of fuel, lack of access to transport/means of self-evacuation, no insurance, amongst others.", "Heat stress is conditional to people being outdoors or not using air conditioning systems indoors as well as the environmental conditions expressed by the index.", "Nevertheless, uncertainty for a past event can be assessed in terms of spatial accuracy of hotspot location and temporal span in which a dangerous signal can be predicted.", "A preliminary assessment of the utility of these new layers was made in collaboration with various stakeholders (see Table 2 for the full list), amongst them is the Instituto Português do Mar e da Atmosfera (IPMA) who collaborated with decision makers in the 2017 event in Pedrógão Grande.", "Positive feedback was recorded, with the Portuguese Institute stating: “we liked the new layers, they identify well areas at risk, [...] they would have been useful during the event” [IPMA, Lourdes Bugalho, personal communication].", "This statement highlights the importance, for stakeholder, to correctly identify hotspots of danger.", "It is also important that forecasts provide enough notice to allow preparedness and, in case of emergency, efficient allocation of resources.", "In January 2019, a workshop for the ARISTOTLE-2 project (http://aristotle.ingv.it/tiki-index.php) was held at ECMWF headquarters and focused on delivering effective “consensus advice” to the Emergency Response Coordination Centre in Europe.", "One of the main feedbacks from the workshop was about the timing of the issued forecasts.", "Fire emergency responders from IPMA and CIMA Research Foundation agreed they would benefit from reliable forecasts 3 days ahead but 5-day notice would be ideal (see list of feedbacks in Table 2, extract from the minutes of the workshop).", "These feedbacks are important to set baseline product expectations that will be discussed in the next section.", "It must be noted that, in the previous section, maps are generated assuming that a significant combined hazard occurs where/when both indices exceed the very high danger threshold.", "This is, however, a simplification due to the need to generate static maps for this publication.", "In the context of a MH-EWS, an interactive map would be, by far, more suitable.", "A basic example of web application using interactive maps was developed for testing purposes and made publicly available at the following URL: http://rpubs.com/clavitolo/fire_heat (see also screenshot in Fig. 9).", "This application was particularly useful when requesting IPMA's feedback as officers were able to zoom into particular regions and check against their event records.", "Ideally, a MH-EWS platform is envisaged to provide user-controlled tools, like radio buttons and sliders, for the interactive maps to allow users to switch between and explore different hazard combinations and days in the forecast horizon.", "Another option would be to generate animations of all the possible combinations so that users have an overview of the sensitivity of the tool to the tuning of the combined hazard thresholds.", "Wildfires and heat stress are commonly occurring natural hazards that lead to loss of lives and livelihoods and which are increasingly impacting upon our society.", "They are routinely monitored as individual hazards and rely on forecasters and civil protection authorities to detect any potential spatio-temporal overlaps.", "When simultaneously affecting the same region, wildfires and heat stress can generate substantial impacts “as the impacts of one hazardous event are often exacerbated by interaction with another” (Marzocchi et al. 2009).", "There is therefore the need for spatio-temporal information layers that are tailored to identify the concurrence of multiple hazards, especially in the context of Early Warning Systems.", "In this work we demonstrate a methodology to generate various multi-hazard information layers based on the reanalysis, high resolution and probabilistic medium-range forecasts of two hazard indices: FWI representing fire danger and UTCI representing heat stress.", "The procedure is, to some extent, hazard-agnostic in the sense that any hazard index could potentially be integrated.", "The only prerequisite is that the indices should be binnable into the same categories of danger.", "This categorization is a very important step as it ensures that the different danger types become comparable to each other.", "We suggest defining categories of danger based on climatological percentiles (in the case study we used 37 years of reanalysis data) and generating maps of danger thresholds that could inform users which danger index values to consider unusual for a particular area and time of the year.", "Based on these thresholds, calculated at each pixel, we are able to assess whether the danger measured or forecasted on a given date is low, moderate, high, or extreme.", "This binning can be applied to both reanalysis and forecast data.", "Based on feedbacks from stakeholders, it seems the definition of danger thresholds is a rather controversial matter.", "Some stakeholders value the climatological information and prefer a statistical approach (consistent with what is presented in this work), others prefer to use a fixed set of thresholds at the pan-European scale and rely on forecasters experience and expertise to interpret them in the context of the local fire regime (consistent with the EFFIS approach).", "Motivations to opt for one approach or the other are deeply ingrained in the stakeholder's best practise, therefore the consensus is to have access to both information.", "Problems may arise if the two sources provide diverging information.", "How to consistently interpret the two approaches remains an open question.", "The second layer we presented is a map of hotspots of combined danger.", "This is obtained by spatial overlap of binned danger maps that have been converted into binary maps based on whether they exceeded a given threshold or not.", "A map of hotspots of combined danger can be used to analyse the extent of a past event and its spatial correlation with other observed variables (working in retrospect, this makes use of reanalysis data) or to make a prediction for the future (using forecast data).", "The confidence in the identification of combined danger in a given area is provided by a third layer: a map of probability of occurrence that is generated by spatial overlap of fire danger and heat stress ensemble forecasts.", "Lastly, an overview of the evolution of the forecasts is provided by the monthly forecast summary plot.", "Although the above mentioned layers have been designed with users' needs in mind, the optimal use and visualisation of multi-hazard layers would require significant co-production: through collaboration with stakeholders, developers can identify strengths and weaknesses and improve the products by incorporating feedback.", "Amongst the feedbacks received during the January workshop and with regards to fire danger forecast, IPMA stated that “the monthly forecast summary was very informative to track the spatio-temporal evolution of the forecasts and can also be very useful as managing tool” (see Table 2).", "Despite fire danger and heat stress being highly correlated (temperature and wind speed are common precursors/predictors), we found there is scope combining them for the benefit of decision makers.", "An increase in relative humidity can play a mitigating role for fire danger, while has an exacerbating effect on heat stress (Climate Communication - Science and Outreach, 2012).", "In the future, therefore, it could be interesting to investigate humid and dry heat waves separately and their occurrence in relation to droughts and wildfires.", "This would also imply the definition of a threshold on the value of relative humidity, which would not be a trivial task.", "With regards to June 2017 in Europe, the signal of an important combined danger was detectable 10 days before the major wildfire in Pedrógão Grande (Portugal) started, demonstrating the usefulness to local responders (who need 3–5 days notice to efficiently allocate resources) while being relevant on a pan-European scale.", "Collaboration with stakeholders was extremely useful to set their expectations and assess our data products accordingly.", "Collaborations are currently ongoing and we plan to have more workshops and opportunities to exchange experiences in the future.", "This study is limited to the combination of two hazards which are expected to occur simultaneously.", "Further work is needed to understand how to best use this type of information in the context of Multi-Hazard Early Warning Systems and support first responders and decision makers.", "In the future, the dynamic update of this multi-hazard zonal classification combined with the integration of other concurrent or lagged hazards could permit the identification of the most impacted and/or multi-hazard prone areas and shape environmental policies in Europe.", "It is also important to point out that this study focuses on a multi-hazard framework, while decision makers and first responders have to operate within a multi-risk setting.", "The latter would require an extension of the analysis including cross-correlation of risk factors and components (such as population density), which would lead to an improvement of the system.", "This is particularly important as projected changes in the climate will lead to a significant increase in the variability of temporal and spatial patterns of extremes which are linked to rising temperatures (such as the two hazards presented in this study) as well as a potential increase in the magnitude and frequency of these hazards (Forzieri et al., 2016).", "An increasing use of multi-hazard and multi-risk frameworks is an essential component of an efficient and effective adaptation strategy to a changing climate.", "The scientific advances of this study aim to support operational programs such as Copernicus.", "Copernicus is the European Programme for the establishment of a European capacity for Earth Observations with a strong focus on services to exploit satellite and in-situ data.", "It is currently divided into six separate services (Atmosphere, Land, Climate, Marine, Emergency, Security) which span multiple hazards.", "For example, fire hazard is part of the atmosphere service as fires impact air quality, part of the land service as it requires information about vegetation cover, integral to the climate service as it is driven by reanalysis and seasonal forecasts, and an important component of the emergency service in terms of forecasting.", "A similar argument can be made for the heat stress used in this study.", "We demonstrated that not only are the different services interconnected through hazards but also that the individual hazards are correlated and connected.", "Thus a future Copernicus service needs to be structured not only from the point of view of the earth observation provider but also should be strongly led from the emergency responder perspective and thus have a stronger focus on cross-cutting, user perspective elements.", "When multiple hazards affect the same region simultaneously, they can generate substantial impacts, as impacts of one hazardous event are often exacerbated by interaction with another.", "This suggests the need for spatio-temporal information layers that identify hotspots of combined danger.", "We suggest a methodology to generate such layers based on the reanalysis, high resolution and probabilistic medium-range forecasts of two hazard indices: FWI representing fire danger and UTCI representing heat stress.", "The maps of hotspots created for June 2017 highlighted that despite fire danger and heat stress being highly correlated, an increase in relative humidity can play a mitigating role for fire danger, while having an exacerbating effect on heat stress.", "Therefore the key to identify areas prone to combined danger is to understand these dangerous trade-offs.", "Probabilistic medium-range forecasts are used to quantify the uncertainties related to the spatial and temporal occurrence of these events.", "We also introduced the monthly forecast summary plot to track the temporal evolution of the forecast and ease comparison with past forecasts.", "The summary plot for June 2017 over Portugal showed the signal of an important combined danger in Portugal was detectable 10 days before the major wildfire in Pedrógão Grande (Portugal) started.", "According to stakeholders, 5 day notice or more would allow sufficient time for planning and allocating resources.", "This work can be used within a Multi-Hazard Early Warning System to provide improved early warning information on concurrent natural hazards, which will support operational programs such as Copernicus." ]	[ "Overestimating the correlation between heat stress and forest fires danger could lead to underestimating potential impact.", "A simple methodology to map areas prone to concurrent hazards, using layers designed for Multi-Hazard Early Warning Systems.", "These layers could be particularly valuable in disaster risk reduction and emergency response management." ]
EU methodology for critical raw materials assessment: Policy needs and proposed solutions for incremental improvements	Raw materials form the basis of Europe's economy to ensure jobs and competitiveness, and they are essential for maintaining and improving quality of life. Although all raw materials are important, some of them are of more concern than others, thus the list of critical raw materials (CRMs) for the EU, and the underlying European Commission (EC) criticality assessment methodology, are key instruments in the context of the EU raw materials policy. For the next update of the CRMs list in 2017, the EC is considering to apply the overall methodology already used in 2011 and 2014, but with some modifications. Keeping the same methodological approach is a deliberate choice in order to prioritise the comparability with the previous two exercises, effectively monitor trends, and maintain the highest possible policy relevance. As the EC's in-house science service, the Directorate General Joint Research Centre (DG JRC) identified aspects of the EU criticality methodology that could be adapted to better address the needs and expectations of the resulting CRMs list to identify and monitor critical raw materials in the EU. The goal of this paper is to discuss the specific elements of the EC criticality methodology that were adapted by DG JRC, highlight their novelty and/or potential outcomes, and discuss them in the context of criticality assessment methodologies available internationally.	[ "ECON", "ENVI", "SOCI" ]	[ "Economic importance", "Import reliance", "Recycling", "Resource criticality assessment", "Supply chain", "Supply risk", "Trade barriers" ]	[ "2002", "2308", "3308", "3312" ]	[ "Raw materials form the basis of Europe's economy to ensure jobs and competitiveness, and they are essential for maintaining and improving our quality of life.", "Securing reliable, sustainable, and undistorted access of raw materials and their circular use in the economy is, therefore, of growing concern within the EU (EC, 2014, 2011; Vidal-Legaz et al., 2016) and globally (Coulomb et al., 2015).", "Recent years have seen a tremendous increase in the amount of materials extracted and used (Krausmann et al., 2009) together with a significant growth in the number of materials used in single products (product complexity) (Greenfield and Graedel, 2013).", "Global economic growth coupled with technological change (e.g., low-carbon energy and transportation systems, modern defence and communication systems) will increase the demand for many raw materials in the future (Blagoeva et al., 2016; Pavel and Tzimas, 2016).", "“Criticality” combines a comparatively high economic importance with a comparatively high risk of supply disruption (Buijs et al., 2012).", "In 2008 the U.S. National Research Council proposed a framework for evaluating material “criticality” based on a metal's supply risk and the impact of a supply restriction (NRC, 2008).", "Since that time, a number of organizations worldwide have built upon that framework in various ways (BGS, 2012; DOD, 2013; EC, 2014; Graedel et al., 2015; IW Consult, 2011; Morley and Eatherley, 2008; NSTC, 2016; Skirrow et al., 2013).", "Even though all raw materials are important (EC, 2010, 2012, 2015), some resources are obviously of more concern than others.", "The list of CRMs for the EU (EC, 2014, 2011) and the underlying criticality methodology (Chapman et al., 2013; EC, 2010) are therefore key instruments in the context of the EU raw materials policy.", "Such a list is a precise commitment of the Raw Material Initiative (RMI) (COM, 2008; EC, 2008) and subsequent updates.", "The EU criticality methodology was developed between April 2009 and June 2010 with the support of the European Commission's (EC) Ad-Hoc Working Group on Defining Critical Raw Materials (AHWG-CRM) within the RMI in close cooperation with EU Member States (MS) and stakeholders (EC, 2010).", "The EC criticality methodology has already been used twice; to create a list of 14 CRMs for the EU in 2011 (EC, 2011) and an updated list of 20 CRMs in 2014 (EC, 2014).", "Given the intense and active dialogue with multiple stakeholders, the use of best available data reflecting the current situation and recent past (non-speculative and non-forward looking approach), and considering that fully transparent datasets and calculations were made available to a large group of experts, the EC criticality methodology is generally well accepted in the EU, as well as considered reliable and robust.", "After the two releases of the list and considering several policy documents that make explicit reference to CRMs (EC, 2015, 2012, 2008), it can certainly be stated that the EC criticality methodology is a well consolidated and reliable tool, which represents a cornerstone of the raw materials policy in the EU.", "In view of the next update of the CRMs list (every three years according to the RMI), the EC is considering to apply again the same methodology.", "This choice of continuity is synonymous with giving priority to comparability with the previous two exercises, which is in turn correlated to the need of effectively monitoring trends and maintaining the highest possible policy relevance.", "Nevertheless, some targeted and incremental improvements of the existing EU criticality methodology are required, taking into account the most recent methodological developments in the international arena (BGS, 2015; Graedel et al., 2015; NSTC, 2016; Roelich et al., 2014), evolving raw materials markets at international scale, and considering explicit requests from the European industry and changing policy priorities and needs, e.g., on trade (OECD, 2014).", "A valuable support also came from recent projects funded by the EU under different schemes, which tackled specific aspects of criticality (e.g., CRM_InnoNet, 2015; ERECON, 2014; EURARE, 2017) and/or contributed to generate European data on flows and stocks of CRMs (BIO by Deloitte, 2015).", "As the EC in-house science service, the Directorate General (DG) Joint Research Centre (JRC) provided scientific advice to DG GROWTH in order to assess the current methodology and identify parameters that could be adjusted to better address the needs and expectations toward the methodology of capturing issues of raw materials criticality in the EU.", "This work was conducted in close consultation with the ad hoc working group on CRMs, who participated in regular discussions with DG GROWTH and other EC services and provided informed expert feedback.", "The analysis and subsequent revisions started from the assumption that the methodology used for the 2011 and 2014 CRMs lists proved to be reliable and robust and, therefore, the JRC mandate was focused on fine-tuning and/or targeted incremental methodological improvements.", "The goal of this paper is to present key new or modified elements of the EU criticality methodology, to highlight their novelties and/or potential outcomes, and to discuss them in the context of criticality assessment methodologies available internationally.", "A comprehensive presentation of the revised EC methodology is not a goal of the present paper, but will be presented in a future EC publication or communication in view of the third revised list expected in 2017.", "CRMs are both of high economic importance to the EU and vulnerable to supply disruption.", "Vulnerable to supply disruption means that their supply is associated with a high risk of not being adequate to meet EU industry demand.", "High economic importance means that the raw material is of fundamental importance to industry sectors that create added value and jobs, which could be lost in case of inadequate supply and if adequate substitutes cannot be found.", "Bearing the above concepts in mind, criticality has two dimensions in the EC methodology: (1) Supply Risk (SR) and (2) Economic Importance (EI).", "A raw material is defined as being critical if both dimensions overcome a given threshold (EC, 2014).", "The SR indicator in the EU criticality assessment (EC, 2014, 2011) is based on the concentration of primary supply from countries and their level of governance.", "Production of secondary raw materials (recycling) and substitution are considered as risk-reducing filters.", "In this formula, SR stands for supply risk; HHI is the Herfindahl Hirschman Index (used as a proxy for country concentration); WGI is the scaled World Governance Index (used as a proxy for country governance); EOLRIR is the End-of-Life Recycling Input Rate; and SI is the Substitution Index (EC, 2014).", "The importance of a raw material to the economy of the Union is assessed by the indicator “Economic Importance (EI)”.", "This indicator relates to the potential consequences in the event of an inadequate supply of the raw material.", "In previous criticality assessments (EC, 2014, 2011), EI was evaluated by accounting for the fraction of each material associated with industrial megasectors at EU level and their gross value added (GVA).", "In the above formula As is the share of demand of a raw material in a megasector and Qs is the megasector's Gross Value Added (EC, 2014).", "The EC criticality methodology considers both abiotic and biotic raw materials.", "The 2011 assessment considered 41 non-energy, non-agricultural raw materials (EC, 2011), while the 2014 assessment considered 54 candidate materials (EC, 2014).", "As a precise policy mandate, in order to maximise comparability with the 2011 and 2014 CRMs lists, the current methodology is to be retained, except for specific aspects for which there were policy and/or stakeholder needs on the one hand to introduce alterations, or strong scientific reasons for refinement of the methodology on the other.", "These will be discussed in the next sections.", "The EC criticality methodology, since the publication of the first list of CRMs in 2011, has responded to the needs of governments and industry to better monitor the raw materials situation and inform decision makers about how the security of supply of raw materials can be achieved through diversification of supply, i.e., from different geographical sources but also from primary sources, recycling and substitution and to prioritise needs and actions.", "For example, at the EU level the list serves as a supporting element in negotiating trade agreements and challenging trade distortion measures, and in programming the research and innovation funding for technological solutions for sustainable production of CRMs or their substitution under the Horizon 2020.", "We note that criticality is a screening tool to highlight issues of concern which can subsequently be followed up with more detailed studies and assessments (NSTC, 2016).", "Again, the results of criticality assessments should be considered a call for attention, not a source of “panic”, or even trigger exaggerated or unbalanced countermeasures (e.g., systematic elimination of CRMs in given categories of products).", "The above clarifications hold true even more in the EU policy context, where the EC criticality methodology has a very wide scope in regard to the large number of candidate critical raw materials included (presently around 80) and with its ambitious objective of capturing the economic importance of raw materials using an EU economy-wide approach.", "In such a context, it is very unlikely that all aspects that could influence criticality can be included in a screening methodology, while keeping the calculation equations short, simple, and objective.", "Also based on an intense discussion with stakeholders, priority was given to the following policy needs, where an improvement or update of the current methodology appeared to be highly desired.", "Under the supply risk dimension, four policy needs were prioritized: (1) incorporate trade barriers and agreements, (2) adopt a more systematic supply chain approach, (3) take into account import dependency and a more accurate picture of the actual supply to the EU and (4) maintain a prominent role for recycling and improve the quality and representativeness of data for the EU.", "Trade barriers are increasingly distorting international raw material markets (OECD, 2014).", "Several cases such as China's export ban on Rare Earth Elements (REEs) showed that impact of trade barriers on the security of supply can be substantial at times.", "However, current criticality methodologies do not explicitly capture trade distortion risk and this is addressed in the revised EC methodology as described in later sections of this paper.", "A systematic supply chain approach was considered important, because of possible shifting of criticality along the supply chain (e.g., a supply chain bottleneck present at the smelter stage instead of the mining stage).", "This issue was already identified in the previous EC study (EC, 2014) and highlighted as a priority by stakeholders, and it is also incorporated into other criticality assessments (Graedel et al., 2015).", "Moreover, the current EC methodology (Chapman et al., 2013) bases the supply risk calculation on the global suppliers mix only and a need was expressed during stakeholder discussions to use a more accurate picture of the actual suppliers to the EU, and introduce import dependency in the calculations.", "Finally, in line with the EU Circular Economy action plan (EC, 2015), a clear mandate was given to the JRC to assess the prominent role of recycling as a risk reducing factor in the supply risk calculation and, moreover, identify data more representative of the EU recycling situation taking into account recently published material stocks and flows data (BIO by Deloitte, 2015).", "For the Economic Importance (EI) dimension, two policy needs were prioritized: (1) a more detailed and transparent allocation of raw materials uses to their corresponding NACE (Statistical Classification of Economic Activities in the European Community) sectors, and (2) use of a raw materials-specific substitution index in the calculation of EI to allow for a reduction in the potential consequences to the European economy due to inadequate raw materials supply.", "The fact that the EC methodology is based on an economy-wide approach creates even higher methodological difficulties, as opposed to assessments where some selected technologies and the related raw materials are targeted.", "In fact, it is virtually impossible to measure the importance of all applications in which the vast majority of all possible raw materials are used.", "Moreover, the choice of measuring the EI downstream practically obliges to adopt substantial simplifications.", "Finally, some stakeholders specifically suggested to introduce substitution in the calculation of EI.", "The following elements summarise the framework and scope of the revision of the EC methodology for criticality assessment:", "Ensure the highest possible level of comparability with the 2011 and 2014 CRM lists;", "Intense and active dialogue with stakeholders from an early-stage in the revision;", "Non-forward looking approach in the assessment based on available data, i.e., criticality is seen as a “snapshot in time” of the current (or past) raw materials situation;", "Use of best quality data reflecting the average of data available for the last 5 years.", "A particularly important aspect in the revision of the EC criticality methodology is a clear separation between backward-looking and forward-looking approaches.", "The revised EC criticality assessment methodology can be considered a snapshot of the current situation, based on the recent past.", "Potential further analyses of future-orientated options, or forecasts, are not integrated.", "The sharp distinction between backward-looking and forward-looking approaches can also be presented in terms of the separation between criticality and resilience (Dewulf et al., 2016), where resilience is focused on the future response of the systems in the context of inadequate supply of a given material, the consequences, and options to reduce these.", "Export restrictions on raw materials have become more frequent in recent years.", "They cause higher prices and volatile market conditions and can affect the security of supply.", "Although there are many ways to restrict raw material exports, the main restrictive measures are export quotas, export taxes, and licensing requirements.", "The impact of export restrictions on raw materials is stronger if combined with high supplier country concentration.", "Fig. 1 presents the number of export restriction measures for raw materials introduced between 1961 and 2012 and that were still in place in 2012.", "Export restrictions reported in the OECD Inventory of Restrictions on Trade in Raw Materials for minerals at HS 6 level1 are presented (OECD, 2014).", "This consists of the different types of restrictions such as, e.g., export taxes, export quotas, export prohibitions, and license requirements.", "While some of these export restrictions have been in place for decades, Fig. 1 highlights that more than 50% of the restrictions that were active in 2012 were introduced only after 2009, and almost 25% were introduced in 2012.", "Only 12 out of the 72 countries for which the OECD reported export restriction measures did not introduce any measures between 2009 and 2012, while the other 60 countries introduced at least one restriction during this time.", "Fig. 2 presents the proportion of global supply subject to export restrictions for a selection of raw materials.", "This selection includes the 20 critical raw materials for the EU (OECD, 2014), and other materials essential to the EU economy.", "In this figure, production is considered to be subject to export restrictions if any of the main restrictive measures, i.e., export quotas, export taxes, or licensing requirements, were applied in the producing countries between 2009 and 2012.", "One case that received a lot of media attention was the Chinese export restriction on rare earths, tungsten, and molybdenum, for which the EU, USA, and Japan brought a case to the World Trade Organization's (WTO) Dispute Settlement Body in 2012.", "Following the WTO's ruling, China dropped its export restrictions in January 2015.", "Alongside the governance quality in the supplying countries, measured by the World Governance Index (WGI), the restrictions imposed worldwide on exports of certain raw materials (RMs) can increase the supply risk.", "On the other hand, concluding trade agreements with the supplying countries can result in more secure supply.", "However, previous criticality assessments (EC, 2014, 2011) did not consider aspects of export restrictions and trade agreements in the evaluation of supply risk.", "To our knowledge, export restrictions have not yet been integrated into criticality studies published elsewhere (BGS, 2012; DOD, 2013; EC, 2014, 2011; Graedel et al., 2015; IW Consult, 2011; Morley and Eatherley, 2008; NSTC, 2016; Skirrow et al., 2013).", "An assessment of the WGI and other country-level indicators suggested that, although WGI is the most robust indicator to capture the level of governance in a country in the context of the criticality assessment, such an index does not capture risks due to export restrictions or the mitigating effects on risk as a result of international trade agreements.", "Therefore, a specific adjustment for WGI was proposed as an additional trade-related variable.", "Modifications were tested for some selected raw materials and subsequently discussed with a representative group of experts and stakeholders (AHWG-CRMs) and then adopted.", "In the revised methodology, three types of restrictions imposed, i.e., export taxes, physical quotas, and export prohibitions are considered if these are applied at least once in the reference period 2010–2015.", "In respect to export taxes, there are three possible cases:", "1) In case a country does not apply export taxes, there is no impact and, hence, no adjustment is needed as the risk is comparable to the EU internal market, for which a 20% reduction of the HHIWGI is applied.", "In case a country applies an export tax, there is an increased supply risk which is either:", "2) Mitigated by a bilateral agreement with the EU (if the raw material concerned is covered by the provision on export duty in the agreement) and the supply risk returns to the level as if no export duty was applied;", "3) Not mitigated in absence of a trade agreement and the supply risk increases.", "The case of export quotas remains a separate element of supply risk, with a specific approach for the calculation of the trade adjustment, as a Free Trade Agreement cannot guarantee their removal.", "Export prohibitions are considered an extreme case for quotas, i.e., quota is zero.", "A worked example with preliminary calculations for tungsten is presented in Table 1.", "The 2014 criticality report (EC, 2014) addressed the comments of the ad-hoc working group of CRMs, which suggested 'examining not only the mining stage but also the smelting and/or refining stages of the supply of raw materials'.", "In the report, data are provided on the spatial distribution of bauxite mines and aluminium refineries, manganese (mine vs. refinery), nickel (mine vs. refinery), and zinc (mine vs. smelter).", "The report concluded that assessing the supply risk at different stages of the value chain, i.e., mining and refining, can lead to different results based on changes in the concentration of production.", "Based on the above remarks, as a proposed solution, the JRC recommended to systematically adopt a supply chain approach by means of a bottleneck screening in order to assess the likelihood of more than one stage being critical (i.e., mining or refining).", "In other words, for the calculation of supply risk, the weakest point in the supply chain is considered, i.e., the stage with the highest supply concentration.", "A similar approach is also applied in other criticality studies (Graedel et al., 2015).", "Moreover, this approach also reinforced the conclusions on WGI in respect to alternative indexes, as WGI is applicable to different life-cycle stages (e.g., mining and refining), whereas other indicators (e.g., the Resource Governance Index (RGI) or Policy Potential Index (PPI) may focus only on a single life-cycle stage (e.g., mining) or a specific type of material (e.g., metals and metalloids).", "In the previous criticality assessments (EC, 2014, 2011), the supply risk was calculated based solely on the mix of global supplier countries.", "However, this does not capture the fact that Europe may depend on a combination of supplier countries different from the global supply mix.", "In addition, the share of imports to the EU is not fully captured, whereas it is fundamental to make a distinction among RMs that are essentially imported and RMs that are essentially sourced domestically.", "In the revised methodology, in order to calculate a more representative measure of the risk for the EU, the global suppliers mix is used in combination with the actual supply to the EU, i.e., the mix of domestic production plus import, which reflects the actual sourcing of the supply to the EU (EU-sourcing).", "An example for tungsten is presented in Fig. 3.", "However, considering data availability and quality for a wide range of materials covered in the EU criticality assessment, a final decision was taken to use both the global supply and actual EU sourcing in combination.", "In fact, although it is not a true measure of the risk specific to the EU, the risk calculated using global supply is more reliable in terms of data availability and quality (data are reported by various sources, e.g., BGS, 2014; USGS, 2015).", "Moreover, the mix of global suppliers is sometimes more stable in time, whereas the exporters to the EU might change more rapidly and trade data are known to have significant data quality issues, as well as need to be derived using a combination of different data sources, not always fully compatible.", "For instance, the EU supply mix can be calculated from trade statistics (EUROSTAT, 2016a, 2016b; United Nations, 2014) and is available partly from other sources (BIO by Deloitte, 2015).", "Yet, sometimes an exporter country is not the original source for the material shipped to EU28, but just an importer and re-exporter.", "Therefore, in the revised methodology a balanced approach between a more representative measure of the risk, but of lower quality (based on EU sourcing), and a more reliable measure, but less representative (based on global supply), is used in the evaluation of supply risk.", "The driver to balance the two measures of the supply risk, i.e., the one based on global supply and the one based on the actual sourcing, is the Import Reliance (IR), which is the ratio of net import to apparent consumption.", "In the revised methodology, when IR is 100%, i.e. import equals apparent consumption, the risk is the average of the two measures, i.e., 50% based on global supply and 50% based on actual EU sourcing.", "In the few cases where EU is independent from import, i.e. IR is 0%, the global supply mix is disregarded and the risk is entirely calculated based on the actual sourcing.", "Several reviews about methods to evaluate raw material supply risk are available (Achzet and Helbig, 2013; Ciacci et al., 2015; Dewulf et al., 2016; Erdmann and Graedel, 2011).", "Beyond the EC criticality methodology, in three out of 15 studies available on criticality, recycling is used in the calculation of supply risk (Erdmann and Graedel, 2011; Graedel et al., 2015; NRC, 2008), but with a less prominent role in comparison to the EC methodology (EC, 2014, 2011) both in terms of visibility and impact on the results.", "In the previous criticality assessments (EC, 2014, 2011), recycling is accounted for as ‘the ratio of recycling from old scrap to European supply of raw material’ and referred to as ‘end of life recycling input rate (EOL-RIR)’.", "EOL-RIR is used as a mitigation factor that can lower the risk of supply, which depends solely on primary supply (HHI and WGI).", "The underlying rationale, already adopted in the previous EC criticality studies, is that the portion of the EU supply of raw materials that is generated via recycling of end-of-life products is assumed to be more secure than other sources of supply (e.g., primary supply from mining/harvesting).", "Against this background, even though recycling is not riskless in terms of supply, for the purpose of the assessment it is considered that the recycling parameter EOL-RIR identifies a flow of supply with significantly lower risk, which can in turn be subtracted from the overall supply to the EU when calculating the risk.", "In the revised methodology, the prominent role of recycling as a risk-reducing filter of supply risk remains unchanged.", "Efforts are focused on using European data readily available for 28 raw materials assessed in the Raw Material System Analysis (MSA) study commissioned by DG GROW (BIO by Deloitte, 2015) as well as data from the UNEP report ‘Recycling Rates of Metals’ (Graedel et al., 2011; UNEP, 2011).", "Guidance on how to update and improve the quality of EOL-RIR is provided via the following options (to be considered in a cascading fashion):", "Use the Raw Material System Analysis (MSA) data (BIO by Deloitte, 2015) when available.", "Use of data from UNEP's report ‘recycling rate of metals’ (Graedel et al., 2011; UNEP, 2011), when data from the Raw Material System Analysis (MSA) are not available.", "Use of recycling rates from the previous EC criticality assessments (EC, 2014, 2011), or data available from other sources as sectorial reports and expert judgement when MSA and UNEP data are not available.", "Fig. 4 illustrates the system boundaries and flows for the calculation of the EOL-RIR when using MSA data.", "The first part of the figure represents the life cycle stages of a raw material in the rest of the world (ROW), while the life cycle stages of a raw material in Europe are represented by the brown boxes.", "The system boundary is represented in pink dashes.", "Flows used for the calculation of the EOL-RIR are represented in green (primary material), yellow (processed material), and purple (secondary material).", "EOL-RIR is to be calculated by applying the following formula:", "In this formula, SR stands for supply risk; HHI is the Herfindahl Hirschman Index (used as a proxy for country concentration); WGI is the scaled World Governance Index (used as a proxy for country governance); t equals the trade adjustment (of WGI); IR is the Import Reliance; EOLRIR is the End-of-Life Recycling Input Rate; and SISR = Substitution Index related to supply risk.", "The importance of a raw material to the economy of the Union is assessed by the indicator Economic Importance (EI).", "This indicator relates to the potential consequences in the event of an inadequate supply of the raw material.", "In previous criticality assessments (EC, 2014, 2011), EI was evaluated by accounting for the fraction of each material associated with industrial megasectors at EU level and their gross value added (GVA).", "However, megasectors combine several 3- and 4-digit NACE sectors with each other and therefore represent GVA at a high level of aggregation.", "In order to link raw materials to the corresponding manufacturing sectors at higher levels of sectorial resolution, the JRC examined the classification of product groups, economic activities, and NACE sectors in which raw materials are generally used.", "The resulting revised approach allows for a more detailed allocation of raw material uses to the corresponding NACE sectors.", "The allocation of uses could, e.g., be done using the PRODCOM product groups (EUROSTAT, 2016b) and the 5-/6-digit CPA classes corresponding to each type of use.", "In the cases in which the identification of a CPA category is not possible, the shares could be allocated directly to the corresponding 4-, 3- or 2-digit NACE sectors.", "At NACE 2-digit level, statistical identification of uses turned out to be easier.", "Allocation of the identified end uses to the NACE 2-digit level sectors is facilitated by the Eurostat's statistical correspondence between CPA, NACE 3-/4-digit and NACE 2-digit.", "In the previous criticality assessments (EC, 2014, 2011), substitution was only addressed as a filter to decrease the supply risk (SR).", "Expert judgment was used to determine the substitution/substitutability indexes.", "However, substitution can also alter the potential consequences of a supply shortage to the European economy and should therefore also be considered in the economic importance (EI) component.", "Substitution of raw materials is addressed in the majority of criticality studies (e.g., Achzet et al., 2011; AEA Technology, 2010; BGS, 2015; Duclos et al., 2010; Graedel et al., 2015; Morley and Eatherley, 2008; Rosenau-Tornow et al., 2009; USDOE, 2011, 2010), in a qualitative or semi-quantitative manner.", "Expert elicitation is indispensable for such qualitative estimations.", "A slightly more detailed approach is adopted in the Yale methodology (Graedel et al., 2015; Harper et al., 2015).", "In the revised EC methodology, substitution is considered to reduce the potential consequences in the case of a supply disturbance.", "Substitution is to be incorporated, therefore, into the economic importance (EI) dimension.", "Nevertheless, given that the availability of substitutes could also mitigate the risk of supply disruptions (SR), as it might decrease demand for a given raw material, it was recommended to also consider substitution in the estimation of SR.", "In summary, two different substitution factors are used, one in the EI and one in the SR.", "Since the scope of the EC assessment focuses on the current situation, only proven substitutes that are readily-available today (snapshot in time) and that could subsequently alter the consequences of a disruption are considered.", "As a result, only substitution, and not substitutability or potential future substitution, is considered in the revised methodology.", "A comprehensive presentation of the revised EC methodology in respect to the underlying calculation of the substitution indexes is not a goal of the present paper, but will be presented in a future EC publication or communication.", "In summary, the two main alterations of the refined EI component include: (1) A more detailed and transparent allocation of RM uses to their corresponding NACE sectors, and (2) introduction of a dedicated substitution index SIEI deemed to be a reduction factor for the EI.", "The EC criticality methodology is a well consolidated and reliable tool, which represents a cornerstone of the raw materials policy in the EU.", "However, due to changing policy priorities and needs, some targeted and incremental improvements were seen as necessary.", "As the European Commission's in-house science service, its Directorate General Joint Research Centre provided scientific advice to DG GROWTH in order to assess the current methodology and identify aspects that could be modified to better address the needs and expectations of the list of CRMs.", "In view of the next update of the CRMs list foreseen in 2017, the JRC mandate was focused on a fitness check of the current methodology and the introduction of some methodological improvements.", "This choice of continuity is synonymous with giving priority to comparability with the previous two exercises, which is in turn correlated to the need of effectively monitor trends and maintain the highest possible policy relevance.", "Original contributions in respect to specific elements of criticality assessment were proposed and tested by the JRC, which also highlighted the novelty and potential outcomes.", "A comprehensive and detailed presentation of the revised EC methodology is not given in the present paper, as possible fine-tuning might still take place during implementation.", "Under the supply risk dimension, the main novelties of the revised methodology, in response to the corresponding policy needs include: (1) incorporation of trade barriers and agreements, (2) adoption of a more systematic supply chain bottleneck approach, (3) inclusion of import dependency and a more accurate picture of the actual supply to the EU and (4) confirmation of the prominent role for recycling and a substantial improvement the quality and representativeness of data for the EU.", "The two main novelties of the refined economic importance component include: (1) A more detailed and transparent allocation of RM uses to their corresponding NACE sectors, and (2) introduction of a dedicated substitution index.", "The authors declare no competing financial interests." ]	[ "Raw materials form the basis of Europe's economy to ensure jobs and competitiveness.", "The list of critical raw materials (CRMs) for the EU is key to the EU raw materials policy.", "Next update of the CRMs list for the EU coming in 2017.", "DG JRC identified key aspects of the EU criticality methodology to be adapted." ]
Data describing lack of effects of 17α-ethinyl estradiol on mammary gland morphology in female mice exposed during pregnancy and lactation	Ethinyl estradiol (EE) is a synthetic estrogen used in pharmaceutical contraceptives. In many studies evaluating estrogenic endocrine disruptors, EE is used as a positive control for estrogenicity. However, the effects of EE often differ from the effects of other xenoestrogens, suggesting that these other compounds might act via distinct mechanisms. Reported here are data describing the effect of low doses of EE during pregnancy and lactation on the morphology of the lactating mammary gland in CD-1 mice. The data suggest that these low doses have few if any discernable effects on mammary gland morphology. Alterations to cell proliferation and the expression of estrogen receptor (ER)α were also not observed. These companion data were collected from the same females analyzed for effects of EE on maternal behavior and brain recently published in Reproductive Toxicology (Catanese & Vandenberg, 2017).	[ "MULT" ]	[ "Endocrine disruptor", "Lactation", "Mammary gland", "Maternal", "Morphometric", "Xenoestrogen" ]	[ "1000" ]	[ "The mammary gland whole mounts and histological sections displayed in Fig. 1A are representative images from female CD-1 mice exposed to vehicle, 0.01 or 1 μg ethinyl estradiol (EE)/kg/day from pregnancy day 9 through lactational day 20.", "Quantification of mammary gland intensity, a measure of the epithelial density, reveals modest but non-significant decreases in the amount of mammary epithelium in EE-treated females (represented by higher intensity values) (Fig. 1B).", "To further investigate the effects of EE on morphology of the lactating mammary gland, we evaluated two histological characteristics in fixed tissue at lactational day 21: the volume fraction of the mammary gland comprised of lobuloalveolar structures and lobule size (Fig. 2A,B).", "There was no effect of EE treatment on either parameter (Fig. 2C,D).", "Finally, we examined the effect of EE treatment on cell proliferation (evaluated by quantifying the number of cells expressing Ki67, Fig. 3A) and the number of cells positive for estrogen receptor (ER)α (Fig. 3B).", "Quantification of these data revealed no effect of EE treatment on either parameter (Fig. 3C,D).", "Expression of Esr1, the gene encoding ERα, was also unaffected by EE treatment (Fig. 3E).", "Timed pregnant female CD-1 mice (Charles River Laboratories, Raleigh, NC) were housed as described previously [1].", "All experimental procedures were approved by the University of Massachusetts Institutional Animal Care and Use Committee.", "On pregnancy day 8, dams were randomly assigned to treatment groups.", "From pregnancy day eight until weaning (on lactational day [LD] 21), pregnant females were weighed daily and fed a small wafer (Nabisco, East Hanover, NJ) treated with ethinyl estradiol (EE) or vehicle alone (70% ethanol) every day.", "Wafers were dosed with solutions to deliver 0, 0.01 or 1 µg EE/kg/day (Sigma Aldrich, St. Louis, MO; >98% purity; n=12–16 for each dose).", "EE dose was adjusted for dam body weight daily.", "Litters were culled to 10 pups on LD1, and pups were weaned on LD21.", "The dams evaluated in this study were euthanized at LD21.", "The 0.01 µg/kg/day dose was selected because it has previously been shown to disrupt estrogen-sensitive endpoints in exposed offspring [2].", "The higher 1 µg/kg/day dose was selected because it can induce uterotrophic responses in pubertal females and is ~2x higher than the concentrations in prescription birth control pills [3].", "Other results from these same females, including effects on behavior and brain, have been submitted for publication elsewhere [4].", "On LD21, dams were euthanized via CO2 inhalation.", "From every dam, the right fourth inguinal mammary gland was dissected from the skin, spread on a glass slide (Fisher Scientific, Pittsburgh, PA) and fixed in neutral buffered formalin (10%) (Fisher Scientific) overnight (standard whole mount preparation).", "The right fifth inguinal mammary gland was fixed in neutral buffered formalin (10%) overnight for histology.", "After fixing in neutral buffered formalin, whole-mounted mammary glands were processed through an alcohol series, defatted with toluene, stained with Carmine-alum, dehydrated in an alcohol and xylene series, and preserved in k-pax heat sealed bags (Fisher Scientific) with methyl salicylate (Acros Organics, Morris Plains, NJ) [5].", "Digital images of whole-mount mammary glands were obtained using a Zeiss AxioImager dissection microscope (Carl Zeiss Microscopy, Jena, Germany) at 6X magnification and a Zeiss high-resolution color camera.", "To evaluate the density of epithelial structures in the whole mount, four identical boxes (each 5 mm2) were placed to the anterior, posterior, and lateral sides of the lymph node.", "ZEN software (Carl Zeiss Microscopy) was used to measure the intensity of the mammary tissue in each box; higher numbers indicate less-dense mammary tissue.", "Mammary glands were processed for histological and immunohistochemical evaluation using methods described previously [5].", "Digital images were collected using a Zeiss Axio Oberserver.Z1 inverted microscope, a 20X objective, and a high-resolution color camera (Carl Zeiss Microscopy).", "To quantify the fraction of the mammary gland comprised of adipose tissue versus lobuloalveolar units (lobules), three images were taken at random from non-overlapping areas of the histological section.", "An identical 10×13 grid was placed on each image, and the tissue type located at each crosshair (lobule, adipose, blood vessel, other connective tissue, etc.) was recorded.", "To quantify the average lobule size, a minimum of three non-overlapping images were collected using a 40X objective.", "For each image, an identical 5×5 grid was placed and all lobules that fell on crosshairs were measured for area.", "A minimum of 9 lobules were evaluated to calculate the average lobule size in each gland.", "Expression of two markers was evaluated using standard methods for immunohistochemistry [5] and commercial antibodies including rabbit anti-ERα (EMD Millipore, Cat# 06–935, Temecula, CA) and rabbit anti-Ki67 (Fisher Scientific, Cat# RM-9106-S1), a marker of proliferation.", "Sections were incubated with primary antibodies (diluted 1:1000), secondary antibody (goat anti-rabbit, Abcam, Cat# ab64256) and streptavidin peroxidase complex (Abcam, Cat# ab64269).", "Diaminobenzidene (DAB) chromogen (Abcam, Cat# ab64238) was used to visualize reactions.", "Sections were counterstained with Harris’ hematoxylin (Fisher Scientific).", "Two non-overlapping images were taken of each sample for each marker of interest (ERα, Ki67) with a Zeiss Axio Observer.Z1 inverted microscope.", "Expression of each marker was evaluated by counting at least 200 epithelial cells.", "Expression of each marker was expressed as a percent ratio of the total number of epithelial cells evaluated.", "Total RNA was extracted from mammary glands of individual mice using Trizol reagent (Ambion, Carlsbad, CA) and a BeadBug microtube homogenizer (Sigma Aldrich, St. Louis, MO).", "Total RNA was quantified by UV spectrophotometry (Nanodrop 1000; Thermo Scientific).", "One microgram of RNA from each sample was reverse transcribed to cDNA using reverse transcriptase (Applied Biosystems, Inc).", "The FastStart Universal SYBR Green Master kit (Roche Diagnostics Corporation, Indianapolis, IN) was used for qPCR along with 1 μL of cDNA and 300 nM forward and 300 nM reverse primers for each target gene.", "β-actin was used as a housekeeping gene.", "Every sample was run in duplicate for each gene target.", "The thermal profile was as follows: 10 min at 95 °C; 40 cycles of 15 s at 95 °C, 30 s at 60 °C, and 15 s at 72 °C; a melting-curve analysis was conducted to identify nonspecific products.", "Relative quantification was determined using the ΔΔCt method to correct for differences in β-actin [6].", "All analyses were conducted by observers blind to the treatment groups.", "Data were analyzed using SPSS Version 23 using 1-way ANOVA General Linear Model analyses with treatment as the independent variable, followed by Bonferroni post hoc tests.", "Data were considered statistically significant at p<0.05.", "Graphs illustrate means ± standard error." ]	[]
Seasonal dynamic thinning at Helheim Glacier	We investigate three annual mass-balance cycles on Helheim Glacier in south-east Greenland using TanDEM-X interferometric digital elevation models (DEMs), bedrock GPS measurements, and ice velocity from feature-tracking. The DEMs exhibit seasonal surface elevation cycles at elevations up to 800 m.a.s.l. with amplitudes of up to 19 m, from a maximum in July to a minimum in October or November, concentrated on the fast-flowing areas of the glacier indicating that the elevation changes have a mostly dynamic origin. By modelling the detrended bedrock loading/unloading signal we estimate a mean density for the loss of 671±70kgm-3 and calculate that total water equivalent volume loss from the active part of the glacier (surface flow speeds >1 m day-1) ranges from 0.5 km3 in 2011 to 1.6 km3 in 2013. A rough ice-flux divergence analysis shows that at lower elevations (<200 m) mass loss by dynamic thinning fully explains seasonal elevation changes. In addition, surface elevations decrease by a greater amount than field observations of surface ablation or surface-energy-balance modelling predict, emphasising the dynamic nature of the mass loss. We conclude, on the basis of ice-front position observations through the time series, that melt-induced acceleration is most likely the main driver of the seasonal dynamic thinning, as opposed to changes triggered by retreat.	[ "EART" ]	[ "Dynamics", "Glaciology", "Helheim Glacier", "Mass balance", "TanDEM-X" ]	[ "1901", "1906", "1908", "1912" ]	[ "Ongoing mass loss from the GrIS is superimposed on a seasonal cycle in ice-sheet mass balance which is dominated by the SMB processes of winter accumulation and summer ablation (van den Broeke et al., 2009; Bamber et al., 2012).", "This cycle is also detected in time series of gravity anomalies from the GRACE system (Velicogna, 2009; Ewert et al., 2012; Wahr et al., 2013), and in the vertical displacement solutions to bedrock-located continuous Global Positioning System (GPS) receivers (Bevis et al., 2012).", "Seasonal loading/unloading of ice causes the Earth to respond elastically (Farrell, 1972), resulting in vertical elastic surface displacement of the crust (Wahr et al., 2013).", "The magnitude of the displacement is proportional to the mass of the load and inversely proportional to the distance between the load and the observing point (Nielsen et al., 2013).", "The processes that concentrate long-term dynamic mass loss on the outlet glaciers also influence the seasonal mass-balance cycle, as meltwater and runoff can affect flow velocities (Andersen et al., 2010; Sole et al., 2011; Joughin et al., 2008), submarine melt (Motyka et al., 2003; Jenkins, 2011), and iceberg calving (O'Leary and Christoffersen, 2013).", "Supraglacial melt is able to rapidly reach the bed at elevations below 1000 m via the development of moulins and crevasses (Clason et al., 2014; Bartholomew et al., 2010, 2011), and once it enters the subglacial hydrologic system it can reduce the effective pressure at the ice-bed interface to promote faster sliding.", "For example, the onset of melt has been observed to cause early season flow acceleration on many glaciers in western Greenland (Sole et al., 2011; Ahlstrøm et al., 2013).", "Evidence of melt-enhanced flow for glaciers in the south-east is weaker, but small variations in daily summer surface velocity on Helheim Glacier in 2007 and 2008 were found to be strongly correlated with daily melt (Andersen et al., 2010).", "At marine-terminating outlet glaciers meltwater and runoff will eventually reach the fjord.", "Once discharged to the fjord the melt-season runoff can amplify submarine melting by forming bouyant plumes (Jenkins, 2011).", "These plumes drive an estuarine circulation within the fjords which, combined with the shelf-forced intermediary circulation, have a strong impact on the stratification of water within the fjord (Sutherland et al., 2014).", "Frontal ablation and the nature of waters present within the fjord therefore both depend significantly on meltwater discharge (Sciascia et al., 2013).", "The importance of submarine melt lies not only in the first-order process of frontal mass loss via melt but also on the potential impact on calving.", "Modelling has shown that submarine melt could lead to undercutting of an ice front and hence increase the rate of calving (O'Leary and Christoffersen, 2013), though a more recent study has shown that this might not be the case in a time-evolving stress field (Cook et al., 2014).", "Seasonal melt water may also directly affect calving rates via crevasse hydrofracture processes.", "Where a crevasse-depth calving criterion is used to model calving rates, rates have been shown to be highly sensitive to water depth within the crevasses (Cook et al., 2012).", "Both submarine melting and increased calving can lead to terminus retreat and a subsequent reduction in resistive stresses at the terminus.", "This force imbalance may result in faster ice-flow and glacier thinning which rapidly propagates upstream (Howat et al., 2005).", "Thus meltwater reaching the bed and progressing to the fjord may cause an increase in ice flow through two mechanisms — reduced basal friction and reduced terminal backstress, both of which will lead to dynamic thinning of a glacier during the melt season.", "Backstresses at the terminus may also vary seasonally owing to the formation and clearance of an ice mélange within the fjord (Howat et al., 2010; Amundson et al., 2010).", "In this study we investigate these processes by employing an unprecedented series of interferometric digital elevation models (DEMs) from June 2011 to May 2014.", "Using these data we map the timing and distribution of seasonal ice loss over Helheim Glacier below 800 m a.m.s.l. (above mean sea level) through three annual cycles.", "We calculate volume change using the DEM time series and associate this with mass loss by considering GPS measurements of relative vertical bedrock displacements from 2011 to 2014 at a permanent site located a few hundred meters from the Helheim Glacier (Fig. 1).", "Feature tracking of ice-flow is used to consider the relative contributions of SMB and dynamic effects to the annual mass-balance cycle, and the importance of ice-front processes versus meltwater penetration in driving dynamic processes.", "Synthetic Aperture Radar (SAR) data from the TanDEM-X satellite system were used to generate a series of DEMs of the lower portion of the Helheim Glacier catchment.", "The TanDEM-X mission is a public/private partnership between the German Aerospace Center (DLR) and EADS Astrium GmbH to generate a highly accurate consistent global DEM (Krieger et al., 2013).", "We use GAMMA Remote Sensing software to generate interferometric DEMs using the bistatic stripmap mode Co-registered Single look Slant range Complex products (CoSSCs).", "These experimental data are available over Helheim Glacier from June 2011 to May 2014 and have a spatial resolution of approximately 2 m which we multilook by a factor of 4.", "We rely solely on the provided orbital vector data to geolocate the images and to calculate the phase scaling.", "The DEMs are tied in the vertical dimension using a ground control point from the Danish Geodata Agency (http://gst.dk/emner/landkort-topografi/groenland/ground-control-greenland/, July 2014).", "The control point is at an elevation of 468.55 m above mean sea level in the GR96 datum system with a second point (347.10 m) used as a validation point (Fig. 1).", "All elevations quoted in this work will be in the GR96 datum system.", "The main error sources in generating interferometric DEMs from TanDEM-X data include errors in orbit or baseline information, unwrapping, and geolocation and orthorectification; only DEMs with less than a 2.5 m error relative to the validation point are included in this analysis.", "Estimates of orbit accuracies (Krieger et al., 2013) suggest that we cannot expect relative elevation accuracies better than 2 m.", "Volume changes are calculated by differencing DEMs, only including ice-covered areas down to the most retreated frontal position.", "Area-mean surface elevation changes are calculated by dividing volume changes by the relevant area.", "To estimate site coordinates from GPS measurements, we use the GIPSY OASIS 6.3 software package (Zumberge et al., 1997) developed at the Jet Propulsion Laboratory (JPL) and using IGS orbits, earth orientation parameters, and clock products.", "The data were processed as described by Khan et al. (2010a) using the Precise Point Positioning (PPP) strategy and the solutions were aligned with the IGS08 frame (Altamimi et al., 2011).", "To focus on the seasonal mass variability of the Helheim Glacier, we remove the trend, which is due to long-term mass loss over southern portions of Greenland (Khan et al., 2010b).", "We compare the observed bedrock displacements with predicted displacements obtained by convolving high-resolution spatial seasonal ice volume estimates from the TanDEM-X DEMs (converted to mass change estimates) with the Green's function for vertical displacements derived by Jean-Paul Boy (Petrov and Boy, 2004) for the Preliminary Reference Earth Model (Dziewonski and Anderson, 1981).", "We convert the seasonal ice volume estimates to mass assuming a density equal to that of ice (917 kg m−3).", "However, the observed bedrock displacements are caused by mass changes from a greater area than that covered by the DEMs.", "To include the area outside the DEMs, we estimate ice-sheet-wide seasonal elevation changes during 2003–2009 using Ice, Cloud and land Elevation Satellite (ICESat) laser altimeter data (Zwally et al., 2011b).", "With this model, overall Greenland mass change matches the GRACE seasonal mass change (Velicogna, 2009).", "SAR data are highly sensitive to the presence of meltwater on the glacier surface in the same way that scatterometer data are (e.g. Steffen et al., 2004), backscatter is high when the surface is frozen and low when meltwater is present.", "Here we use 934 earlier (July 2005 to September 2011) Envisat ASAR Wide-Swath-Mode images to determine typical spring and autumn transitions between surface melt and freeze states at a point approximately 4 km back from the ice front.", "WSM images have a spatial resolution of 150 m and an acquisition frequency of about 3 days at this latitude (66.4°N).", "Midday air temperatures from the coastal station of Tasiilaq (85 km to the south of Helheim Glacier) are consistently positive from mid to end April and vary little from year to year (Fig. 2), justifying the use of earlier time series of surface melt to estimate melt onset for the years under consideration in this study.", "A total of 40 DEMs were included in the following analyses with a root mean square (r.m.s.) error relative to the validation point of 1.3 m.", "The good agreement between DEMs and the validation height gives us high confidence in the DEM differences especially at lower elevations close to the control point where phase unwrapping is initiated.", "We estimate that SAR penetration depths on the glacier are negligible during the summer following studies that show C-band SAR penetration depths on the Geikie ice cap, just north of Helheim Glacier, to be 0 m in the soaked snow zone between 1600 m and 1900 m (Dall et al., 2001), and 1–2 m on bare ice on Jakobshavn Isbrae, west Greenland (Rignot et al., 2001); the higher frequency X-band TanDEM-X SAR will have a lower penetration depth than C-band SAR.", "Microwave backscatter values show that melt onset is typically around April or May (Fig. 3) and field studies (Andersen et al., 2010) confirm that the area under consideration in this study, i.e. below 800 m a.m.s.l., is within the ablation zone.", "Mapped differences between DEMs (Fig. 4(a)) show that changes are concentrated on the fast-flowing regions of the glacier (Fig. 4(b)).", "We now calculate elevation changes only where surface flow speeds exceed 1 m day−1.", "Mean surface elevations, in 100 m elevation bands from 100 m to 800 m a.m.s.l., show annual cycles with a maximum amplitude in 2013 of 19 m between 25/03/2013 and 22/11/2013 in the lowest 100 m elevation band (Fig. 5(a)).", "Rapid surface lowering begins around July and lasts until October or November.", "In spite of the quality control applied to DEM selection we see four discontinuities in height change trends in August 2011, February 2012, March 2013 and February 2014.", "On these occasions we see a reversal of an otherwise monotonic increase or decrease in surface elevation which features in all altitude bands.", "The jumps are less than 1 m in magnitude and well within our accuracy expectations; they are much less than would be expected from unwrapping errors, as the height of ambiguity is around 48 m, and may therefore be a consequence of errors in satellite orbit information.", "We include these DEMs in subsequent analyses as we have no objective reason to remove them and also because it is not possible to identify which of two consecutive DEMs contains the error.", "Throughout the period between June 2011 and May 2014 the velocity close to the front of the glacier (black star in Fig. 4(b)) varies between 16.5 m day−1 (February 2013) and 20.6 m day−1 (July 2013) (Fig. 5(c)).", "5.7 km further up glacier velocities are only slightly lower and follow the same pattern, although the acceleration in 2013 is less pronounced.", "The ice-front location spans a 3.2 km range, being most advanced in February 2013 and most retreated in August 2013, with calving occurring all year round.", "There are two periods in the latter half of the record where the glacier advances with little or no calving events and its velocity falls steadily.", "The first of these periods in early 2013 ends with a 5 month period of retreat and acceleration.", "Elevation changes for Helheim Glacier catchment show a clear seasonal signal, with rapid lowering commencing after July and lasting until October or November.", "The height change is concentrated on fast-moving (>1 m day−1) areas with no measurable change taking place on the stagnant ice sheet, suggesting that dynamic thinning is a significant contributor to the change.", "This concentration of height change on the active glacier reflects a similar pattern to the dynamically induced interannual surface lowering observed on Helheim (Pritchard et al., 2009).", "An attempt to use interpolated grids of glacier thickness supplied by the Center for Remote Sensing of Ice Sheets (CReSIS) to calculate a complete flux divergence field revealed large unrealistic variations.", "The anomalies are a result of fine-scale features in the velocity field caused by bed topography which is not captured by the sparse flightlines of airborne radar sounding; an effect that has been encountered before, for example on Nioghalvfjerdsfjorden (79 north) Glacier (Seroussi et al., 2011).", "Thus, the increase in flux divergence from winter to summer on the lower elevations of the glacier is more than sufficient to account for the surface lowering we observe, which is of the order of 0.1 m day−1 in summer, without taking any seasonal melt into account.", "However, there are many assumptions and approximations in the calculation.", "For example, we do not allow for the probability that flux divergence is a result of crevasse expansion which would not manifest in elevation change, a process that is very likely to be occurring along the lower reaches of the glacier where the stress balance under large velocity gradients will promote crevasse opening and fracture propagation.", "Our method of calculating elevation change via volume differences between relatively high resolution DEMs may capture some change due to crevasse opening, but some will remain due to unresolved crevasses and due to the SAR DEMs not capturing the full crevasse depth.", "We can compare surface elevation changes with field measurements of surface ablation rates.", "Measurements using sonic-rangers found that ablation rates in 2007 and 2008 on Helheim, at an elevation of 650 m, were of the order of 3.2 cm/day over a 27 day period from the 27th July (Andersen et al., 2010).", "Over the whole melt period and between 600 m and 700 m, we observe a mean rate of thinning of 6.5 cm/day in 2013, and 5.0 cm/day in 2012.", "At Tasiilaq in 2007 there were 198 positive degree days (PDDs) and in 2013 there were only 165 (Fig. 2); use of a PDD model to determine surface melt would indicate that surface melt in 2013 was less than 2007.", "The additional surface lowering we observe is, therefore, most likely due to dynamic thinning (which would not be measured with the sonic-ranger method) plus any basal melt.", "The seasonal dynamic effects are more clearly observed in the time series and maps of surface elevation change than in glacier surface velocity; only from July 2013, when glacier thinning was most pronounced, do we observe a clear increase in flow speed which precedes the thinning.", "The record of flow speeds presented here continues that in Bevan et al. (2012) which showed that annual peaks in velocity are common but not observed in every year.", "Whether seasonal velocity increases on outlet glaciers occur because of basal lubrication following meltwater penetration to the bed or because of ice-front retreat changing the force balance at the terminus is still unresolved.", "Although surface melting can have a significant impact on ice-sheet flow (Zwally et al., 2002; Joughin et al., 2008; Das et al., 2008; Palmer et al., 2011), the net impact on the dynamics of outlet glaciers of enhanced seasonal melt is usually found to be small, i.e. less than 15% (Joughin et al., 2008).", "Meltwater has been observed to briefly accelerate flow but the development of a more efficient subglacial drainage system, as melt volumes increase, can subsequently cause abrupt slow down (Howat et al., 2010), resulting in a very small overall increase in annual flow speed (Sole et al., 2011).", "Brief speed-ups have been observed to follow episodes of enhanced surface melting on Helheim (Andersen et al., 2010), but are less significant than those following calving episodes (Nettles et al., 2008; Sundal et al., 2013).", "However, whereas seasonal changes in front position are found to influence flow speeds for accelerating and retreating glaciers (Nick et al., 2009) such as Jakobshavn Isbrae in west Greenland (Joughin et al., 2008), they do not appear to have a dominant influence on the dynamics of stable outlet glaciers (Howat et al., 2010).", "In this instance, whilst we see a clear signal of dynamic origin in the glacier surface elevation, we do not observe a corresponding signal in the ice-front position; in fact, from August 2013 when the glacier is thinning the ice front is advancing.", "It therefore seems most likely that it is surface melt, penetrating to the bed, which causes the dynamic component of glacier seasonal thinning rather than any acceleration as a result of changes in back stress at the glacier terminus.", "Long-term mass-balance trends on the Greenland Ice Sheet are driven by disruptions to the seasonal mass balance cycle; understanding the seasonal cycle, especially on fast-flowing marine-terminating glaciers, underpins any attempt to predict long-term mass loss.", "We investigated three full years of seasonal mass-balance cycles on Helheim Glacier in south-east Greenland using bedrock GPS measurements and a series of 40 TanDEM-X interferometric digital elevation models (DEMs).", "We conclude that seasonal mass loss by dynamic thinning is of the same order of magnitude as our observed decreases in surface elevation and that, on the basis of ice-front position observations through the time series, melt-induced acceleration is most likely the main driver of the thinning, as opposed to changes triggered at the terminus.", "The calculation of large-scale flux divergence quantities, which would enable more precise estimates of the glacier-wide dynamic component of thinning, was found to require a much finer resolution ice-thickness grid than is currently available.", "The magnitude of seasonal changes in ice-surface elevations highlights the importance of selecting consistent measurement dates when using altimetry to assess long-term temporal change in glacier thickness." ]	[ "TanDEM-X DEMs are used to reveal seasonal thinning on Helheim Glacier.", "Feature tracking for surface speeds ties the thinning to areas of fast-moving ice.", "Bedrock GPS measurements are used to estimate an effective density for the ice loss.", "Annual water equivalent volume loss ranges from 0.5 km3 in 2011 to 1.6 km3 in 2013.", "We conclude that the thinning is dynamic and is driven by melt penetration to the bed." ]
One-pot synthesis of copper nanoconjugate materials as luminescent sensor for Fe3+ and I− detection in human urine sample	A simple synthetic approach of water-soluble and stable fluorescent copper nanoclusters (Cu NCs) at room temperature was proposed. The cysteamine functionalized nanoconjugate materials (CA-Cu NCs) exhibited variety of exceptional properties, including good water-solubility, photostability, low cytotoxicity and acting as an excellent cell imaging probe. Based on the electron transfer and aggregation induced fluorescence quenching mechanism, CA-Cu NCs were utilized in the selective determination of ferric (Fe3+) and iodide (I−) ions. The detection limits of Fe3+ and I− ions were calculated as 423 nM and 2.02 μM, which are lower than the maximum level of Fe3+ and I− ions, allowed in drinking water by the U.S. Environmental Protection Agency. The assay method of paper strip was also successfully applied for the detection of Fe3+ and I− ions in live cells and human urine.	[ "BIOC", "COMP", "ENGI", "MATE" ]	[ "Cell imaging", "Copper nanoclusters", "Cysteamine", "Fe3+ and I− detection", "Fluorescent assay", "Paper strip" ]	[ "1305", "1711", "2208", "2504" ]	[ "Luminescent metal nanoclusters (M NCs) based sensors are of research interest due to potential applications in bio-detection and bio-imaging fields [1,2].", "In general, the size of functional M NCs tend to be approximately 2 nm in diameter and also do not display plasmonic properties in nanoscale [3–5].", "Various factors such as luminescent stabilizers, quantum yield (Φ), photostability and biocompatibility may influence the applicability of M NCs [6].", "Hence, these factors are often first optimized before moving on to chemo- or bio- sensory applications.", "With this approach in mind, M NCs based probes for the detection of various analytes have been developed by others.", "Among the reported functional M NCs, including Au NCs, Ag NCs, Pt NCs and Cu NCs, templated Cu NCs based sensory assays are of great interest as non-noble species [7–10].", "Stabilizer templated Cu NCs have been effectively applied in diverse chemo- and bio- sensory studies.", "However, determination of metal ions and anions are not yet thoroughly established and pose as an exciting field of research [11].", "For example, ferrous/ferric [Fe(II)/ Fe(III)] systems are a well-known redox pair involved in the electron transport for a variety of enzymatic reaction in biological processes (e.g. respiratory functions).", "An unbalanced concentration of Fe3+ in a physiological system may lead to many diseases [12–15].", "I− ions are important micronutrients for human growth and metabolism.", "I− deficiencies during prenatal and early growth periods may lead to malfunctions leading to spontaneous abortion, increased infant mortality, hypothyroidism and cretinism [16–18].", "Therefore, quantification of Fe3+ and I− ions are required studies that may assist in improving the quality of human healthcare.", "Various stabilizer templated Cu NCs have been reported for the assay of Fe3+ or I− ions.", "However, a single species capable of detecting both Fe3+ and I− ions has not yet been established.", "Recently, Boonmee et al., used cysteamine capped Cu NCs as a probe for the “turn-on” determination of Al3+ ions, synthesized using CuSO4 as a precursor under base conditions.", "The demonstrated selectivity was not ready for application.", "Notably, in this work, the PL emission peak for Cu NCs was fixed at 380 nm, which may vary with precursor and reaction conditions [19].", "In Bao's work, they synthesized cysteamine capped Cu NCs for determination of picric acid (PA), using CuSO4 as a precursor as well, and successfully applied in real water samples.", "However, the probe was easy to be interfered by biomolecules, which has a strong background for UV absorbing [20].", "With improving on previous researches in mind, we developed cysteamine functionalized Cu NCs (CA-Cu NCs) by using CuCl2 as a precursor and ascorbic acid as a reducing agent.", "The obtained CA-Cu NCs revealed strong emission at 467 nm and acted as a probe for the detection of both Fe3+ and I− ions.", "Herein, we report the synthesis and sensing properties of a low toxicity species, cysteamine functionalized Cu NCs (CA-Cu NCs), with blue emission and luminescent quantum yields of up to 16%.", "The CA-Cu NCs based sensory studies revealed selective detection of Fe3+ and I− over other ions even when elevated amounts of other ions were present.", "Moreover, the applicability of CA-Cu NCs was verified by demonstrating urine analysis, cell imaging and paper strip tests.", "UV–vis absorption spectrums were obtained by the HITACHI, U-3310.", "Transmission Electron Microscope [TEM] and Fourier transform Infrared spectroscopy (FTIR) analysis were done by JEOL-JEM-2100 and Perkin Elmer-100 FT-IR SPECTRUM ONE spectrometer, respectively.", "X-ray Photoelectron Spectroscopy (XPS) analysis was done using the Microlab-350 (Thermo Electron Corporation).", "Powder X-Ray Diffraction (XRD) peak spectrums were obtained by the BRUKER AXS D2 Phaser.", "The mass spectra of CA-Cu NCs were collected in the linear positive mode with α-cyano-4-hydroxycinnamic acid as the matrix using a MALDI-TOF-MS analyzer (Bruker Autoflex 111).", "The samples were prepared by mixing CA-Cu NCs with matrix in 1:1 and from this 0.8 μL of sample was used for spotting.", "The luminescence quantum yield of CA-Cu NCs were calculated by using riboflavin monophosphate in water as reference (Φ =26.8%).", "CA-Cu NCs were synthesized via one-pot reaction as represented in Scheme 1.", "0.5 mL of 100 mM CuCl2 and 2 mL of 10 mM cysteamine aqueous solution was added dropwise into 6 mL of water under stirring at room temperature.", "CA-Cu NCs were obtained by adding 450 μL of 200 mM ascorbic acid.", "The unreacted cysteamine was separated by centrifugation at 3500 rpm for 30 min.", "The obtained product was stored in a refrigerator at 4 °C for further use.", "100 μL of as-prepared CA-Cu NCs solution was diluted in 800 μL of HEPES buffer [pH -7] to form the sensing probe solution.", "Afterwards, varied concentrations of target ion species in 100 μL DI water was added to the sensing probe solution and mixed thoroughly.", "Immediately after mixing, the PL spectra was recorded at room temperature with the excitation wavelength set at 385 nm.", "Sensing targets of Ba2+, Na+, Fe3+, Cr3+, Ca2+, Mg2+, Fe2+, In3+, Ga3+ and Al3+ metal cations were synthesized by dissolving their respective chloro- and perchlorate- compounds in DI water; Co2+, Zn2+, Pb2+, Mn2+, and Hg2+ metal cations were dissolved with their respective acetate salts; CH3COO−, Br−, NO3−, ClO4−, H2PO4−, OH– SO42− and I− anionic species were dissolved with respective tetra-butyl ammonium salts.", "The CA-Cu NCs practical application of Fe3+/ I− detection was investigated in human urine samples by using the standard testing method, adopted by most qualified publications [21].", "Dilution of the urine sample 150 times with distilled water was done to reduce the influence of other species during the detection of Fe3+/ I− in urine sample.", "Analysis was carried out without further pretreatment.", "To acquire the desired titration concentrations, Fe3+/ I− in different amounts of known concentrations were added to the urine sample.", "The detection limit of solution was further obtained by PL titrations.", "CA-Cu NCs layered paper strip assay was established on Whatman filter paper.", "The filter paper was cut into strips of identical dimensions and drenched in CA-Cu NCs solution for 30 min then dried in air.", "The resulting CA-Cu NCs coated paper strips were further dipped in the Fe3+/ I− solutions with variable concentrations and dried in air.", "Color and luminescence was assessed by visual comparison.", "Cytotoxicity evaluation of CA-Cu NCs was carried out using methyl thiazolyl tetrazolium (MTT) assay.", "In a 96-well plate, HeLa cells were seeded with a population of 1.5 × 104 cells per well.", "Cells were permitted to spread for 24 h to get 80% confluence.", "The media was then substituted with CA-Cu NCs of various concentrations.", "Then, the cells were incubated in a humidified incubator with 5% CO2 at 37 °C up to 24 h. Afterwards, 10 μL MTT (5 mg/mL) was added to each well and further incubated under 5% CO2 at 37 °C for 4 h.", "After the elimination of the MTT solution from plates, yellow precipitates (formazan) were dissolved by 180 μL DMSO and 25 μL Sorenson's glycine buffer (0.1 M glycine and 0.1 M NaCl).", "Lastly, each well was subjected to absorbance measurement at 570 nm through a Multiskan GO microplate reader.", "Cell viability percentage was estimated as follows,", "Cell viability (%) = (mean of absorbance value of treatment group) / (mean of absorbance value of control group).", "HeLa cells were seeded in confocal dish and cultured in DMEM containing, 10% fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 units/mL) at 37 °C in a humidified atmosphere of 5% CO2 for 12 h.", "The cells were then rinsed with fresh phosphate buffer solution (PBS, 10 mM, pH = 7.4) for three times.", "30 μL of the prepared storage CA-Cu NCs solution was then added to 1 mL cell culture, and cells incubated for 5 h at 37 °C.", "The Cu NCs stained cells were further incubated with or without Fe3+ (100 μM) solution for 30 min.", "After washing, fluorescent imaging (excitation at 405 nm) was recorded using confocal laser scanning microscopy.", "In this study, the CA-Cu NCs were synthesized via one-pot reaction as represented in Scheme 1.", "To achieve the highest emissive intensity of the CA-Cu NCs.", "Various factors were obtained through the optimization of following parameters (a) Reaction time, (b) pH of the solution, (c) Reaction temperature, (d) Cysteamine concentration, (e) CuCl2 concentration, and (f) Ascorbic acid concentration.", "Best results were obtained at (a) 4 h reaction time, (b) pH = 7, (c) Room temperature, (d) 200 mM of cysteamine, (e) 200 mM of CuCl2, and (f) 200 mM of ascorbic acid.", "The synthesized CA-Cu NCs were stored straightway in the refrigerator at 4 °C and stabilized for at least 60 days.", "To show the morphology and size of CA-Cu NCs, HR-TEM investigations were undertaken.", "In Fig. 1A, HR-TEM image analysis showed the particle diameter of CA-Cu NCs ranging between 1–5 nm [22], with a majority of diameters around 3 nm (inset of Fig. 1A).", "Moreover, from Fig. 1B, a diffraction value of 0.207 corresponded to the 111 directional distance of Cu atoms, indicating the formation of Cu NCs [23].", "To support TEM particle size calculations, DLS studies were conducted, estimating particle sizes of CA-Cu NCs as 4.1 ± 3.0 nm, shown in Fig. 1C, which agreed with HR-TEM results.", "Lastly, to justify stability as well as applicability, the zeta potential (ζ) of CA-Cu NCs were obtained as −8.28, shown in Fig. 1D, which allowed the Cu NCs particles to disperse in polar solvents such as water via –Ve repulsive forces [24].", "FTIR investigations were carried out to verify ligand attachment [25] in Fig. S1 of supporting information.", "The free thiol (-SH) group signal at 2528 cm−1 completely vanished for CA-Cu NCs, indicating the conversion to CuS bonds holding the cysteamine onto copper atoms [26].", "Additionally, after attachment onto copper atoms, the asymmetric and symmetric –NH stretching of cysteamine at 1495 and 1612 cm−1 downshifted to 1631 and 1748 cm−1, respectively.", "The broad FTIR band between 3050–3500 cm−1 corresponded to the free –NH2 group, which also slightly decreased.", "MALDI-TOF analysis was also done to establish ligands presence at the surface of the copper atoms.", "As given in Fig. S2, CA-Cu NCs had high m/z values at 1385.823, 1449.053 and 1512.568, which could be attributed to Cu9 L11 + H –Na+, Cu10 L11 + H –Na+ and Cu11 L11 + H –Na+ configurations.", "L represents the cysteamine, confirming cysteamine linkage on the copper surface [27].", "The EDX spectrum of CA-Cu NCs in Fig. S3 displayed the presence of Cu (42.48%), C (46.52%), N (3.12%) and S (7.88%) atoms at the surface, corresponding with the capping of CA over copper atoms.", "XPS data of CA-Cu NCs showed Cu2p peaks at 935.2 eV (2p3/2) and 955.2 eV (2p1/2) in Fig. S4a [28,29].", "An absence of the satellite peak (due to Cu+) around 941.9 eV confirmed the formation of Cu NCs [30].", "The S2p, N1 s and C1s peaks at 168.7, 405.2 and 287.9 eV confirmed the existence of S, N and C atoms on copper atoms in Fig. S4b–d. XRD data of CA-Cu NCs in Fig. S5 displayed the amorphous nature of nanocluster copper, while slight appearances of cysteamine peaks (e.g. 29.64°) established the orderly-structured cysteamine over copper atoms.", "CA-Cu NCs UV–vis absorptions in Fig. S6 exhibited a bathochromic shift to around 365 nm with respect to both cysteamine and Cu2+ ions, due to additional absorption states [27,31].", "In Fig. S7, CA-Cu NCs displayed PL peaks at 467 nm by excitations ranging from 365 to 415 nm, with maximum luminescence under 385 nm irradiation, which was selected as the optimal excitation wavelength [32].", "The CA-Cu NCs luminescent quantum yield (Φ) of 385 nm excitation was calculated as 16%.", "Next, a pH value for titration studies was selected for optimal sensory results [33].", "Shown in Fig. S8, by comparison of PL intensities in different pH value solutions at room temperature, CA-Cu NCs revealed elevated emissions in pH values of 2 to 7 and decreased emission intensities in basic pH value (8 to 14) solutions.", "Therefore, the pH working condition for sensory studies was selected as pH 7 at room temperature [34].", "The sensing probe solution was prepared by diluting the CA-Cu NCs solution in a HEPES buffer of pH 7.", "1 mM of various metal ions (Ba2+, Na+, Fe3+, Cr3+, Ca2+, Mg2+, Fe2+, In3+, Ga3+, Al3+, Co2+, Zn2+, Pb2+, Mn2+, and Hg2+) or 10 mM of anions (CH3COO−, Br−, NO3−, ClO4−, H2PO4−, OH– SO42− and I−) were added to the sensing probe solution and fluorescence changes recorded subsequently [34,35].", "Of the fluorescence responses of individual ion species, Fe3+ and I− ions induced significant fluorescent quenching of CA-Cu NCs as shown in Fig. 2A and B. Even when added higher concentrations of initially unresponsive analytes of 10 mM (for metal ions) and 100 mM (for anions), luminescence still did not change.", "This revealed, in Fig. 2C and D, the highly specific sensing of CA-Cu NCs towards Fe3+ and I− ions.", "Further, to prove the selectivity of CA-Cu NCs Fe3+ and I− sensing, dual analytes studies were also done.", "In Fig. S9, after added a with mixture of Fe3+ (1 mM) + other metal ions (10 mM) or I− (10 mM) + other anions (100 mM), CA-Cu NCs sensor responses were near identical to that of single analyte studies.", "Stern-Volmer Quenching Constant (KSV) calculations [36–38] were done to prove the exceptional selectivity of CA-Cu NCs to Fe3+ and I− ions.", "As listed in Tables S1 and S2, the KSV values of CA-Cu NCs + Fe3+ and CA-Cu NCs + I− were approximately 4.21 × 10−3 and 1.73 × 10−3 M.", "In contrast, remaining metal ions had KSV values of 10−4–10−6 M, and KSV for remaining anions in the order of 10−6.", "With the sensing results, selective detection of Fe3+ and I− by CA-Cu NCs was successfully demonstrated.", "Among different functionalized copper nanocluster probes, CA-Cu NCs were the only species that detected both Fe3+ and I− ions, shown in Table S3.", "Fluorescence quenching of CA-Cu NCs with different concentrations of Fe3+ and I− ions were established through PL titrations as follows.", "Upon incremental addition of Fe3+ ions (0–1000 μM), the fluorescent peak at 467 nm decreased linearly in Fig. 3A. Similarly, incremental addition of I− ions (0–10 mM) showed linear fluorescent quenching as depicted in Fig. 3B. From titration results, linear regressions were adopted to calculate the limit of detections (LODs) in Fig. 3C and D. Linearly fitted equations estimated the LODs of Fe3+ and I− ions to be 0.423 μM and 2.02 μM, respectively.", "The mechanism behind detection of Fe3+ and I− ions has been initially established by TEM studies.", "In the presence of responsive analytes, the CA-Cu NCs led to aggregation as evidenced in Fig. S10A and 10B.", "Notably, with Fe3+ ions, CA-Cu NCs aggregated in nanorod form as exposed in Fig. S10A.", "On the other hand, CA-Cu NCs + I− revealed the diverse-structural aggregation in Fig. S10B.", "The difference in aggregation with Fe3+ and I− ions might have been due to the structure of those analytes and involved binding forces.", "To further investigate the aggregation forms, DLS interrogations were considered.", "As shown in Fig. S11, from DLS of CA-Cu NCs + Fe3+ system, the particle size was estimated as 138.4 ± 36.8, which was 34 times higher than that of dispersed CA-Cu NCs.", "Similarly, as exposed in Fig. S12, the particle size of CA-Cu NCs + I− by DLS was 70.8 ± 19.2 (17 times higher than CA-Cu NCs).", "Both DLS studies supported the occurrence of an aggregation based mechanism from TEM deductions.", "Moreover, the calculated particle sizes also agreed with the TEM data.", "Hence, aggregation of CA-Cu NCs during the recognition of Fe3+ and I− ions was well proved.", "To support the detection of Fe3+ and I− ions, zeta potential changes were accounted.", "The zeta potential value ζ of CA-Cu NCs shifted largely in the existence of Fe3+ ions to +Ve charges from −8.28 to 15.02, as depicted in Fig. S13.", "In contrast, Fig. S14 showed that in the presence of I− ions, CA-Cu NCs + I− has ζ value of −54.0 (a higher –Ve value than CA-Cu NCs).", "These zeta potential variations also validated the Cu NCs reactions with Fe3+ and I – ions.", "XPS data established the existence of Fe3+ and I− ions.", "As exposed in Fig. S15a–d, after the addition of Fe3+ ions, XPS peaks of Cu2p at 935.2 eV (2p3/2) and 955.2 eV (2p1/2) completely diminished and broadened.", "S2p, N1 s and C1s peaks shifted to 169.8 eV, 402.5 eV and 285.8 eV respectively, which may have been due to the involvement of those atoms in the formation of CA-Cu NCs + Fe3+ complexations.", "The existence of Fe3+ ions was authenticated by the XPS peaks at 712.5 eV (Fe2p3/2) and 725.9 eV (Fe2p1/2) as illustrated in Fig. S15e.", "In Fig. S16a–d, after I− detection, the XPS pattern of Cu2p had slight deviations to 934.5 eV (2p3/2) and 954.7 eV (2p1/2).", "Meanwhile, the S2p, N1 s and C1s peaks shifted to 170.5 eV, 404.3 eV and 287.4 eV with the formation of CA-Cu NCs + I− complexations.", "Shown in Fig. S16e, the existence of I− ions was confirmed by XPS peaks at 620.6 eV (I3d5/2) and 632.2 eV (I3d3/2).", "In order to find the binding forces involved in Fe3+ and I− ions assay, the FTIR investigations were undertaken.", "In the presence of Fe3+ or I− ions, the broad –NH2 bands intensified with sharp band centers at 3387 cm−1.", "This might have emerged from the bonding between –NH2 and Fe3+ or I− ions.", "On the other hand, the symmetric –NH stretching at 1748 cm−1 completely vanished along with downshifted asymmetric stretching bands from 1631 to 1635 cm−1 as shown in Fig. S17.", "Hence, the involvement of –NH2 groups in the complexation with Fe3+ or I− ions was well confirmed, however, other atoms such as Cu, C and S may also support the complex formation as revealed by XPS analysis.", "To demonstrate the applicability of CA-Cu NCs based assays of Fe3+ and I− ions, select applications multiple sorts were tested in this section.", "Titrating CA-Cu NCs with human urine samples containing 0–1000 μM of Fe3+ ions, PL intensities and linear regression was plotted with Vs fluorescent intensity and Fe3+ concentrations, estimating the LOD as 0.92 μM, shown in Fig. 4A and B; titration for I− ions (0–10 mM) in human urine samples also led to PL intensities and linear regression plotted in Fig. 4C and D, the LOD was calculated as 3.01 μM.", "Hence, CA-Cu NCs mediated Fe3+ and I− ions detection in human urine samples was validated.", "Next, for cell imaging of Fe3+ and I− ions, the cytotoxicity of CA-Cu NCs was first obtained.", "The MTT assay of CA-Cu NCs demonstrated cell viability of 80% at even 25 μg/mL, proving low toxicity [40,41] as in Fig. S18A.", "Further, the IC50 value of CA-Cu NCs is calculated as 75.74 μg/mL as displayed in Fig. S18B.", "Therefore, low toxicity of Cu NCs was ensured for safe use in cell imaging studies (Fig. 5A).", "Incubation of CA-Cu NCs with HeLa cells exhibited bright blue luminescence as visualized in Fig. 5B.", "The luminescent cells were then stained with Fe3+ ions and further incubated for 30 min, which resulted in quenched luminescence in cells as seen in Fig. 5C.", "In particular, when the above procedure was operated with I− ions, widespread cell death occurred, possibly due to toxicity of I− ions.", "In order to demonstrate alternative methods of CA-Cu NCs based assays of Fe3+ and I− ions, a paper test is also done by following the standard testing procedures [42].", "Absorbent and luminescent changes of CA-Cu NCs in the presence of Fe3+ and I− ions could be seen in Fig. 6A and B. Importantly, Fig. 6B showed significant luminescent changes, authenticating the applicability of CA-Cu NCs towards Fe3+ and I− ions.", "In summary, we demonstrated a simple one-pot method for the synthesizing of CA-Cu NCs at room temperature.", "CA-Cu NCs exhibited low toxicity and acted as capable cell imaging probes.", "The detection of Fe3+ and I− ions could be done with significant luminescence decreases of CA-Cu NCs in the presence ions with nano- and micro- molar detection limits, respectively.", "The detecting process could be performed quickly at room temperature by electron-transfer induced fluorescent quenching and aggregation of CA-Cu NCs.", "Moreover, CA-Cu NCs successfully performed the visualization of Fe3+ ion concentrations in live cell imaging.", "The luminance changes of CA-Cu NCs on paper script was great after Fe3+ or I− ions added.", "The proposed system was applied for detection of Fe3+ and I− ions in human urine samples.", "The Cu NCs based analytic detections in this work may assist further researches towards diverse bio-analysis." ]	[ "Facile one-pot synthesis of copper nanoconjugates.", "Nanoconjugate can selectively determine ferric and iodide ions.", "Cu nanoconjugate exhibits blue emission with quantum yield up to 16%.", "Visualization of ion concentration with nanoconjugate from live cell imaging." ]
Subregional volume reduction of the cholinergic forebrain in subjective cognitive decline (SCD)	Subjective cognitive decline (SCD) patients are considered as a risk population for preclinical Alzheimer's Disease (AD). Supporting this idea, previous studies in SCD populations report subtle alterations in various cognitive and neuroimaging biomarkers that are typically affected during AD progression. To extend these observations, the present study examined whether SCD patients show atrophy of cholinergic basal forebrain nuclei (chBFN), analogous with recent findings in prodromal and clinical AD patients. We assessed volume reductions of the chBFN in 24 SCD subjects compared to 49 matched controls on 3D-T1-weighted MR images based on a postmortem derived atlas. Furthermore, we assessed whether chBFN atrophy was linked with cognitive, structural and metabolic biomarker alterations we previously reported in this SCD cohort: Using correlation analyses we tested for associations between the volumes of the chBFN with the hippocampal gray matter volume, and posterior medial glucose consumption, and the trajectory of verbal memory performance. The SCD cases showed a significant total volume reduction of the chBFN, with largest effect sizes in the Ch1/2 and Ch4p subdivisions of the chBFN. The latter was associated with a reduced glucose metabolism in the precuneus for the SCD group only. These data show an early involvement of the cholinergic basal forebrain nuclei in SCD predominantly in Ch1/2 and Ch4p which supports the conceptual link between SCD and preclinical AD.	[ "MEDI", "NEUR" ]	[ "Alzheimer", "Basal forebrain", "Glucose metabolism", "Nucleus basalis Meynert", "Precuneus", "Subjective cognitive decline" ]	[ "2728", "2741", "2805", "2808" ]	[ "The research focus of early disease detection in Alzheimer's disease (AD) is moving to the preclinical stage, where cognitive performance levels are still in the normal range (Sperling et al., 2014; Sperling et al., 2011).", "Frequently, identification of these cognitively normal at-risk individuals is based on biological markers, either genetic risk factors (e.g. familial predisposition, or APOE epsilon 4 carriers), or by direct measurement of amyloid depositions using cerebral spinal fluid (CSF) assays or positron emission tomography (PET).", "Another, clinically-based approach is the examination of individuals who experience subjective cognitive decline (SCD) while their objective cognitive performance level is still in the normal range, which may reflect incipient AD (Jessen et al., 2014).", "Indeed, accumulating evidence suggests that SCD individuals are at an increased risk of future dementia (Dufouil et al., 2005; Glodzik-Sobanska et al., 2007; Jessen et al., 2010; Reisberg et al., 2010; van Oijen et al., 2007), and frequently display incipient alterations of AD-related neuroimaging biomarkers (Amariglio et al., 2012; Erk et al., 2011; Perrotin et al., 2012; Visser et al., 2009; Wang et al., 2013).", "For example, structural MRI investigations with SCD subjects show reduced volumes in areas that are typically affected in clinical AD, i.e. hippocampus and entorhinal cortex (Jessen et al., 2006; Saykin et al., 2006; Scheef et al., 2012; van de Pol et al., 2009).", "A recent study (Peter et al., 2014) observed that SCD patients did not only show a higher similarity of gray matter atrophy patterns with clinical AD patients than controls, but that SCD patients with more AD-like atrophy patterns showed stronger decline in longitudinal episodic memory tests.", "The same SCD population provided initial FDG PET evidence for reduced brain metabolism in the right precuneus (Scheef et al., 2012) which overlapped with the more extensive patterns of temporoparietal hypometabolism frequently observed in prodromal and clinical AD (Chetelat et al., 2003; Drzezga et al., 2003; Minoshima et al., 1997).", "It is well established that AD is associated with cell loss of the cholinergic basal forebrain nuclei leading to a reduced cholinergic innervation of the cortex (Davies and Maloney, 1976; Lehericy et al., 1993; McGeer et al., 1984; Perry, 1980; Ruberg et al., 1990; Whitehouse et al., 1981).", "The cholinergic denervation contributes to the development of clinical symptoms and is the basis for the efficacy of acetylcholine esterase inhibitors in the treatment of cognitive impairment in AD (Dumas and Newhouse, 2011; Ellis et al., 2006; Furey, 2011).", "MRI-based volumetry has shown that gray matter volume of the chBFN declines from prodromal to clinical stages of AD (Grothe et al., 2012; Kilimann et al., 2014), which is in line with neuropathological findings (i.e.(Lehericy et al., 1993)).", "While some extent of structural basal forebrain atrophy can also be observed in healthy aging, chBFN atrophy is more pronounced in individuals with early AD, especially in those showing a clinical progression over time (Grothe et al., 2013).", "The earliest affected part of the cholinergic system is the Nucleus basalis Meynert (NBM), especially the posterior part (Ch4p), showing volume reductions already in patients with mild cognitive impairment (MCI) as a supposed prodromal disease stage of dementia due to AD (Grothe et al., 2010; Kilimann et al., 2014).", "Furthermore, basal forebrain atrophy in MCI patients shows cross-sectional correlations with atrophy of other cortical brain regions implicated in AD, such as hippocampus and precuneus, as well as with alterations in connecting fiber tracts (Grothe et al., 2010; Teipel et al., 2011), which may reflect an early involvement of basal forebrain degeneration is AD pathogenesis.", "Actually, longitudinal data from the ADNI cohort show that NBM atrophy in mildly amnestic individuals precedes entorhinal degeneration and can predict longitudinal memory impairment (Schmitz et al., 2016).", "Critically, a correlation of basal forebrain volumes with amyloid deposition in cognitively normal and mildly impaired non-demented elderly suggests that chBFN alterations may already be present in preclinical at-risk individuals with confirmed AD pathology (Grothe et al., 2010).", "Moreover, an earlier study found reduced basal forebrain volume in cognitively unimpaired subjects who subsequently developed dementia due to AD (Hall et al., 2008).", "Together, these previous findings suggest that atrophy of the basal forebrain is an early event in AD pathogenesis that is already emerging in preclinical AD.", "Thus, if SCD frequently reflects preclinical AD, we should expect similar incipient chBFN atrophy patterns.", "In this study, we measured chBFN volumes in a previously reported sample of SCD subjects who predominantly report subjective memory impairment (Peter et al., 2014; Scheef et al., 2012).", "Based on high-resolution T1-weighted MRI data, we compared the chBFN volumes in SCD subjects with a matched group of elderly non-SCD individuals.", "We hypothesized that the SCD cohort will show reduced chBFN volumes, most pronounced in the posterior part Ch4p.", "To examine their relationship with other putative biomarkers of AD progression, we assessed the association of basal forebrain volumes with (a) cross-sectional and longitudinal memory performance over the course of up to 4 years (Peter et al., 2014), and with (b) right hippocampal volume reduction, and right precuneal hypometabolism (Scheef et al., 2012): In our previous report, these regions did not only show the most robust group differences, but also presented correlations with subsequent memory decline, making them the most plausible candidates for AD-related neurodegeneration in this sample.", "We expected that this atrophy would be associated with metabolic and structural markers of AD-typical neurodegeneration, as well as with declining memory performance over time.", "Individuals with SCD (n = 24) were recruited by the memory clinic of the Clinical Treatment and Research Center for Neurodegenerative Disorders (KBFZ), Departments of Psychiatry and Neurology, University Hospital Bonn.", "The definition of SCD was based on the fact that subjects were referred to the memory clinic for work-up of memory complaints and on a standard question: ‘Do you feel like your memory is becoming worse?’", "Possible answers to this question were: ‘no’, ‘yes, but this does not worry me’ and ‘yes, this worries me’.", "To be classified as SCD in this study, the subjects had to answer with ‘yes, this worries me’.", "We only included subjects whose memory worsening was confirmed by others (Vellas et al., 2008).", "In all cases, the informant was the spouse or a close relative (usually those who accompanied the patient with SCD during the visit of the memory clinic).", "In addition, only patients with an onset of experienced memory impairment within the last 10 years were included, in order to exclude life-long ‘memory complainers’.", "The control cohort (n = 49) was recruited from the general population.", "Participants were included as controls if they denied any memory worsening or if a memory decline was only reported on active inquiry and was considered of no concern.", "For all subjects, normal cognitive function was defined by performance on the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neuropsychological battery (Morris et al., 1989).", "For both groups, exclusion criteria were: (1) past or present neurological disorders; (2) significant medical diseases; (3) medication that may interfere with cognition, including any psychotropic medication; (4) any other detectable causes of memory impairment.", "Current and life-time psychiatric disorders were assessed with the German version of the Structured Clinical Interview for DSM-IV, SCID (Wittchen et al., 1997).", "Subjects with any psychiatric disorder at present or in the past were excluded, with the exception of a single depressive episode >10 years ago, which was reported by two patients with SCD and four subjects in the control group.", "In addition, the Beck Depression Inventory (BDI) was completed by all subjects (Beck et al., 1961) (Table 1).", "The study protocol was approved by the ethics committee of the medical faculty of the University of Bonn.", "It was performed in accordance with the Declaration of Helsinki.", "All participants provided written informed consent.", "The neuropsychological test battery was designed to test verbal and visual memory, processing speed and executive functions, and was administered at baseline and at follow-up visits.", "Verbal memory was assessed with the Verbal Learning and Memory Test (VLMT) (Helmstaedter et al., 2001), the German version of the Rey Auditory Verbal Learning Test (Rey, 1964).", "Visual memory was tested with the Rey Complex Figure test (Osterrieth, 1944).", "Processing speed and executive functions were tested with the Trail Making Test (TMT) A and B (Reitan, 1958) and with a semantic lexical 2-min verbal fluency task (Aschenbrenner et al., 2000).", "Because episodic memory is the most severely and earliest affected cognitive domain in MCI and AD, we focus in this paper on VLMT only (Rabin et al., 2009).", "Both groups returned to the memory clinic for three follow-up visits including neuropsychological testing (see below).", "On average, the SCD group returned 14 ± 3.0 months, 32.5 ± 6.1 months, and 45.1 ± 5.6 months after baseline, while the control group returned after 15.5 ± 2.9 months, 33.3 ± 6.1 months, and 41.7 months.", "Twenty-two SCD subjects had at least one follow-up, 16 had two follow-up visits and 11 had three follow-up visits.", "Forty-five participants of the control group returned for at least one follow-up visit, 36 participants had two follow-up visits, and 15 had three follow-up visits.", "Structural data were obtained from 24 SCD subjects and 49 controls.", "All scans were performed on a 3 Tesla MR system (Philips Achieva, Philips, Best, Netherlands), equipped with an 8-channel SENSE head coil.", "For each participant, four high-resolution T1-weighted data sets were acquired consecutively, using a 3D turbo field echo (TFE) sequence (SENSE reduction factors 2.5AP, 1.5RL, TE/TR/flip angle = 3.6 ms/7.6 ms/8°, field of view 256 × 256 mm2, matrix size 320 × 320, number of slices = 170, spatial resolution 0.8 × 0.8 × 0.8mm3).", "Preprocessing was performed using the VBM8 toolbox (C. Gaser, www.dbm.neuro.uni-jena.de/vbm8/).", "Preprocessing included realignment and averaging of each participant's four structural data sets, tissue class segmentation, and spatial normalization to MNI standard space using the high-dimensional registration algorithm DARTEL (Ashburner, 2007).", "The total amounts of GM, white matter (WM), and cerebrospinal fluid (CSF) were kept constant during the normalization by multiplying each voxel value of the tissue-class-specific probability map with the determinant of the Jacobian matrix of the local deformation matrix (Good et al., 2001).", "The individual probability maps for GM, WM and CSF derived from the normalization procedure were summed up to estimate the total intra-cranial volume (ICV).", "Based on the finding from Scheef et al., 2012, we chose the brain region that showed the nominally largest effect size in local gray matter volume group difference and was associated with memory decline.", "The rationale for this approach was on one hand to increase the likelihood to reveal any existing association between those imaging markers and the cholinergic forebrain volumes, and on the other hand to be as close as possible to the clinical aspect of the SCD group, i.e. memory decline.", "The region that meets both conditions was the right hippocampus.", "Region of interest analysis (ROI) of gray matter volume for the right hippocampus was performed following a protocol described previously (Scheef et al., 2012) calculating the volume using the normalized unsmoothed GM partition.", "The ROI templates were taken from the WFU Pick Atlas (Maldjian et al., 2003).", "The individual ROI volumes were calculated by adding together all voxel intensity values of the GM partition within a ROI.", "The ROI volumes were normalized by dividing the individual ROI volumes with the individual ICV and multiplying the result with the mean ICV.", "The cholinergic basal forebrain is composed of four different cell groups, which form partially overlapping ‘subunits’ (Ch1–Ch4), and project to different anatomical brain structures (Mesulam, 2004b; Mesulam and Geula, 1988; Mesulam et al., 1983a; Mesulam et al., 1983b).", "Ch1 reflects the medial septal nucleus, Ch2 the vertical nucleus of the diagonal band of Broca, Ch3 the horizontal limb of the diagonal band nucleus, and Ch4 refers to the cholinergic component of the nucleus basalis Meynert (NBM) (Mesulam, 2004b).", "The latter can be further subdivided into anterior lateral (Ch4al) and medial (Ch4am), intermediate (Ch4i), and posterior regions (Ch4p).", "A lateral extension of the Ch4al division has been termed Nucleus subputaminalis (NSP), or Ayala's nucleus, and has only been described in humans and anthropoid monkeys so far (Boban et al., 2006; Simic et al., 1999).", "The main cholinergic innervation of the hippocampus is provided by Ch1 and Ch2.", "Ch3 projects into the olfactory bulb, and Ch4 provides the cholinergic innervation of the cortex (Mesulam, 2004b; Mesulam et al., 1983a).", "Basal forebrain volumes were extracted using a cytoarchitectonic map of chBFN in MNI standard space (Kilimann et al., 2014).", "In short, the chBFN masks were derived from a post-mortem brain.", "This brain was scanned ‘in cranio’ as well as dehydrated before histologically processed.", "The histologically delineated chBFN subnuclei ROIs were then transferred onto the MRI of the dehydrated brain.", "The latter was transformed into the space of the in cranio MRI by applying a 12-parameter affine transformation and high-definition normalization and warped into MNI-space using DARTEL (Ashburner, 2007).", "The ROIs were transformed into the MNI-space by applying the derived transformation information on the chBFN ROIs in ‘dehydrated brain space’.", "An overview on the spatial location, and the extent of the chBFN in MNI-space is given in Fig. 1.", "Due to the small size, Ch1 and Ch2 were not considered separately and will be referred to as ‘Ch1/2’ in the following.", "We also combined Ch4am, Ch4al without NSP, and Ch4i into one volume (Ch4).", "The posterior region (Ch4p) and NSP were considered separately, and the ROIs were combined across both hemispheres for all of the nuclei.", "As described for the hippocampal volume, the individual chBFN volumes were calculated by summing the voxel intensity values of the GM partition across the chBFN ROIs.", "The total volume of the basal forebrain nuclei (chBFNtot) was calculated by adding up the volumes of all subdivisions.", "All volumes were normalized by the ICV as described above.", "All [18F] fluoro-2-deoxyglucose PET (FDG-PET) scans were performed on a PET/CT scanner (Biograph, Siemens Medical Solutions, Inc.).", "Imaging started 30 min after injection of 150 MBq FDG.", "The acquisition started with low dose CT-scan for attenuation correction (19 mAs, 110 keV, 1 cm slice thickness).", "PET emission data acquisition was performed over 20 min.", "The subsequent attenuation correction for PET was based on a rescaling of the CT image and scatter correction using a single scatter model.", "Emission data were reconstructed with an in-plane spatial resolution of 1.3 × 1.3 mm2 and a slice thickness of 2.4 mm.", "The intrinsic resolution of the Biograph system is 9.3 mm (Prieto et al., 2010).", "The preprocessing of the FDG-PET data was performed with SPM8; Wellcome Department of Cognitive Neurology, Institute of Neurology, London, UK.", "Data were transformed, normalized into a common stereotactic space, resliced with a voxel size of 2 × 2 × 2 mm3, and smoothed with a 12-mm kernel.", "Global counts were normalized by proportional scaling to a mean value of 50 mg/100 ml/min and by applying a threshold masking of 80% of maximum intensity.", "Following the approach described for the MRI-ROI selection, we chose the right precuneus as ROI for the PET analysis, because this region showed the highest effects strength in the direct group comparison and was associated with the memory decline over time (Scheef et al., 2012).", "The mean regional glucose metabolism for the right precuneus ROI was extracted, averaged, and additionally weighted by the mean ROI-GM volume for correlation analyses as described previously (Scheef et al., 2012).", "As for the MRI-analysis, the ROI templates were taken from the WFU Pick Atlas (Maldjian et al., 2003).", "χ2 or t-test statistics were used to test for group differences in demographic data, CERAD data and in the neuropsychological battery.", "The main outcomes are displayed in the Tables 1 and 2.", "All subjects -except one SCD subject- participated in longitudinal neuropsychological follow-up.", "In this study, we focused on the VLMT performance only, which showed a significantly different trajectory for the groups, as reported previously (Peter et al., 2014), and whose decline is an established clinical predictor of AD (Bastin and Salmon, 2014).", "In order to characterize VLMT performance at T0 and follow-ups, the different VLMT sub-scores were reduced to one single factor (‘memory total score’) by an exploratory and confirmatory factor analysis of the four subscores (immediate recall, immediate recall after interference, delayed recall after 30 min, and recognition).", "To accommodate varying numbers of follow-up visits per subject and irregular interval length, growth curve modeling with linear growth factors was used to characterize the individual trajectory of the memory total score.", "The slope extracted by the growth curve model describes the individual course of memory performance over time.", "Both approaches, the factor analytical approach of the VLMT and the linear growth curve model, are described in detail elsewhere (Peter et al., 2014).", "To test whether the SCD group showed a decreased total volume of the basal forebrain nuclei (chBFNtot) we used a one-sided independent two sample t-test with the a priori hypothesis of smaller volumes in the SCD group.", "In a second step, we tested which subnucleus was affected and determined the respective effect sizes.", "The Shapiro-Wilk test was used to test for normal distribution, and the Levene test was used to test for equality of variance (Levene, 1960; Shapiro and Wilk, 1965).", "All further analyses were restricted to chBFNtot and those nuclei showing a significant group effect.", "As discussed below, groups differed significantly regarding BDI scores (see also: (Scheef et al., 2012)).", "To explore potential linear relationships of this confounded covariate with our variables of interest, we additionally investigated the correlations of the BDI with the cholinergic forebrain volumes for both groups separately.", "In order to investigate the association between chBFN volume reduction and our second putative marker of AD-related degeneration, we performed a correlation analysis in the SCD group between the significantly atrophied basal forebrain nuclei and the right hippocampus.", "For any correlation yielding a significant effect, we conducted complementary analyses for the control group in order to test the specificity of the finding.", "Group differences were tested using linear regression analysis.", "To examine potential associations between chBFN volumes and regional glucose metabolism, similar analytic procedures as for the structural MRI data were applied.", "The right precuneus served as target region.", "All participants scored within 1.5 standard deviations (SD) of the mean according to age-, gender-, and education adjusted norms2 on all subtests of the CERAD test battery.", "Both groups did not differ in terms of gender, age, education, APOE 4 distribution (Hixson and Vernier, 1990, Table 1).", "Follow-up rates did not differ between the SCD and control group (χ2(3) = 2.67, not significant).", "No significant difference with regard to age, gender, and years of education existed between those participants still remaining in the study and those dropping out.", "The SCD group scored slightly lower in the MMSE but was still well in the range of normal cognition (≥24 = no cognitive impairment; Tombaugh and McIntyre, 1992).", "The difference was not statistically significant after correcting for multiple comparisons.", "While a significant group difference was found in the BDI, both groups scored within the normal range, with no participant reaching clinical levels.", "As already reported in Scheef et al. (2012), we did not find any association between BDI scores and any cognitive measure or imaging marker.", "Especially for the cholinergic forebrain, correlations between BDI scores and chBFNtot or its subnuclei were not statistically significant.", "All chBFN volumes were normally distributed for both groups and homogeneity of variances was met according to Shapiro-Wilk and Levene's Test, respectively.", "The (normalized) chBFNtot volume was smaller in the SCD group (mean ± SD = 600 ± 76 mm3) compared with the (normalized) volumes of the control group (mean ± SD = 642 ± 102 mm3).", "This difference of 42 mm3 was significant (t71 = 1.80, p = 0.038), with a small to moderate effect size (Cohen's d = 0.45).", "Following up the significant overall effect, the Ch1/2 and Ch4p nuclei showed significantly smaller volumes in the SCD group as compared with the control group, with moderate effect sizes (81 ± 14 mm3 vs. 91 ± 19 mm3; t71 = 2.29, p = 0.013, d = 0.57 for Ch1/2, and 99 ± 15 mm3 vs. 106 ± 15 mm3, t71 = 2.01, p = 0.024, d = 0.50, for Ch4p, respectively).", "The analyses for the other nuclei yielded no significant effect, although a smaller group difference in Ch3 volume approached significance (t71 = 1.61, p = 0.056, d = 0.38).", "The main results are summarized in Table 3.", "Modeling memory performance over time using a growth curve model revealed a significant memory decline over time for the SCD group (slopeSCD = −0.033 ± 0.017), compared to a slight improvement for the control group (slopeControl = 0.150 ± 0.123).", "The group difference was significant (t70 = 10.2, p <0.000).", "Neither for the control group nor for the SCD cohort we found any significant correlations between chBFN volumes and the memory performance over time.", "No significant correlation was found between Ch1/2 and right hippocampal volume (Table 4).", "For both groups, the Ch4p volume was highly correlated with the gray matter volume of the right hippocampus (Table 4).", "However, this correlation did not differ significantly between groups.", "In an explorative approach, we also correlated the individual Ch1/2 and Ch4p volumes with the individual total hippocampal volumes.", "This did not yield a different result.", "No significant correlation was found for Ch1/2 and hippocampal volumes but the correlation between the Ch4p the hippocampal volume was highly significant.", "As for the unilateral ROI, the correlation coefficients did not differ significantly (Control: rCh4p,hippo = 0.647, p <0.001; SCD: rCh4p, hippo = 0.656, p = 0.001; z = 0.06, p =0.9; all tests were performed two sided).", "Ch1/2 did not show any significant correlations with metabolism in the respective ROI for any single group, or after pooling the data across both groups.", "Meanwhile, Ch4p showed a significant association with right precuneus metabolism for the SCD group only (Table 4) and yielded a significant group effect (right precuneus: Tgroup = 3.739, p = 0.0004; see also Fig. 2 and Table 4).", "Following the same explorative approach described above for the hippocampus, the results were almost paralleled.", "Pooling the precuneus metabolism across both hemispheres did not reveal a significant correlation between Ch1/2 volume and metabolism for any of both groups.", "The same holds true for the correlation between Ch4p for the control group.", "The correlation between Ch4p volume and bilateral precuneus metabolism showed a strong trend towards significance (p =0.053).", "The difference of the correlation coefficient was significant (Control: rCh4p, precuneus = −0.153, p =0.3; SCD: rCh4p, precuneus = 0.418, p = 0.053; z = −2.14, p =0.03; all tests were performed two sided).", "In the present study, we used in vivo volume measurements of cholinergic forebrain nuclei in SCD and correlated the findings with memory trajectory over time, hippocampal volume and glucose metabolism of the precuneus.", "Consistent with the idea that SCD patients show a higher risk of preclinical, AD-related neuroimaging and cognitive alterations, we were able to show that the cholinergic basal forebrain volume was smaller in SCD compared to controls matched for age, gender and ApoE status, consistent with previous findings in MCI and AD dementia.", "Even though almost every nucleus showed atrophy, greater effect sizes were observed for Ch1/2 and Ch4p than for Ch3.", "While these atrophic changes were not predictive for the cognitive trajectory of the SCD patients, they were related to other AD-related neuroimaging markers that we previously found to be altered in this cohort (Scheef et al., 2012).", "We showed a significant association between the volume of the Ch4p subregion of the Nucleus basalis Meynert and right precuneal hypometabolism, a brain region that is known to show alterations already in early stages of AD development.", "Importantly, the latter association was only present in the SCD group.", "The observed basal forebrain atrophy is in line with postmortem and in vivo studies in prodromal and clinical AD patients: Neuropathological data show that AD is associated with a loss of cholinergic neurons of the chBFN (Arendt et al., 1985; Vogels et al., 1990), and a consecutive loss of cortical cholinergic innervation (Geula and Mesulam, 1989).", "Neuronal loss seems to follow a specific pattern.", "It is most pronounced in the posterior parts of the NBM, especially Ch4p, whereas the rostral parts are less affected (Arendt et al., 1985; Doucette et al., 1986; Liu et al., 2015; Vogels et al., 1990; Wilcock et al., 1983).", "Using structural MRI it was not only possible to replicate the histopathological data in AD, showing an atrophy of the whole chBFN in AD dementia (Grothe et al., 2012; Kilimann et al., 2014), but to demonstrate that atrophic changes in the NBM (Schmitz et al., 2016) and most pronounced in its posterior subdivision (Grothe et al., 2013; Grothe et al., 2012; Grothe et al., 2014) are already detectable in MCI patients.", "Notably, recent data show that basal forebrain volume in cognitively normal individuals is negatively associated with brain amyloid load (Grothe et al., 2014), suggesting that incipient atrophy already develops in preclinical AD.", "Consistent with the idea that SCD frequently reflects preclinical AD, our findings may extend the latter observations towards SCD patients by also showing a significantly reduced Ch4p (and Ch1/2) volume in this cognitively normal sample.", "A relationship between the Ch4p alterations in our SCD patients and AD-related neurodegenerative mechanisms is further supported by the observed correlation between Ch4p volume, and the glucose metabolism the right precuneus, which was only observed in the SCD group.", "The lower the volume of the Ch4p the lower the glucose consumption in the right precuneus.", "The exploratory data analysis using a bilateral precuneus ROI revealed a similar result.", "Even though the effect seems to be driven by the right precuneus a larger group size might be needed to further elucidate any laterality effect.", "However, the correlation between the Ch4p volume and the right precuneus metabolism is a remarkable finding, because lower precuneus glucose metabolism is known to be a highly sensitive and early marker for the development of AD (Minoshima et al., 1999; Minoshima et al., 1997).", "A detection of a decreased metabolism in the posterior cingulate (PC) and precuneus region was associated with a conversion rate of >90% within two years from MCI to AD (Chetelat et al., 2003; Drzezga et al., 2003; Minoshima et al., 1997).", "Even though it is tempting to assume that a reduced cholinergic innervation of the parietal lobe triggers the observed reduction of the glucose metabolism in the medial parietal cortex, it is not very likely that the reported relationship between metabolism and Ch4p deterioration is causal.", "The main cortical projection site of the Ch4p subdivision is assumed to lie in the superior temporal gyrus and temporal pole (Jones et al., 1976; Mesulam and Geula, 1988; Mesulam et al., 1983a), whereas the precuneus has shown to be mainly connected to anterior parts of the chBFN (Mesulam, 2004b; Parvizi et al., 2006).", "The link may be indirect, i.e. depend on the mediation (or common causation) of unidentified additional brain mechanisms.", "Here, integration with connectivity-based methods like resting-state fMRI (e.g. Hafkemeijer et al., 2013; Verfaillie et al., 2018) may help to explore underlying system-level mechanisms.", "While the observed associations of basal forebrain volumes with our second putative neuroimaging marker for AD neurodegeneration, right hippocampal GM volume, seem to provide further support for this claim at first sight, caution is warranted, since significant correlations were observed in both SCD and control participants: Accordingly, they may reflect a common non-AD process, e.g. aging.", "Despite the agreement of the presented Ch4p/NBM atrophy in SCD with findings reported in MCI and AD, and the relation with local glucose metabolism, our data also show distinct differences regarding the role of Ch1/2 with respect to the literature.", "While reports on MCI and early-stage clinical AD published so far (Fujishiro et al., 2006; Grothe et al., 2012; Kilimann et al., 2014; Vogels et al., 1990) support the idea of a later spreading atrophy from the posterior to anterior parts of the cholinergic forebrain (Mesulam, 2004a), we did already find an early involvement of the Ch1/2 complex.", "Moreover, although the Ch1/2 volume was significantly smaller in the SCD group, it did not show an association with local glucose metabolism or local gray matter volume in AD-relevant brain regions.", "In summary, these Ch1/2 findings do not fit well with an AD-related neurodegenerative process, but may reflect non-AD pathologies, although CSF or PET biomarkers of amyloid (or other) neuropathology would be needed to further elucidate this issue (see: Limitations).", "On the other hand, Ch1/2 provides the main cholinergic innervation for the hippocampus which serves as a core structure for episodic memory, and Butler and colleagues have already shown that the volume of this region is predictive for memory performance in healthy adults (Butler et al., 2012).", "This is also confirmed by a recent study where Ch1/2 volume showed a much stronger association with memory performance than the other chBFN (Wolf et al., 2014).", "Therefore, findings of Ch1/2 atrophy in a population of SCD patients whose complaints were primarily characterized by symptoms of memory decline, show face validity.", "Yet, as we could not replicate the previously reported correlations between Ch1/2 atrophy and memory decline, functional interpretations have to be cautious.", "The reported data in this study are based on in vivo MRI measurements of structures that do not have a sufficient MR contrast to its neighborhood, and thus the derived chBFN volumes rely on a recently developed cytoarchitectonic atlas that was based on postmortem data of a single subject (Kilimann et al., 2014).", "Given that the ground truth is based on a single subject, the reliability of such an indirect measure can be called into question.", "Reliability is determined by two factors: 1.", "accurate definition of the ROIs, and (2) accuracy of the volumetric method, including GM segmentation and spatial normalization.", "Recent data show that the chBFN delineated based on the single subject template are in good agreement with chBFN delineated by a cytoarchitectonic probability map which was derived from 10 subjects (Grothe et al., 2010; Zaborszky et al., 2008).", "The center of gravity coordinates for the different chBFN of the Zaborszky probabilistic atlas project well onto the chBFN atlas published by Kilimann et al. (2014).", "Therefore, one can assume that the used atlas sufficiently represents the chBFN.", "As a consequence, the reliability is basically limited by the volumetric method itself.", "The consistent findings across imaging modalities, cohorts, in vivo and postmortem measurements in MCI and AD argue for the validity of the method.", "However, one should still keep in mind that this method provides an indirect volume measure only, which cannot distinguish between glial or neuronal loss, or between cell shrinkage or cell death.", "Considering the very small size of the chBFN, the method may also be more susceptible to unspecific normalization effects based on global group differences, but the employed high-dimensional normalization algorithm has been shown to reduce those effects to a minimum (Klein et al., 2009).", "The observed specificity of the atrophy pattern (showing the strongest differences for Ch1/2 and Ch4p), as well as the observed covariation with local GM volume in other brain regions further argues against unspecific normalization effects.", "One might argue that groups differences in basal forebrain volumes were actually driven by confounding group differences in depression level, which represent an independent, non-neurodegenerative influence on SCD symptomatology.", "In general, all participants' BDI scores were within the normal range, and none of the participants reached clinical levels.", "Moreover, we did not find any significant correlation between the BDI scores and any cognitive measures or imaging marker (see: Scheef et al., 2012), including BF total and subregional volumes, making it is very unlikely that the group difference in BDI had any relevant influence on the study outcomes.", "In fact, there is a paucity of studies pointing towards basal forebrain volume reductions even in clinically depressed patients.", "The sample size can be considered as an additional limitation.", "Even though the group sizes were sufficiently high to detect statistically significant differences in chBFN volumes, a larger group size might have allowed to elucidate further the role of the Ch1/2 atrophy within the SCD group.", "As an additional point one might argue that the tests used to compare the volume differences between both groups were one-sided and the reported data are not corrected for multiple comparisons.", "However, considering histology data and imaging data at preclinical stages, testing for atrophy only can be justified.", "In addition, the main findings of this paper, the atrophy of the cholinergic basal forebrain and significant correlation between Ch4p volume and precuneus hypometabolism in SCD, would still hold if one would only test for changes of the BF-total volume - ignoring the substructure of the cholinergic system- and perform the correlation analysis with those subregions showing the nominally largest volume reduction (i.e. Ch1/2–11%; Ch4p ~7%).", "Also, the irregular numbers of follow-up visits and intervals in-between are not optimal.", "However, by using a growth curve model to determine the individual memory trajectory, we addressed this problem adequately.", "Unfortunately, the observation time was still too short to clearly associate the selected cognitive and neuroimaging parameters with individual conversion to dementia which would prove the underlying assumption that they represent actual biomarkers for preclinical AD progression.", "For this, the required observational time periods are estimated to be in the range of 10–15 years in samples that start at the SCD stage (Reisberg et al., 2008; Reisberg et al., 2010).", "Of course, confirmation of preclinical AD in SCD patients does not require clinical long-term follow-up but could be achieved by in vivo measurements of AD pathology, i.e. CSF or PET examinations of amyloid burden.", "Unfortunately, this sort of information is not available for the present cohort.", "Yet, our initial findings may provide a starting point for further testing in larger-scale longitudinal study cohorts which also provide additional amyloid measures, e.g. the Harvard Aging Brain Study (Amariglio et al., 2015).", "To the best of our knowledge, the presented data show for the first time that chBFN atrophy is already present in SCD which would support the idea of SCD as an at-risk population for preclinical AD.", "This notion is further supported by an association between Ch4p atrophy and decreased glucose metabolism in the right precuneus in the SCD group.", "These findings may provide a good starting point for larger-scale studies which also take into account direct measures of AD pathology, and longer follow-up durations to monitor the long-term clinical outcomes of this risk population." ]	[ "Cholinergic forebrain shows atrophy in SCD.", "Ch12 and posterior part of Nucl.", "Basalis Meynert (Ch4p) show largest effect size.", "Atrophy of the Ch4p correlates with reduced glucose metabolism in precuneus." ]
Psychiatry & the psychedelic drugs. Past, present & future	The classical psychedelic drugs, including psilocybin, lysergic acid diethylamide and mescaline, were used extensively in psychiatry before they were placed in Schedule I of the UN Convention on Drugs in 1967. Experimentation and clinical trials undertaken prior to legal sanction suggest that they are not helpful for those with established psychotic disorders and should be avoided in those liable to develop them. However, those with so-called ‘psychoneurotic’ disorders sometimes benefited considerably from their tendency to ‘loosen’ otherwise fixed, maladaptive patterns of cognition and behaviour, particularly when given in a supportive, therapeutic setting. Pre-prohibition studies in this area were sub-optimal, although a recent systematic review in unipolar mood disorder and a meta-analysis in alcoholism have both suggested efficacy. The incidence of serious adverse events appears to be low. Since 2006, there have been several pilot trials and randomised controlled trials using psychedelics (mostly psilocybin) in various non-psychotic psychiatric disorders. These have provided encouraging results that provide initial evidence of safety and efficacy, however the regulatory and legal hurdles to licensing psychedelics as medicines are formidable. This paper summarises clinical trials using psychedelics pre and post prohibition, discusses the methodological challenges of performing good quality trials in this area and considers a strategic approach to the legal and regulatory barriers to licensing psychedelics as a treatment in mainstream psychiatry. This article is part of the Special Issue entitled ‘Psychedelics: New Doors, Altered Perceptions’.	[ "NEUR", "PHAR" ]	[ "Clinical trials", "Psychedelics", "Psychiatric disorders" ]	[ "2804", "3004" ]	[ "The classical psychedelic drugs include mescaline, psilocybin, dimethyltryptamine (DMT) and d-lysergic acid diethylamide (LSD).", "Coined by psychiatrist Humphrey Osmond in a letter he wrote to Aldous Huxley in 1956, the word ‘psychedelic’ is derived from the ancient Greek words psychē (ψυχή, translated as “soul” or “mind”) and dēlein (δηλειν, translated as “to reveal” or “to manifest”).", "Therefore, psychedelic literally translates as ‘mind manifesting’ or ‘soul revealing’ (Osmond, 1957).", "Other terms such as ‘hallucinogen’ and ‘psychotomimetic’ are less favoured, perhaps because they place too much emphasis on individual elements of a multi-faceted subjective state.", "Psychedelics were used long before the Western world was introduced to them in 1897, when Arthur Heffter isolated mescaline from the peyote cactus.", "The earliest direct evidence for use of psychotropic plants dates back 5700 years in the north eastern region of Mexico (Bruhn et al., 2002), where carbon-dated buttons of peyote cacti and red beans containing mescaline were found in caves used for human habitation.", "The Eleusian ceremonies of ancient Greece were likely based around some form of psychedelic compound (Wasson et al., 2008).", "Psilocybin containing ‘magic’ mushrooms, which grow all over the world, appear to have been used ubiquitously (Akers et al., 1992; Letcher, 2008).", "Mescaline is still used in Native American Church ceremonies (Stewart, 1987).", "In Brazil (McKenna et al., 1984) and the broader Amazonian basin (Schultes and Hofmann, 1979), ritual healing practices and spiritual ceremonies are practiced using ayahuasca, a drink which combines plant derived DMT and β-carboline monoamine oxidase inhibitors that allow it to be used orally.", "The archetypal psychedelic in modern Western society, LSD, was first synthesised in 1938 by Albert Hofmann as part of a systematic investigation of compounds derived from the ergot alkaloids at the Sandoz laboratories in Switzerland (Hofmann, 2013).", "The ergot alkaloids, which include lysergic acid and its derivatives, were known to be responsible for episodes of mass poisoning in medieval Europe from stocks of grain spoiled with the parasitic fungus Claviceps Purpurea.", "In smaller doses, a specific ergot alkaloid (ergometrine) was also known to be effective for treating bleeding in women after childbirth due to its vaso- and utero-constrictive properties.", "LSD was the 25th derivative of lysergic acid that Hofmann synthesised, explaining why it is sometimes referred to as ‘LSD-25’ (Hofmann, 2013).", "In animal testing, LSD was found to be physiologically unremarkable and the compound was shelved.", "Hofmann describes how he decided to resynthesize LSD in 1943 on the basis of a ‘peculiar presentiment … that this [compound] might possess properties beyond those established in the first pharmacological studies’ (Hofmann, 2013).", "On April 16th of that year Hofmann accidentally contaminated himself with a small amount and, noticing some unusual psychic effects, purposefully ingested 250 mcg 3 days later, whereupon the full, and remarkably potent, effects of LSD on the psyche became apparent for the first time.", "Further investigation of LSD by Sandoz found that, despite its potency, it was a notably non-toxic compound physiologically.", "Recognising that its psychoactive properties were likely to be of interest both to psychiatrists and academics, it was marketed in 1947 under the trade name ‘Delysid’ and made freely available to those interested in researching its properties.", "Hofmann also isolated and synthesised the active component of psilocybe ‘magic’ mushrooms, psilocybin, in 1958 (Hofmann et al., 1959).", "This was marketed by Sandoz under the brand name ‘Indocybin’.", "At a time when psychiatry lacked effective medical therapies, the discovery of LSD was of interest, with some key features noted.", "Firstly, acute intoxication appeared to mimic some of the symptoms of acute psychosis, particularly ego-dissolution, thought disorder and visual misperceptions (although not, notably, auditory hallucinations).", "Secondly, there appeared to be an increased awareness of (and emotional connection to) repressed memories and other elements of the subconscious, which was thought to be potentially useful in those failing to make progress in psychotherapy.", "Physiological toxicity was not observed, even after very large overdose.", "However, initial testing of psychedelics in patients with schizophrenia showed that they were not helpful, exacerbating psychotic symptoms and failing to lead to clinical improvement.", "Trials in depressive, anxious, obsessive and addictive disorders were more encouraging, with the psychedelics noted to have therapeutic potential within psychologically supportive contexts (Eisner and Cohen, 1958) and a low risk of toxicity (Cohen, 1960).", "By the end of the 1960s, hundreds of papers described the use of mescaline, psilocybin and (most frequently) LSD in a wide variety of clinical populations with non-psychotic mental health problems (Grinspoon and Bakalar, 1981).", "However, as the psychedelics diffused into wider society and recreational use increased, some individuals reported a variety of ongoing symptoms including visual distortions, flashbacks and other symptoms that occurred long after the drugs had left the body.", "This came to be classified as ‘Hallucinogen Persisting Perceptual Disorder’ (Halpern and Pope, 2003).", "Unethical and covert use of psychedelics along with a general hardening of socio-political attitudes towards drug use contributed to the decision to place psychedelics in Schedule I of the 1967 UN Convention on Drugs.", "Medical use ceased and research dwindled until the turn of the millennium, since when there has been a steady renaissance of clinical and academic interest in the psychedelic drugs, reflected by a socio-political narrative that has increasingly questioned the relative benefits and harms of the so-called ‘war on drugs’ (Godlee and Hurley, 2016; Hari, 2015).", "This paper presents a synopsis of selected clinical studies with psychedelics performed before 1970 and all major clinical studies since the turn of the millennium.", "We discuss the controversies and practical considerations in designing modern clinical trials with psychedelics and review the formidable legal and regulatory hurdles that must be overcome if psychedelics are to become licensed medicines in psychiatry again.", "The first medical report of use of a classical psychedelic in Western medicine was made by Prentiss and Morgan in the United States in 1895, who reported the ceremonial use of buttons of the peyote cactus by indigenous people in Central America (Prentiss and Morgan, 1895).", "Mitchell, reporting in the British Medical Journal in 1896, reports self-experimentation with peyote, describing closed-eye visual experiences and commenting that ‘for the psychologist this agent should have value’ (Mitchell, 1896).", "This was a view repeated by Havelock Ellis in 1897, describing the experience as ‘ … mainly a saturnalia of the specific senses, and chiefly an orgy of vision … it is of no little interest to the physiologist and psychologist’ (Ellis, 1897).", "Whilst Arthur Heffter isolated the active component of peyote (mescaline) in 1897, there is limited further mention of it in the English medical literature until 1913, when Alwyn Knauer administered mescaline by subcutaneous injection to himself and other volunteering physicians (Knauer and Maloney, 1913).", "Knauer had worked as an assistant to the psychiatrist Emil Kraeplin who, aside from his involvement in the inception of psychiatric diagnosis, was also interested in the effects of psychoactive drugs in producing psychopathology.", "However, his experimentations were restricted, according to Knauer, to ‘substances, which … produce mental states which have little similarity to actual insanities’.", "Administering mescaline repeatedly both to themselves and other volunteering physicians, they commented, ‘Soon after the onset of the visual hyperesthesia, to nearly all the investigated persons, out of total darkness, kaleidoscopic pictures appeared.’", "Whilst commenting on the ‘ … vividness of the [visual] hallucinations … they came unsought, they were uncontrollable … ’ they also noticed that ‘the independence of the hallucinations to thought and will was never quite absolute.", "On the nature of the conscious experience under mescaline, they noted that it ‘ … remained practically unclouded … somewhat similar to … consciousness in hypnosis’ (Knauer and Maloney, 1913).", "Whilst the nature of hallucinations in psychosis remained opaque, Knauer and Maloney recognised that the mental state induced by mescaline bore some similarity to the psychotic state.", "Heinrich Kluver, in 1926, again experimenting on himself, commented, in addition to the established visual imagery, on changes in ‘object-awareness’ and ‘self-awareness’ (referred to in the original German ‘gegenstandsbewusstsein’ and ‘ichbewusstsein’):", "‘My body and its organs seemed to be most of the time non-existent or detached from me as a perfectly functioning machine.", "While speaking I seemed to listen to a speech apparatus … In general, the line of demarcation drawn between ‘object’ and ‘subject’ in normal state seemed to be changed.", "The body, the ego, became ‘objective’ in a certain way, and the objects became ‘subjective’.", "They became subjective not only in the sense that they behaved as visionary phenomena, but also in the sense that they gained certain affective qualities … There is no doubt that these changes in ‘Gegenstands- und Ichbewusstsein’ are comparable to those observed in schizophrenia’.", "(Kluver, 1926)", "In 1936, Erich Guttman, then working at The Maudsley Hospital in London, United Kingdom, raised the possibility of a therapeutic effect of mescaline (Guttmann, 1936).", "He, and a variety of other colleagues, gave the drug to an undisclosed number of ‘medical students’, ‘undergraduates’, ‘normals’, ‘psychopathic patients’, ‘manic-depressives’, ‘schizophrenics’, ‘depressives’, ‘morphinists’ and those suffering from ‘derealisation’ and ‘depersonalisation’ phenomena (Guttmann, 1936).", "Not publishing any sort of objective results, he nonetheless made the first observation of the potential therapeutic utility of the mescaline-induced state in psychotherapy, noting:", "‘There is reason to suppose that patients in such a state may be very susceptible to psychotherapeutic influence … If it is so, the intoxication could be made use of as a sort of forced or concentrated analysis’.", "Similarly, he recognised the importance of the drug for psychiatrists attempting to understand …", "‘ … the complicated interplay of aetiological factors in the origin of psychoses.", "Careful analysis of one intoxication like mescaline promises a reliable basis for knowledge in the field of toxic psychoses generally, and perhaps hints for the solution of the great problem of psychiatry, that of schizophrenia.’", "Perhaps because mescaline was never marketed (it was first synthesised by Ernst Spaff in 1919 and then manufactured by the pharmaceutical company Merck as a research chemical), but perhaps also because of the predominance of psychoanalytic theory at the time, its use by psychiatrists was sporadic and infrequent.", "In contrast, after Hofmann synthesised LSD in 1943, not only did Sandoz provide it free of charge to psychiatrists, but it came at the same time as the emerging idea that psychiatric states might have biological, rather than psychological, aetiologies.", "Moreover, regulation of the medical community, and of medical research, was minimal.", "Growth of interest in LSD was rapid.", "In 1950, Busch and Johnson (1950), working in Missouri, United States, published one of the first studies using LSD in patients.", "They gave 21 patients, mostly hospitalised with schizophrenic or manic episodes, LSD.", "They observed that all patients showed ‘increases in activity’, particularly those with mania.", "Based on these observations they gave LSD to a further 8 patients.", "3 had diagnoses of catatonic schizophrenia, 4 ‘psychoneurosis’ and 1 ‘psychosomatic’.", "Describing the results narratively,", "‘The effect … disturbed the barrier of repression and permitted a re-examination of significant experiences of the past, which sometimes were lived with frightening realism.", "With this, some of the patients were then able to re-evaluate the emotional meaning of some of their symptoms, and improved.", "Most were better able to organize their ideas in relation to real rather than fancied problems and were seen to experience and express relevant emotion.", "Two of the patients [both psychoneurotic] … were improved sufficiently to discontinue treatment … ’", "In a further group of 59 individuals with schizophrenia divided into 17 with 'pseudoneurotic' schizophrenia, 26 with 'undeteriorated' schizophrenia and 16 with 'deteriorated' schizophrenia, Paul Hoch and colleagues variously administered LSD and mescaline (Hoch et al., 1952).", "Not recording any objective data, they commented that the drugs ‘markedly aggravated’ the mental symptomatology of the individuals they studied.", "Those with ‘deteriorated’ schizophrenia showed ‘catatonic withdrawals’ in response to the drugs.", "Whilst noting the propensity of the drugs to uncover new material, the therapeutic value of the drugs in this patient group was considerably doubted.", "This was echoed in studies by Liddell (Liddell and Weil-Malherbe, 1953) and Pennes (Pennes, 1954), who observed worsening in those with undeteriorated and deteriorated forms of schizophrenia, but improvement in some with ‘pseudoneurotic’ forms of schizophrenia.", "‘Pseudoneurotic schizophrenia’ is an archaic term that included a multitude of depressive, anxious and obsessive symptomatology.", "None of these reports include details of follow up.", "Making some attempt at follow up, Herman Denber and Sydney Merlis gave 500 mg mescaline intravenously to 25 patients with schizophrenia.", "Whilst 1 patient achieved ‘complete remission’ from her symptoms (and was well at 1 year follow) and 3 patients showed temporary remission of symptoms, psychotic symptoms were either reactivated or worsened in the remainder (Denber and Merlis, 1955).", "In another study published in 1957, Sidney Merlis gave 24 patients with chronic schizophrenia between 500 and 750 mg of mescaline and noted that 1 ‘improved sufficiently to be discharged’, 7 ‘temporarily’ improved and in the remainder ‘no change’ was noted (Merlis, 1957).", "Reporting of adverse events or those that worsened with treatment was not included in the report.", "He concluded that mescaline was not a clinically effective agent in schizophrenia.", "After the early 1960s, clinical studies of the use of LSD and mescaline in psychosis rapidly diminished as it became clear that the drugs exacerbated the symptoms of most and did not lead to clinical improvement.", "However, the differences between the psychosis characterised by schizophrenia and the state characterised by LSD was still of interest.", "Langs, in 1968, published a detailed questionnaire study comparing schizophrenic patients and healthy controls who were randomly assigned to be given LSD or placebo (Langs and Barr, 1968).", "They noted that those with schizophrenia manifested ‘somatic and persecutory delusions and hallucinations which qualitatively extended far beyond anything reported by our LSD subjects’.", "Nonetheless, ‘‘paranoid schizophrenics’ responses resembled those of LSD-25 subjects in regard to feelings of unreality, loss of controls, changes in the meanings of experiences, and suspiciousness; they did not, however, exhibit the body image changes and elation-related effects found in many of the drug subjects.’", "It was noted that the hallucinatory element of the LSD experience tended to be largely visual in nature, whereas in the schizophrenic state auditory hallucinations predominated.", "Ronald Sandison, then working at the Powick Mental Hospital near Worcester, United Kingdom, published a paper in 1954 in which 36 patients with predominantly ‘psychoneurotic’ disorders were treated with variable doses of LSD given over a variable interval (usually weekly) in the context of psychotherapy (Sandison et al., 1954).", "LSD dosage was started at 25 mcg and then increased until an ‘adequate’ reaction was observed.", "This paper described the ‘psycholytic’ method of psychedelic psychotherapy: using LSD within the context of psychotherapy to ‘loosen’ ego defences and catalyse access to traumatic material.", "27 out of 30 with more classical neurotic and depressive disorders were reported to have benefited from the intervention, although this was a subjective judgement made by the treating clinician.", "There was no control group and no details of those patients who worsened with the treatment.", "Reporting an extension of their research in a publication in 1957, Sandison reported 6 month follow up in 93 patients with ‘severe neuroses’ (Sandison and Whitelaw, 1957).", "Of these 93 (of which 30 were also included in the original 1954 paper), 21 (22.6%) were ‘recovered’, 20 (21.5%) were ‘greatly improved’, 20 (21.5%) were ‘moderately improved’ and 32 (34.4%) ‘not improved’.", "Again, there were no objective measurements, no control group and no information on those who worsened in this study.", "Chandler and Hartman, working in California, published a work in 1960 in which 110 patients with predominantly ‘psychoneurotic’ and ‘personality disorder/trait’ diagnoses were given a total of 690 psychotherapy sessions using LSD, usually given in gradually escalating dosages between 50 mcg and 150 mcg (Chandler and Hartman, 1960).", "They also commented on the therapeutic utility of music in ‘ … helping to bring out affectively charged memories and fantasies’.", "Experiences under LSD were likened to ‘a waking dream’ with the aim of therapy ‘ … to understand it in terms of its emotional meaning rather than to worry about its objective validity.’", "Of these 110 patients, 50 showed ‘considerable’, ‘marked’ or ‘outstanding’ improvement, 38 showed ‘some’ or ‘slight’ improvement and 22 showed ‘little or no change’ or were ‘slightly worse’.", "No patients were judged to be ‘definitely worse’.", "No control group was reported.", "Attempting to provide a control group, Whitaker, working in Australia, described the use of an average of 3.28 LSD psychotherapy sessions in the treatment of 100 patients, comparing those treated with LSD psychotherapy to a group of patients treated in years previous to the study that were similar in terms of diagnosis and duration of illness (Whitaker, 1964a, 1964b).", "Of the 100 patients, 49 had ‘psychoneuroses’, 27 ‘personality disorder’, 21 ‘sexual disorders’ and 3 ‘residual schizophrenia’.", "Outcomes reported were clinician and patient agreements of improvement, divided into ‘successful’, ‘borderline’ and ‘failure’.", "Of the 100 patients undergoing LSD therapy, 47 were judged to be successful outcomes, 18 borderline and 35 failure.", "In the control group 12 were judged to be successful, 30 borderline and 58 failures.", "The rate of success was observed to be higher (75%) in those with the shortest duration of illness (0–2 years) as compared to those with the longest (more than 21 years), where only 37% were classed as successes.", "Of those 35 who were deemed as treatment failures, 19 ‘evaded’ ongoing therapy after their first LSD experience.", "None of the 3 residual schizophrenics were judged to have improved, however ‘a successful result was obtained in more than half the cases of anxiety state, hypochondriasis, hysterical personality, antisocial character disorder, anorexia nervosa and exhibitionism’.", "Outcome data was based on subjective judgements and the results were not analysed statistically.", "Probably the largest studies of the therapeutic utility of psychedelics in the pre-prohibition era were carried out at the Spring Grove State Hospital and the Maryland Psychiatric Research Center, both in Baltimore, Maryland, United States during the 1960s and early 1970s.", "Reporting on a total of 243 patients with a variety of non-psychotic psychiatric diagnoses that included anxiety and depressive disorders, personality disturbances and alcohol addiction, Charles Savage and colleagues also pointed out ‘the crucial importance of non-drug variables as determinants of reactions to chemical agents [which is] not confined to the psychedelics … ’ (Savage et al., 1967).", "They administered LSD without the context of formal psychotherapy.", "LSD at a dose of 200–300 mcg was given, with mescaline at a dose of 200–400 mg given to potentiate the effect in some patients.", "Emotional support and companionship was provided by a male and female sitter, but with no attempt made at interpretation of material.", "Follow up was the next day, then at 1, 2, 4, 8, and 12 weeks and finally at 6 months.", "A questionnaire was then sent, retrospectively, to the first 113 patients in the sample.", "Ninety-three replied.", "83% reported ‘lasting benefit’ and this was found to be ‘highly correlated (tetrachoric r = 0.91) with the report of a greater awareness of an ultimate reality’.", "The claimed improvement rate was 76% at one to three months post LSD, and 85% in the three to six months after LSD, this remaining constant after 12 months.", "Clinician ratings of improvement were made in retrospect by 4 raters, with improvement ratings divided into ‘worse’, ‘none’, ‘some’, ‘substantial’ and ‘marked’.", "Of 243 patients, 197 (81.1%) were judged to have improved to some degree: 35.8% showing ‘some’ improvement, 26.3% showing ‘substantial’ improvement and 18.9% showing ‘marked’ improvement.", "Of the remainder, 16.9% showed no improvement and 2.1% were ‘worse’.", "Clinical studies using LSD in the treatment of alcoholism before 1970 benefited from a more systematic approach than other disorders, probably because drinking behaviour is easier to quantify objectively.", "Several reasonable quality controlled studies were undertaken, particularly during the latter 1960s, however initial studies were usually uncontrolled.", "For example, Maclean and colleagues gave 61 alcoholics and 39 patients with other diagnoses 400–1,500 mcg LSD on an undisclosed number of occasions, following them up for up to 18 months (Maclean et al., 1961).", "Whilst noting that, of the alcoholics, 30 were ‘much improved’, 16 were ‘improved’ and in 15 there was ‘no change’, there was no comparison control group.", "In another study that included a comparison group, Jensen gave LSD to 58 alcoholics on an inpatient unit in Ontario, Canada, comparing this to 35 alcoholics given group psychotherapy and 45 receiving ‘standard’ care from psychiatrists not connected to the study (Jensen, 1962).", "By chi square analysis, they reported a significant difference in rates of abstinence between those given LSD and those given group psychotherapy and standard care.", "However, the control groups were not matched and the authors stated that the group treated with LSD were composed of people who remained in follow up, whereas the control groups included those lost to follow up.", "The methods of statistical analysis, chi square statistic and p value for significance were also not stated.", "More systematic studies were published after 1965, often with non-significant results.", "Smart et al., in 1966, reported the effect of a single LSD experience under controlled conditions on the behaviour of 30 alcoholics who were either inpatients or outpatients in Toronto, Canada (Smart et al., 1966).", "They were randomised to a control group that received standard care including psychotherapy, a group who received standard care plus a 60 mg dose of ephedrine sulphate (a drug chosen because it shares some similar subjective effects to LSD but with no known efficacy in alcoholism), and standard care plus an 800 mcg dose of LSD.", "Drug treatments were administered on a psychiatric ward, with participants kept overnight.", "Otherwise treatment was the same.", "Both participants and therapists were blind to treatment allocation.", "Nonetheless, in 19 out of the 20 drug sessions, the therapists correctly guessed the drug condition.", "Baseline and 6 month follow up alcohol usage data was collected in all groups by a participant- and researcher-rated questionnaire.", "Researchers following up participants at 6 months were blind to treatment and analyses were completed prior to blinding being broken.", "No significant differences were observed between the groups in terms of pragmatic measures of alcohol misuse.", "Hollister et al., working in 1969 at the Veterans Administration Hospital in California, USA, reported the results of a controlled comparison of 72 male inpatients with alcoholism randomised to a single dose either of 600 mcg LSD or 60 mg of dextroamphetamine (Hollister et al., 1969).", "Music and low lighting was provided and a research assistant was available for reassurance, but no attempt at psychotherapy was made.", "Baseline and follow up measurements of drinking and associated social effects were recorded with a scale designed and validated for the trial.", "Follow up data was collected at 2, 6 and 12 months post treatment by a researcher blind to treatment and independent of the treatment programme.", "Of the 72 patients, 20 had dropped out at 2 month follow up (10 in each group) and 27 had dropped out of follow up by 6 months (11 in the LSD group and 16 in the dextroamphetamine group).", "In terms of mean change of questionnaire scores, analysed by ANOVA, those in the LSD group were significantly improved over the dextroamphetamine group (F = 8.5, p < .01), however the difference was not significant at 6 month follow up.", "Those who dropped out did not differ significantly in baseline scores compared to those who did not.", "At 12 month follow up, only 17 patients remained in the LSD group and 12 in the dextroamphetamine group, and the authors considered analysis of this group unproductive.", "Ludwig et al., working at the Mendota State Hospital in Wisconsin, USA, published in 1969 a study in which 176 male inpatients with alcoholism were randomly allocated to one of three LSD treatments and a control condition (44 participants per group) (Ludwig et al., 1969).", "All participants allocated to the LSD groups received 3 mcg per kg of body weight, with the treatments differing by the nature of therapy offered during the LSD experience.", "One group received LSD plus hypnosis plus psychotherapy, a second group LSD plus psychotherapy and a third group LSD alone.", "The fourth ‘control’ group were required to spend an equivalent amount of time in the treatment room by themselves, but no intervention was otherwise given.", "Therapists were not blind to treatment group.", "Evaluation was with a variety of symptomatology, drinking behaviour and social adjustment inventories taken at baseline then immediately post-treatment and at 3, 6, 9 and 12 months after treatment.", "Researchers collecting follow up data were blind to treatment allocation.", "All groups showed consistent improvements and no significant difference was found between the LSD groups and the control groups.", "A similar approach, and non-significant result, was found in two further controlled studies by Bowen (Bowen et al., 1970) and Tomsovic (Tomsovic and Edwards, 1970), with borderline significant results found by Pahnke (Pahnke et al., 1970).", "Meta-analysing the previous 6 studies, Krebs and Johansen found that LSD treatment was significantly associated with maintained abstinence at 1–3 months (OR = 2.07 95% CI, 1.26–3.42; p = .004), but by 6 months statistical significance was lost (OR, 1.42, 95% CI, 0.65–3.10, p = .38) (Krebs and Johansen, 2012).", "Studies of the clinical utility of psychedelics published prior to 1970 were, like many studies of that time, methodologically suboptimal.", "A non-exhaustive list of the obvious problems includes the following:", "Treatment groups were inadequately and inconsistently defined", "Treatments were inconsistently applied amongst groups", "Control groups were often absent", "Attempts to blind study teams were usually absent", "Outcome measures were not validated", "Outcome data was inconsistently reported", "Adverse outcomes were often not reported", "Statistical analysis of results was often absent", "Power calculations were not used to estimate sample sizes needed to detect an effect", "Nonetheless, the pre-prohibition research strongly suggested that psychedelic drugs were not useful for those with established psychotic disorders and should probably be avoided in those liable to develop them.", "Worsening of psychotic symptomatology was observed in most of those with pre-existing schizophrenia and whilst the occasional case was observed to recover, no trial reported improvements that might not otherwise have been attributable to the passage of time.", "Reports were more encouraging in trials with so-called ‘psychoneurotic’ disorders, a term which covers a wide variety of anxious, obsessive and depressive states.", "We have systematically reviewed trials in broadly defined unipolar mood disorder in another work, which showed that nearly 80% of participants in these trials were judged to have ‘improved’ by their clinicians (Rucker et al., 2016).", "The data was not of sufficient quality to meta-analyse and clinical improvement was usually a subjective judgement, rarely based on objective or validated scales.", "Indeed, initial trials were usually little more than case series reported by clinicians who probably had positive expectations about treatment.", "Studies in later years suggest more moderate preconceptions, but still reported subjectively defined efficacy in many cases.", "However, in controlled trials with alcoholism using quantitative measures of alcohol use, borderline or non-significant findings were often reported.", "Given the lack of power calculations, this may reflect a lack of power to detect an effect or a true lack of efficacy.", "Krebs and Johansen's meta-analysis of these studies suggests the former, at least in the use of LSD in the treatment of alcohol dependent individuals (Krebs and Johansen, 2012).", "Side effects, or adverse events, during the experience were generally not reported systematically, if at all.", "Interpretation of an adverse event's aetiology varied according to research team, particularly for mental phenomena.", "This ranged from a directly toxic action of the drug on the brain, which tended to cluster within studies of those with schizophrenia, to the expected expression of repressed trauma, which tended to cluster within studies of those with neuroses and alcoholism.", "This may reflect both the varying perspectives of cliniciansabout the aetiology of mental disorder and the pharmacological effect of the drugs.", "We have listed reported adverse events (non-exhaustively in view of space) in Table 1.", "Immediate adverse events were more often reported than delayed adverse events.", "In no particular order (frequencies were rarely reported), headaches, palpitations, gastrointestinal disturbances, changes in temperature perception, feelings of tremulousness or dizziness, and a variety of other somatic complaints were most commonly reported.", "A consistent theme was a disparity between the degree of subjectively reported physical symptoms and objective clinical signs on medical examination, which tended to reveal only minor increases in pulse rate and blood pressure along with pupillary dilatation and, occasionally, signs of body temperature changes (shivering and piloerection, or sweating).", "The significance of this disparity is uncertain, however may represent both an increased subjective awareness of bodily sensations under the influence of psychedelics, as well as drug induced changes in the autonomic nervous system itself.", "Sidney Cohen, in 1960, attempted to systematically investigate the incidence of adverse events during treatment with psychedelics by sending a questionnaire to 62 investigators who were using LSD or mescaline in healthy subjects or patients (Cohen, 1960).", "44 investigators replied.", "The data covered almost 5000 individuals given LSD or mescaline on a total of more than 25,000 occasions.", "No instance of physiological toxicity was reported.", "Of those with pre-existing psychiatric problems receiving LSD or mescaline, the rate of attempted suicide was 0.12%, completed suicide, 0.04% and psychotic reactions lasting over 48 h, 0.18%.", "The rate in healthy subjects was 0%, 0% and 0.08% respectively.", "No instance of addiction was reported.", "The instances of these serious events appear rather low and the study can be criticised for relying on the recall of clinicians who may have underestimated (or been unaware of) the nature and degree of adverse reactions in their subjects, particularly in those lost to follow up (who may have been at higher risk of experiencing them).", "In a narrative report of nine cases who had suffered a variety of persistent psychotic and neurotic symptoms after recreational use of, or medical treatment with, LSD, Cohen and Ditman concluded that complications were ‘much more likely to occur after the unsupervised or inexpert use of the drug.’", "Whilst the study was unsystematic, the majority of cases occurred using illicit LSD taken within a psychologically destabilising milieu that also included other psychoactive drugs and lack of access to timely medical assistance (Cohen and Ditman, 1963).", "This was a view echoed by Strassman in a comprehensive review of adverse events to psychedelics in 1984 (Strassman, 1984).", "As recreational use of LSD and mescaline increased in the 1960s, so evidence of toxic psychological reactions in sensitive individuals accumulated, with occasional tragic cases (Keeler and Reifler, 1967) accompanied by sensationalist media reporting.", "This paralleled investigation of psychedelics as so-called ‘truth drugs’ or chemical weapons, particularly by the Central Intelligence Agency (Lee and Shlain, 2007).", "An immoral failure, these experiments (and the public outcry over them) accompanied a more general hardening of socio-political attitudes towards psychoactive drugs.", "Moreover, unethical medical use of psychedelics probably occurred in some centres.", "Whilst documentation is patchy, concerns about this issue were reflected in a legal ruling in Denmark, where the ‘LSD Damages Law’ was enacted in 1986.", "This led to a series of 151 patients gaining compensation in the 1980s and 1990s for a variety of psychiatric symptomatology that was presumed (but not proven) to have resulted from LSD treatment that was claimed sometimes to have been given under coercion, or without informed consent (Larsen, 2016).", "A critique of the report of these cases has recently been published (Erritzoe and Richards, 2017).", "In summary, the research conducted prior to 1970 suggests that whilst there was certainly clinical interest in the therapeutic potential of psychedelics in patient populations with non-psychotic mental health problems, a firm conclusion about efficacy and safety was not reached prior to legal sanction that was largely socio-political in motivation, although also reflected medical concerns about the sequelae of recreational use.", "It is within this agnosticism and strict regulatory framework that the modern resurgence of research interest has taken place and to which we now turn.", "In 1967, psychedelics, including mescaline, psilocybin and LSD, were classified under Schedule I of the 1967 UN Convention on Drugs.", "This legally defined them as having no accepted medical use and the maximum potential for harm and dependence.", "Successive national legislation throughout the Western world tended to mimic the 1967 UN Schedules.", "Medical use of psychedelics ceased quickly because doctors were no longer permitted to prescribe them.", "Without a clinical focus, research dwindled almost to a standstill in the late 1980s and 1990s.", "This is depicted graphically in Fig. 1, which shows the annual number of publications listed in the database PubMed where the title refers to a classical psychedelic drug, expressed as a proportion of all PubMed publications, from 1950 to 2016.", "The herald to modern clinical research using psychedelics were three papers investigating the effects of mescaline, dimethyltryptamine and psilocybin in healthy volunteers by, respectively, Leo Hermle et al. in Germany (Hermle et al., 1998), Rick Strassman et al. in the United States (Strassman and Qualls, 1994), and Franz Vollenweider et al. in Switzerland (Vollenweider, 1997).", "These studies formed the basis for a resurgence of further studies in healthy volunteers focussing on neuroimaging (Carhart-Harris et al., 2012, 2016b; Daumann et al., 2010; Muthukumaraswamy et al., 2013; Palhano-Fontes et al., 2015; Preller et al., 2017; Riba et al., 2004, 2006; Vollenweider, 1997), psychopharmacological (Kometer et al., 2012; Preller et al., 2017; Valle et al., 2016; Vollenweider et al., 1998) and neuropsychological (Carter et al., 2007; Gouzoulis-Mayfrank et al., 2005) correlates of the psychedelic state.", "This literature is beyond the scope of this clinically focussed review.", "The first modern clinical trial investigating the safety and feasibility of using a psychedelic drug in a psychiatric patient population was published by Francisco Moreno and colleagues, working at the University of Arizona in the United States (Moreno et al., 2006).", "Nine subjects with treatment resistant obsessive compulsive disorder and no other major psychiatric pathology were given up to 4 different doses (25, 100, 200, 300 mcg/kg body weight) of psilocybin in an open-label design.", "Treatments were separated by at least 1 week.", "29 doses were given in total.", "The Yale Brown Obsessive Compulsive Scale, the Hallucinogen Rating Scale and a visual analogue scale measuring overall symptomatology was administered at 0, 4, 8 and 24 h post dosing.", "Whilst significant reductions in OCD symptoms were observed in all dosing conditions, there was no significant difference between the different dosages of psilocybin, although the trial was likely underpowered to detect an effect.", "No serious adverse events were reported.", "Matthew Johnson and colleagues, working at the Johns Hopkins University in Maryland, United States, published an open-label pilot trial in 2014 using moderate (20mg/70 kg) and high (30mg/70 kg) doses of psilocybin given to 15 otherwise psychiatrically healthy subjects with tobacco addiction undergoing a structured 15-week smoking cessation treatment (Johnson et al., 2014).", "Psilocybin was given at weeks 5, 7, and 13.", "Initial psilocybin dosing was with the moderate dose and the higher dose offered, but not enforced, in the subsequent sessions.", "A total of 19 meetings took place as part of the smoking-cessation programme and psilocybin delivery.", "Biological markers of smoking cessation were assessed at baseline, weekly throughout the treatment intervention and at 6 monthly follow up.", "A total of 42 psilocybin sessions were delivered.", "No clinically significant adverse events were reported during the treatment or follow up.", "12 of 15 (80%) of participants were abstinent from tobacco as measured by biological markers at 6 month follow up.", "Whilst this was highly significant when pre and post treatment self-reported smoking figures were compared statistically, the authors were measured in their interpretations given the open label design and low numbers of participants.", "In a further open-label pilot study in 2015 on addiction, this time to alcohol, Michael Bogenschultz and colleagues at the University of New Mexico, United States, gave psilocybin to 10 alcohol dependent patients (4 women) in addition to standard motivational enhancement therapy (Bogenschutz et al., 2015).", "Psychological support was given before, during and after 2 psilocybin sessions, spaced 4 weeks apart.", "The total treatment intervention was 12 weeks.", "Outcome data was collected at baseline and for 36 weeks in total.", "The dose of psilocybin used was either 0.3 mg/kg or 0.4 mg/kg.", "The primary outcome was the percentage of days spent drinking heavily compared between measures taken at baseline and weeks 5–12.", "9 participants completed follow up.", "Results suggested, in line with studies in the 1960s using LSD in alcoholics, that the acute effects of psilocybin were less strong in this group.", "Large and statistically significant improvements in drinking behaviour immediately after treatment were seen and these correlated with the intensity of the drug effect.", "However, the open-label, uncontrolled design suggests caution is needed in extrapolating the finding.", "No serious adverse events were noted.", "Again, the authors concluded that psilocybin was a safe and feasible treatment to deliver in a clinical trial setting.", "Three pilot studies using psychedelics in major depressive disorder have been published to date in the modern literature (Carhart-Harris et al., 2017, 2016a; Osório et al., 2015; Sanches et al., 2016).", "Our own open-label pilot study gave 2 doses of psilocybin (a 10 mg “test” dose and a 25 mg therapeutic dose) 1 week apart with psychological support before and after the experience to 20 patients with treatment resistant depression who were moderately to severely depressed, but without psychotic features (Carhart-Harris et al., 2017, 2016a).", "Participants were withdrawn from their antidepressant medications prior to psilocybin treatment.", "The primary outcome measure was the mean change in the participant-rated quick inventory of depressive symptoms rating scale from baseline to 1 week after the second psilocybin treatment.", "Follow up was for 6 months.", "No serious adverse events occurred.", "Significant improvements in depression ratings were seen at 1, 2, 3 and 5 weeks, and at 3 and 6 month follow up.", "The maximal effect was seen at 5 weeks (Cohen's d = 2.3).", "Further treatment seeking by participants after 5 weeks, and particularly after 3 months, likely confounded follow up data collected at 3 and 6 months.", "5 of the 20 participants sought and obtained psilocybin again during the follow up period.", "The trial established feasibility in this patient group and initial evidence of safety, but efficacy interpretations are precluded by the open-label, uncontrolled design.", "Flavia de Lima Osorio and colleagues (Osório et al., 2015) and Rafael Sanches and colleagues (Sanches et al., 2016), both working in Sao Paulo, Brazil, reported studies where a single dose of ayahuasca to patients with recurrent depression.", "In Osorio's pilot study, 6 medication and ayahuasca naive participants (4 women) were given 2.2 ml/kg of a standardised preparation of ayahuasca containing 0.8 mg/ml DMT and 0.21 mg/ml of harmine.", "Measurement of depressive symptoms was with the clinician rated Hamilton Depression Rating Scale (HAMD) and the Montgomery Asperg Depression Rating Scale (MADRAS).", "Baseline measurements were compared with measurements taken at 1 day, 1 week, 2 weeks and 3 weeks after treatment.", "Significant reductions in depressive symptoms were seen at 1 day, 1 week, 3 weeks but not 2 weeks after treatment, both with the HAMD and MADRAS.", "The treatment was administered safely without any serious adverse events.", "Sanches and colleagues conducted a follow up to this work with an open-label study on 17 patients with recurrent depressive disorder given the same dose of ayahuasca, with identical outcome measures.", "Again, significant reductions in depressive symptoms were observed up to the three-week end point of follow up.", "The treatment was well tolerated with no serious adverse events.", "The failure to collect participant rated scales of depressive symptoms and the open-label design precludes clinical interpretation beyond feasibility and safety.", "Finally, four separate studies have been published on the use of psychedelics in end-of-life anxiety associated with life threatening illness (Gasser et al., 2014; Griffiths et al., 2016; Grob et al., 2011; Ross et al., 2016).", "Charles Grob and colleagues, working in California, United States, gave 12 subjects (11 women) a moderate (0.2 mg/kg) dose of psilocybin and an active placebo (niacin 250 mg) several weeks apart with psychological support in a double-blind design in which subjects acted as their own control (Grob et al., 2011).", "All subjects had advanced cancer diagnoses and DSM-IV defined acute stress disorder, generalised anxiety disorder, or adjustment disorder with anxiety because of the cancer diagnosis.", "Four subjects were psychedelic naive.", "All 12 completed 3 month follow up.", "Non-statistically significant trends towards improvements in mood were observed.", "The treatment was well tolerated with no serious adverse events.", "Peter Gasser and colleagues, working in Switzerland, gave LSD to 12 patients with anxiety associated with life threatening disease in a double-blind, randomised, active placebo controlled pilot trial (Gasser et al., 2014) with a 12 month qualitative follow up study also reported (Gasser et al., 2015).", "Drug free psychotherapy sessions were supplemented by two LSD assisted psychotherapy sessions given 2–3 weeks apart, in which participants were randomised to receive either 200 mcg or 20 mcg of LSD.", "An open label extension was offered to those randomised to the 20 mcg dose.", "The State Trait Anxiety Inventory was used as the outcome measure.", "Significant reductions were found in state, but not trait, anxiety at 2 months, sustained at 12 months.", "The treatment was delivered safely with no serious adverse events.", "This is the only modern trial to our knowledge that has used LSD in patients.", "Two larger, double blind, randomised, placebo-controlled crossover trials investigating the efficacy of psilocybin in the treatment of anxiety and depression in patients with life-threatening cancer diagnoses were published from two separate groups in the United States in 2016 (Griffiths et al., 2016; Ross et al., 2016).", "Working in New York University, Stephen Ross and colleagues gave 29 patients a single dose of 0.3 mg/kg psilocybin or 250 mg niacin, both in conjunction with psychotherapy.", "Crossover occurred at seven weeks.", "2 therapists worked with each patient and extensive psychological support and therapy was provided.", "A variety of clinician and participant rated measures, including the State Trait Anxiety Inventory and the Beck Depression Inventory, served as primary outcome measures and were collected at 1 day prior to the first dose, 1 day, 2 weeks, 6 weeks and 7 weeks after the first dose, then 1 day, 6 weeks and 26 weeks after dose 2.", "The treatment was delivered safely, with no reports of serious adverse events.", "The group receiving psilocybin showed ‘immediate, substantial, and sustained’ clinical benefits as measured by both clinician and participant rated scales that lasted for the 7 weeks prior to crossover and were also sustained at the final point of the study, 26 weeks after dose 2 (approximately 8 months after dosing).", "The group that received niacin as dose 1 showed transient reductions that were not sustained at 7 weeks.", "After crossover and receiving psilocybin, immediate and sustained reductions in anxiety and depression were observed, with the effect sustained at follow up at 6 and a half months.", "Working in Baltimore, Roland Griffiths and colleagues gave psilocybin using a similar double blind, randomised, crossover design to 51 patients with life threatening cancer and associated anxiety and depressive symptoms (Griffiths et al., 2016).", "In this study, the placebo condition was a very low dose of psilocybin (1 mg or 3mg/70 kg) compared with a high treatment dose of psilocybin (22 mg or 30mg/70 kg) administered in a counterbalanced sequence with 5 weeks between sessions and 6 month follow up.", "Thus, those who received the low dose of psilocybin first received the high dose second and vice versa.", "Extensive psychological support was provided before, during and after the experience and the average length of participation in the study was approximately 9 months.", "The primary outcome measures were the Hamilton Depression Rating Scale and the Hamilton Anxiety Scale, two clinician-administered scales.", "The results showed statistically significant superiority of the high dose versus the low dose in terms of the primary outcome measures and self-reported measures when data at 5 weeks was considered.", "There were no serious adverse events reported.", "Because participants crossed over from low to high dose, and vice versa, at 5 weeks, the blind was effectively broken at this point.", "Significant associations between mystical type experiences and enduring positive changes were observed, reflecting previous research done by this group (Griffiths et al., 2008, 2006).", "Commentary on both studies pointed out that the crossover design and degree of psychotherapy provided around the psilocybin experience may have confounded the effect attributed to psilocybin (Sellers and Leiderman, 2017).", "As ever, trials such as these are often subject to the so-called ‘winner's curse’, whereby effect sizes tend to be inflated in pioneering trials of new treatments due to a variety of subtle effects.", "Future trials are likely to report more modest findings.", "Clinical trials with investigational medicinal products (IMPs) ultimately have one aim: to provide objective data to determine whether the IMP in question is safe and efficacious enough to justify a license.", "In the modern context of research with classical psychedelics, the IMP most likely to be licensed is psilocybin.", "However, the legal, regulatory and commercial hurdles to this are formidable.", "For the remainder of this article we concentrate on this process and discuss, within the context of modern trials so far, a strategic approach to tackling this challenge.", "A license for an IMP results from approval from national medicine's regulatory bodies.", "In the UK, for example, this is the Medicines and Healthcare products Regulatory Agency.", "A license can be gained via various ‘routes’.", "For example, existing drugs may be relicensed with market exclusivity for rare diseases to drive development in areas that would otherwise be commercially non-viable (the ‘orphan’ drug route).", "However, most drugs are developed de-novo for more common diseases based on the commercial potential predicated on a limited period of market exclusivity after licensing.", "Regardless of the route, licensing is based on objective data about the IMP's safety and efficacy in defined patient groups.", "This requires a series of randomised, controlled trials (RCTs) conducted on sufficient numbers of participants in a regulated fashion that seeks to collect valid, objective data about adverse outcomes and to disambiguate the effects of the IMP from other influences on outcome.", "A licensing decision is made based on a balanced judgement of the risks and costs of treatment with the IMP weighed against the risks and costs of the disease itself.", "In the modern era, trials are divided into phases 1, 2, 3 and 4.", "Phase 1 trials are open-label and investigate safety in small numbers of healthy human volunteers.", "Phase 2 trials investigate safety and feasibility in modest numbers of patients.", "Whilst publications that result from phase 2 trials often report data about efficacy they are actually more concerned with safety and feasibility.", "They may be open label or controlled and the data is used to design phase 3 trials, which are are usually RCTs that investigate safety and efficacy in larger numbers of patients.", "Data from phase 3 trials often form the mainstay of evidence used for licensing decisions.", "Phase 4 trials are conducted on very large numbers of patients after a medicine has been approved and marketed and are designed to pick up treatment effects and rare side effects that could not reasonably be detected in phase 3 trials.", "At the time of writing, psilocybin is undergoing phase 2 trials, with phase 3 trials in the late stages of planning.", "This leads us naturally to a discussion about the safety of psilocybin and the feasibility of the clinical trials with it.", "To date, 146 patients with a variety of psychiatric problems have been treated with psilocybin and reported in the modern medical literature.", "There have been no serious adverse events reported in these trials, although the infrequent reports of drop outs suggest absence of complete follow up data.", "A serious adverse event is defined as a reaction that results in death, is life threatening, results in prolonged hospitalisation or persistent or significant disability.", "The absence of this so far is consistent with research using psilocybin in healthy volunteers (Studerus et al., 2011), pre-prohibition research with LSD and mescaline (Cohen, 1960), modern population level data on recreational use of psilocybin mushrooms and other psychedelics (Carhart-Harris and Nutt, 2013; Hendricks et al., 2014, 2015; Johansen and Krebs, 2015; Krebs and Johansen, 2013a; Nutt et al., 2010; Walsh et al., 2016) and toxicology work (Gable, 2004).", "However, modern trials with psilocybin are notable for not collecting adverse event data systematically in a manner that allows aggregated analyses.", "The incidence of adverse events, and particularly any cases of hallucinogen persisting perception disorder, mania, psychosis, self-harm or suicidal behaviour will need to be compared between treatment and control groups, along with assessments about causality.", "A single consolidated database of adverse event information is necessary for regulatory approval.", "A number of groups have recently collaborated in Europe to establish this for trials using psilocybin in treatment resistant depression.", "We have collated the most common immediate and delayed adverse events reported in the literature in Table 1.", "Transient anxiety, nausea, vomiting and mild increases in blood pressure and heart rate are the most frequently observed immediate adverse events.", "Headache is the most common delayed adverse event.", "No cases of prolonged psychosis or hallucinogen persisting perception disorder have been reported in modern trials with psilocybin, ayahuasca or LSD.", "Dropout rates have been low, however in these early trials participants may be a self-selecting group with favourable attitudes towards psychedelics, which may be reflected by the proportion reporting previous use.", "In our trial of psilocybin in treatment resistant depression 35% of participants had a lifetime history of psilocybin mushroom use.", "This is less than a recent worldwide survey of 22,289 recreational drug users, which found a lifetime prevalence of use of 43.1% (Winstock et al., 2013) but more than prevalence figures of 17% for lifetime LSD, mescaline or psilocybin use amongst 21–64 year olds in the 2010 US National Survey on Drug Use and Health (Krebs and Johansen, 2013b).", "There is very limited evidence that psychedelics cause dependence or addiction (Brunton et al., 2011; Morgenstern et al., 1994).", "Euphoria is not a consistent feature of the psychedelic experience, tolerance develops quickly and completely and there is no known withdrawal syndrome (Buckholtz et al., 1985; Cholden et al., 1955; Isbell et al., 1956).", "Psychological dependence appears to be rare, however research in this area is limited (Blacker et al., 1968).", "Thus psychedelics appear to have a low potential for abuse relative to other psychoactive drugs (Fábregas et al., 2010; Gable, 2007).", "Given the above and the fact that psilocybin would be delivered within a controlled healthcare setting rather than the community, the risk of diversion of drug supplies in the context of existing security measures for other controlled drugs used in healthcare settings appears to be low.", "The ascendency of the RCT, which inherently attempts to separate drug effects from their contexts, has its origins in the Kefauver Harris Drug Amendments of 1962, which were developed in response to the thalidomide tragedy.", "However, a common theme in the literature is the opinion that psychedelics are therapeutic within a psychologically supportive context, rather than therapeutic per se.", "Given the disparity between the mechanism of action of psychedelics and the purpose of RCTs, some have argued that RCTs with psychedelics are fundamentally flawed and therefore not feasible (Oram, 2012).", "To exemplify, modern RCTs with psilocybin have shown large effect sizes in distress associated with life threatening disease (Griffiths et al., 2016; Ross et al., 2016), however in these studies the psychological support provided was extensive.", "Criticism of these trials reflects this (Sellers and Leiderman, 2017).", "At the other extreme, Smart et al., in their 1966 study of LSD in alcoholism, tied their participants to a bed with a Posey belt and gave them a very large dose of LSD (800 mcg) before attempting to engage them in a 3-way interview about their alcohol use (Smart et al., 1966).", "Perhaps unsurprisingly (the study was also probably underpowered) negative results were reported.", "A meta-analysis of pre-prohibition trials in alcoholism showed significant evidence of efficacy overall (Krebs and Johansen, 2012), however only one pilot study has been completed so far in the modern era using psilocybin in alcoholism.", "This found an encouraging effect on drinking behaviour, however again psilocybin was given in the context of quite extensive psychological support and a motivational enhancement programme (Bogenschutz et al., 2015).", "It appears that the problem of determining the relative contribution of psychedelic and its context to the putative therapeutic effect is difficult.", "However, given that it would be unethical to give psychedelics without some sort of psychological and emotional support it seems germane to emphasise that this basic milieu is (and morally must be) the treatment being tested.", "Thus, trials that attempt to understand how different contexts interact favourably, or unfavourably, with psychedelics may be of more value than those that attempt to artificially separate them.", "At the same time, the evidence for a licensing decision must include trials that focus primarily on the drug itself.", "Overall, whilst the RCT design is not ideal we conclude that carefully designed clinical trials with psychedelics are feasible and, since it would be unethical not to include a modest degree of psychological support within the design, RCTs with this are, by definition, valid.", "This has been demonstrated practically by modern pilot trials and RCTs with psychedelics, although the nature and extent of psychological support provided will attract differing opinions.", "We remain agnostic about outcome when the RCT design is applied in phase 3 trials of psychedelics, but agnosticism is ultimately why the research is necessary.", "Phase 2 trials are relatively inexpensive in comparison to phase 3 trials, which often cost many millions of dollars and thus generally require profit-driven commercial investment.", "This usually requires the commercial potential inherent from a period of patented market exclusivity for the developer.", "This allows recuperation of development costs and generates income that drives further work.", "However, since the patent on psilocybin (and LSD) has long expired, commercial viability, at first glance, seems doubtful.", "Treating psilocybin as an orphan drug is not an obvious solution to this because the proposed disease areas (such as treatment resistant depression) are not rare enough to fulfil criteria for this route.", "Trial costs are exacerbated further by the security and bureaucratic requirements imposed by Schedule I of the UN Convention on Drugs (Nutt et al., 2013).", "How can phase 3 trials with psilocybin be funded if there is no commercial potential to incentivise that funding?", "The answer to this is multi-faceted.", "Recently, a UK based company announced a multi-center phase 3 trial of psilocybin in 300 patients with treatment resistant depression in Europe, with significant financial backing from investors, suggesting that the commercial potential does exist, at least in principle (“COMPASS - Navigating Mental Health Pathways,” 2017).", "Grant holders in Europe and the US have started to fund trials with Schedule I substances over the last 10 years, perhaps reflecting a subtle shift in socio-political attitudes.", "Psychedelics tend to capture the imagination of the public (as well as the media) and significant sums have been raised through social media-linked crowd funding as well as from charitable and entrepreneurial sources (Emerson et al., 2014; Nichols, 2014).", "So, whilst the commercial potential in psilocybin may be somewhat atypically predicated, there is accumulating evidence that it now exists in sufficient quantity to fund the trials needed to collect evidence to submit to regulators.", "With commercial potential, the practical and bureaucratic burdens imposed by Schedule I are inconvenient, but not insurmountable.", "Special licenses are required to process and administer Schedule I drugs and strict security protocols are necessary that require particular infrastructure.", "For example, pharmacies holding Schedule I drugs in the UK must be monitored by closed circuit television at all times and subject themselves to regular inspections.", "Storage containers holding Schedule I drugs must satisfy certain security standards and be securely fastened to reinforced walls or floors.", "Those teams and institutions that have already conducted trials with psilocybin have implemented these requirements and are thus in a good position to conduct further research.", "With precedent comes familiarity and then replication.", "As the socio-political landscape changes, so do the attitudes of grant funders.", "In combination, this should stimulate other research groups to engage with the practicalities of this fledgling field of research, where there is, after all, plenty of room for expansion.", "Moreover, there is historical precedent of Schedule I drugs being developed for medical use.", "Dronabinol (synthetic delta-9-tetrahydrocannabinol) was approved as an orphan drug for AIDS-related anorexia in 1985 and is now also approved for cancer chemotherapy related nausea and vomiting (Brafford May and Glode, 2016).", "Xyrem (sodium γ-hydroxybutyrate) was approved for the treatment of cataplexy associated with narcolepsy in 2004 (Owen, 2008).", "An extract of cannabis sativa (nabiximols) was licensed in 2010 in the UK for spasticity in Multiple Sclerosis (Lakhan and Rowland, 2009).", "Extracts of cannabis are also being investigated for certain forms of childhood epilepsy (Hussain et al., 2015).", "Generally, the drug development pipeline is similar and a successful licensing application results in the specific formulation of the drug being legally rescheduled.", "At this point, the drug becomes prescribable by medical doctors without the need for a special license, breaking the ‘vicious cycle’ of Schedule I research suppression (Nutt et al., 2013).", "With the force of commercial interest behind it, we anticipate a similar process for psilocybin if efficacy and safety are confirmed.", "If trials using psychedelics are commercially and legally viable, then which patient groups should be focussed on?", "In a costs-driven world, it is likely that this will be those associated with high socio-economic burden, morbidity and mortality and where effective treatments are lacking or burdensome.", "Within psychiatry, the most logical initial focus is probably unipolar depressive disorder, with treatment resistant depression a priority.", "Unipolar depressive disorder is increasingly prevalent (Lopez and Murray, 1998; Murray and Lopez, 1997; “World Health Organisation,” 2017), confers startlingly high socio-economic burden (Greenberg et al., 2015, 1993; McCrone et al., 2008; van Wijngaarden et al., 2004; Wang et al., 2003), is under-researched relative to disease burden, related to poorer outcomes in a wide variety of physical health problems (Moussavi et al., 2007) and is associated with a 20 fold increased risk of completed suicide (Harris and Barraclough, 1997).", "Of those who have a depressive episode, 85% will go on to have another and successive episodes increase the risk still further (Mueller et al., 1999).", "Treatment resistance, defined as failure to respond to at least two antidepressants, is common and longer term, depth psychological and social therapies are sufficiently expensive to deliver to make psilocybin therapy a viable potential alternative, if safety and efficacy is demonstrated.", "Whilst the initial focus of commercially driven trials is likely to be treatment resistant depression, the largest modern trials to date have been in psychological distress associated with terminal illnesses.", "Clinical scenarios involving the use of psychedelics in palliative care have inherent advantages in the process of gaining regulatory approval, because the safety data requirements are not as burdensome in groups where life expectancy is limited.", "Nonetheless, evidence of efficacy is still required and it is not yet clear where the funding for phase 3 trials of psilocybin in end of life care will come from.", "Given the mechanism of action of psychedelics, the potential scope of application could be wide.", "Functional neurological disorders (Bryson et al., 2017) and anorexia nervosa appear interesting candidates for further exploration, for example.", "However, it is anathema to give psychedelics without informed consent, a point that leads us into a final brief discussion of the practical aspects of conducting trials with psychedelics such as psilocybin.", "Whilst their physiological safety is relatively well established, psychedelics elicit acute sensitivity to context and psychologically toxic reactions do occur.", "Rarely, tragic circumstances have occurred (Keeler and Reifler, 1967; Reynolds and Jindrich, 1985), often attracting disproportionate media coverage.", "In the light of this, what practical steps can be taken to minimise risks in psychedelic trials?", "A full discussion of this question has been covered elsewhere (Johnson et al., 2008), but we present some key points, revolving around our experience of using psilocybin in treatment resistant depression.", "Recruitment in trials of psychiatric disorders is inherently difficult as the disorders themselves affect motivation and adherence.", "In addition to this more general problem, psychedelics are stigmatised.", "This combination may lead to selection bias.", "This can be overcome in part by using established clinical databases of patients that have consented to research contact, for example our own at King's College London (Perera et al., 2009; Stewart et al., 2009).", "This allows researchers to approach potential participants, rather than relying on a self-selecting sample of volunteers contacting the research team.", "On the other hand, self-selecting volunteers may be less liable to experience adverse events to psychedelics because of the positive preconceptions that motivate them to volunteer.", "Post hoc comparisons of study samples with case registers can also help determine to what extent study samples may differ to the populations they are drawn from, as well as allowing comparisons with ‘treatment as usual’ cohorts.", "Clinical trials with psychedelics should include adequate procedures to screen out high risk individuals.", "A personal or family history of psychosis, personal history of mania, personal history of repeated violence towards others and a recent personal history of suicide attempt serious enough to require hospitalisation are sensible exclusions, as is current drug or alcohol abuse (unless this is the target for intervention).", "Medical screening should exclude those with serious neurological, renal, liver or cardiac disease.", "Given psilocybin's tendency to modestly increase blood pressure, uncontrolled hypertension should also be an exclusion.", "Women who are pregnant, at risk of becoming pregnant (inadequate contraception) or breast feeding should also be excluded.", "All participants should be registered with a local general or family practitioner and consent to the sharing of their records with the study team.", "Failure to consent to this should raise clinical suspicion about motivations for participation.", "Commonly prescribed psychiatric medications should be withdrawn prior to use of psychedelics.", "Sufficient washout time is necessary, particularly for fluoxetine (Burke et al., 2000).", "Some tricyclic antidepressants, lithium, and the acute administration of selective serotonergic reuptake inhibitors (SSRIs) may increase sensitivity to psychedelics (Bonson and Murphy, 1996), as may haloperidol (Vollenweider et al., 1998).", "Chronic administration of SSRIs (Bonson, 1996; Stolz et al., 1983; Strassman, 1992) and monoamine oxidase inhibitors (Bonson and Murphy, 1996) appear to reduce sensitivity to psychedelics.", "Antagonists of the 5-HT2A receptor (mirtazapine and most antipsychotic drugs) attenuate response to psychedelics, as do benzodiazepines, particularly in acute use.", "The effect of antiepileptic drugs is not known.", "A psychiatrist with an appropriate Schedule I license is required to prescribe and administer the psilocybin, manage other medication, medically monitor the treatment and provide assessment and management of mental state and risk during the participant's journey through the trial.", "At least one session of psychological preparation is required for all participants and is probably most effectively delivered by psychotherapists, psychologists or counsellors that have experience of the psychedelic state and appropriate training (Phelps, 2017).", "Likely effects of the psychedelic should be discussed and attention given to the possibility of long forgotten, unknown or emotionally charged material surfacing.", "On the day of the treatment, participants should be accompanied at all times, preferably by those who provided psychological preparation.", "Onset with psilocybin starts at about 30 min, peaks after about 90 min and subsides after 4–6 h, making day case treatment viable.", "A comfortable, supportive environment with easy access to the lavatory is recommended.", "Music is often used to accompany the experience and has been shown to enhance the emotional response to psychedelics (Kaelen et al., 2015, 2016).", "A psychiatrist should be available at short notice, but need not necessarily be present.", "Dysphoria, confusion, anxiety, agitation, panic and paranoia are all expected reactions in a proportion of psychedelic experiences.", "They are usually mild and respond to simple reassurance and attendance to physical pain or discomfort.", "Rescue medications are a last resort, given under the supervision of the psychiatrist.", "A short-acting benzodiazepine such as lorazepam or midazolam is recommended.", "If rescue medications are used, it may be necessary to arrange overnight stay and monitoring in the hospital or clinical research facility.", "Otherwise, participants can leave the research facility accompanied by a friend or relative once the clinical team is satisfied that this is safe.", "Follow up of participants should include at least one session of psychological support with the therapist that has accompanied the participant through the trial.", "The question of ‘how much is enough’ is difficult.", "In a world where cost-efficiency is prioritised by most healthcare providers and given the requirements of regulatory bodies, we suggest that support should be minimal.", "We acknowledge that some may strongly disagree with this idea, but believe it strikes the right balance between idealism and pragmatism in the context of clinical trials.", "The trial itself will be a support if it includes regular follow up for collection of outcome measure data and psychiatric monitoring.", "In common with many trials of psychoactive drugs, effective blinding of psychedelic therapy is hard to achieve because the subjective effect is often obvious to participants and observers.", "This creates expectancy effects in both trial participants and researchers that can bias outcome measures and inflate effect sizes.", "This suggests certain elements of trial design.", "Firstly, ratings of primary outcome measures should be taken by trained raters who are blind to treatment allocation and preferably independent of the trial.", "Ratings should focus simply on the clinical state over the time period required for the rating and should not enquire about the nature and extent of the psychedelic experience.", "Videos of such ratings allow quality control, comparisons between raters and calculation of inter-rater reliability scores.", "Secondly, active placebos should be considered.", "Active placebos can be subthreshold doses of the investigational drug or a different drug with a similar (but non-therapeutic) psychoactive effect.", "Subthreshold dosages of the investigational drug have the advantage of simplicity and may be particularly effective if participants are not told the different dosing regimens in a trial (although this approach may raise ethical issues).", "However, subthreshold dosages may still be psychoactive, potentially reducing statistical power and increasing the likelihood of a type II error during analysis.", "Use of other psychoactive drugs as active placebos, such as methylphenidate, a benzodiazepine, or niacin, has been performed.", "However, this introduces the difficulty of choosing a drug that has a similar subjective effect but is known not to have a therapeutic effect.", "Crossover trials have been performed, but these are practically difficult and create problems with statistical analysis because of the putatively extended therapeutic effect of psychedelics.", "There is little consensus on which strategy is preferred.", "Psychedelics have a long history of use and yet they attract emotive and often polarised opinions in modern Western society.", "History suggests they may have a place in the treatment of refractory neurotic disorders, particularly depressive disorder, anxiety disorders, addictions and in the psychological challenges associated with death and dying.", "Psychedelics appear to have a context-dependent effect.", "This mandates carefully designed trials within safe and comfortable settings staffed with psychotherapists and psychiatrists familiar with their use.", "Whilst modern pilot studies (largely using psilocybin) have shown promise, treatments with classical psychedelics will need to stand up to the scrutiny of the RCT design, which itself poses significant challenges.", "The money to finance RCTs with psychedelics will likely come from a mixture of profit-driven commercial enterprises, charitable organisations, crowd-funding and government.", "The aim of RCTs is to demonstrate safety and efficacy.", "If safety and efficacy is confirmed, licensing and rescheduling will likely follow.", "At this point, psychedelics will need to demonstrate deliverability and cost-effectiveness if they are to become established and accepted treatments.", "Many treatments fail these tests.", "Delivery of psychedelics in real-world healthcare is likely to be expensive relative to other interventions, underlining our opinion that they are best investigated as options for those with socioeconomically costly psychiatric problems (such as treatment resistant depression) that are refractory to cheaper and more established therapies.", "JR wrote the article with contributions from JI.", "DN reviewed and commented on the article.", "All authors have seen and approved the final version of the manuscript before submission.", "JR has no conflicts of interest to declare.", "JI has no conflicts of interest to declare.", "DN has no conflicts of interest to declare.", "This study presents independent research part-funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King's College London.", "The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.", "The authors of this article acknowledge funding from the UK Medical Research Council (MR/J00460X/1)." ]	[ "Psychedelics may be useful for treating resistant depression, anxiety & addictions.", "Pilot trials with psilocybin in depression show early evidence of safety & efficacy.", "The legal & regulatory hurdles to approval are formidable, but surmountable." ]
Pyrolysis and spontaneous ignition of wood under transient irradiation: Experiments and a-priori predictions	Wood is a material widely used in the built environment, but its flammability and response to fire are a disadvantage. Therefore, it is essential to have substantial knowledge of the behavior of wood undergoing external heating such as in a fire. The majority of studies in the literature use constant irradiation. Although this assumption simplifies both modelling and experimental endeavors, it is important to assess the behavior of materials under more comprehensive heating scenarios which might challenge the validity of solid-phase ignition criteria developed previously. These criteria are evaluated here for the spontaneous ignition under transient irradiation by combining experimental measurements and a-priori predictions from a model of heat transfer and pyrolysis. We have applied a two-step transient irradiation in the cone calorimeter in the form of a growth curve followed by constant irradiation. We use white spruce samples of size 100×100 mm and thickness of 38 mm. We measure the temperature at different depths and the mass loss. A one dimensional model written in the open source code Gpyro is used to predict the pyrolysis behavior. The model has a chemical scheme in which the components of wood (hemicellulose, cellulose, lignin) become active, then decompose in two competing reactions: one reaction to char and gas, and one reaction to tar. The kinetic parameters, as well as the thermal properties of the wood and char are taken from the literature, while ρ and moisture content are measured experimentally. A priori predictions of the temperature, made prior to the experiments, show excellent agreement with the measurements, being within the experimental uncertainty range. The mass loss rate (MLR) predictions are qualitatively similar to the measurements, but there is a large uncertainty in the measurements. For a-posteriori simulations, certain parameters are changed after having access to the measurements to improve the simulations. Also, we perform an evaluation of the solid phase ignition criteria used in the literature, and find that neither criteria is a consistent indicator of ignition. These results help understand the spontaneous ignition of wood subjected to transient irradiation and identify strengths and gaps in the topic.	[ "CHEM", "ENGI", "MATE", "PHYS" ]	[ "Heat transfer", "Ignition", "Modelling", "Pyrolysis" ]	[ "1600", "2213", "2500", "3100" ]	[ "Wood is one of the oldest building materials and, because of its cost effectiveness and environmental advantages, it has now become a viable alternative to concrete and steel for modern structures.", "However, fire is a major risk for wood structures.", "Therefore it is essential to have a substantial understanding of how wood behaves when subjected to a fire, especially when and how it will ignite.", "Ignition is a complex phenomenon which includes thermochemical processes in both solid and gas.", "A full numerical representation of ignition requires multiple complex calculations, making it impractical to solve ignition problems in most applications related to solid-phase response.", "One solution to this issue is to eliminate consideration of the gas phase, and instead adopt ignition criteria, which are values linked to thermal response parameters such as surface temperature and mass loss rate [1].", "The majority of ignition studies in the literature use the assumption of a constant irradiation from a heat source.", "While this assumption is convenient due to its simplicity, it is singular and does not reflect a realistic fire scenario, for which irradiation tends to grow over time.", "Transient irradiation is a more comprehensive case, thus analyzing dynamic heat transfer effects and their impact on solid-phase ignition criteria is a relevant topic [2].", "Previous studies have used transient heating scenarios for piloted ignition of different materials such as polymers and wood.", "However, very few works look at spontaneous ignition under transient irradiation.", "DiDomizio et al. [3] evaluated a number of models in the literature that predict the spontaneous ignition of wood under time-varying exposure, but the current study is the first work that assesses the performance of solid-phase ignition criteria for wood under transient irradiation.", "In this paper, all these criteria are evaluated for the spontaneous ignition of wood under transient irradiation by combining experimental measurements and a-priori predictions obtained from a one dimensional model of pyrolysis.", "A-priori predictions are results of simulations run prior to performing the experiments, so they are not affected by bias [8].", "They differ from a-posteriori predictions, which are the most used type of predictions in literature, that are done after the experimental measurements have been analyzed.", "In this paper, a-posteriori simulations are used as sensitivity analysis to show the influence of parameters such as heat of pyrolysis, reaction order, and radiative absorption coefficient.", "A series of experiments were conducted using a cone calorimeter apparatus on commercially available Canadian white spruce, a typical softwood used in construction.", "The specimens were conditioned to a 9% moisture content, and the wet density measured to be between 490 and 520 kg/m3.", "The sample size was 100×100 mm, with a thickness of 38 mm.", "For temperature measurements, 3 thermocouples were placed in the sample, located at 3 mm below the top surface, the middle of the sample, and 3 mm above the bottom surface.", "The thermocouples were stainless steel sheathed K-type (unexposed junction) with a 1.6 mm outer diameter.", "Three 1.98 mm diameter holes were drilled into one of the sides of the specimen at depths of 3, 19, and 35 mm.", "The instrumented specimen was placed on a 25 mm thick layer of ceramic fiber insulation, which was then placed on top of the standard cone calorimeter sample holder, filled with an additional 25 mm layer of insulation, as shown in the left side Fig. 1.", "This thick layer of insulation was used to ensure that the boundary condition on the bottom surface of the specimen could be approximated as adiabatic.", "To this end, an additional thermocouple was placed between the two layers of insulation in order to quantify heat losses through the bottom surface.", "The sample holder and sample were then wrapped in a layer of 25 mm thick ceramic fiber insulation on the outside, which was affixed in two locations with 20 gauge mechanics wire.", "Mass and temperature measurements were taken for 30 min in the case of no ignition or a few minutes after in case of ignition.", "As this study investigates spontaneous ignition of wood, the spark igniter was removed.", "After the sample assembly was placed on the load cell, the cone heater was used to expose the top surface to a two-step transient irradiation.", "The cubic curve of the irradiation replicates a growing fire, which becomes steady after a certain time, at the threshold value.", "The transient irradiation scenarios were obtained by setting the initial temperature of the heater, initially ambient temperature, then increasing it using a digital temperature controller at a constant rate of approximately 1 °C s−1 up to a setpoint value.", "The constant threshold value was applied until the experiments reached the 30 min mark, unless ignition occurred, in which case the experiment was stopped shortly after ignition.", "Five temperature setpoints were selected in this study, corresponding to steady-state irradiation values of 25, 27, 28, 30 and 50 kW/m2.", "The threshold values of 27–28 kW/m2 are of particular interest in the present work because ignition was less likely, but still possible, as seen in previous work [3] and indicated by the a-priori simulations.", "Prior to the experiments, the transient irradiation scenarios were first characterized using a water cooled Schmidt-Boelter heat flux gauge.", "The cone heater temperature was also recorded, in both characterization tests as well as the experiments, to ensure that the transient heating profiles were identical in both cases.", "The irradiance over the surface of the specimen was uniform, although minor decreases towards the corners of the specimen were observed (less than a 5% decrease from the center value).", "The chemical scheme used in this model is based on the scheme first used by Koufopanos [14], using the kinetic parameters from Bellan [15].", "As shown in Fig. 2, the first reaction consists in the transformation of the virgin components of wood (hemicellulose, cellulose, lignin) into active species (marked as (1) in Fig. 2), then each component decomposes into char and gases (marked as (2)) and tar (marked as (3)).", "This chemical scheme has been used frequently in the literature and has proven to be versatile in predicting a variety of experiments on wood materials.", "The kinetic parameters, namely the pre-exponential factor, activation energy and heat of pyrolysis for each reaction are given by Bellan [15] and were first implemented in Gpyro by Richter [16].", "They are summarized in Table 1.", "Two sets of simulations were produced.", "A-priori simulations were done after the experiments were devised using material properties for wood [15], as shown in Table 2, but before the experiments were run, thus avoiding bias.", "All 3 components of wood have the same thermal and physical properties.", "The thermal conductivity and specific heat capacity were taken at room temperature.", "Also, the model does not predict ignition, but simulates pre-ignition pyrolysis behavior.", "The ignition times are taken from the experiments and the model results after experimental ignition occurs are disregarded.", "The results of the a-priori simulations and experimental measurements are presented in Fig. 3 for the 30 kW/m2 threshold and in Fig. 4 for the 50 kW/m2 threshold scenarios.", "Because there were two repeats for each scenario, both experimental values are plotted, with an uncertainty cloud between them.", "In all of these four experiments, ignition occurred between 9 and 10 min or shortly after the 10 min mark.", "The temperature predictions show excellent agreement with the measurements, as presented in Fig. 3 and 4, being within the experimental uncertainty cloud.", "The MLR predictions are qualitatively similar to the measurement in the case of the higher irradiation case (50 kW/m2), however for the 30 kW/m2, the measurement drops right before ignition, whereas the prediction shows an increase in mass loss rate, which is generally indicative of ignition.", "One of the explanations is that there was a lot of experimental uncertainty in the mass loss readings which were amplified in the differentiation to obtain the MLR.", "This could be because of the relatively small mass loss compared to the mass of the sample and the sample holder, which is within the range of uncertainty of the scale.", "For the scenario with a threshold at 25 kW/m2, ignition did not occur in either of the repeats, thus the measurements were continued for a period of 30 min, as seen in Fig. 5.", "However, after around 10 min from the start of the experiment, the charring front passed the depth of 3 mm where the topmost thermocouple was placed.", "As this occurred the thermocouple dislodged from the charred surface, which means the recorded measurement was no longer representative.", "Therefore, the measurements at 3 mm depth were not relevant after 10 min.", "Non-piloted ignition did not occur in the scenarios with a threshold of 25 and 27 kW/m2 and in one repeat of the 28 kW/m2, but it occurred in 4 repeats of the 28 kW/m2 threshold scenarios and in the higher heat flux thresholds of 30 and 50 kW/m2.", "The observed ignition times are summarized in Table 4.", "The ignition criteria analyzed are the critical surface temperature, MLR, heat flux, and time-energy squared.", "The a-posteriori simulations were conducted after the experimental measurements were concluded and, while they do not contain any direct experimental results, they were done knowing what the experimental results look like.", "The sensitivity analysis of the modelling was performed with an a-posteriori model.", "The parameters investigated are the heat of pyrolysis, in-depth radiation and reaction order.", "The scenario with a threshold of 50 kW/m2 was used for presenting the sensitivity analysis, although the differences between this scenario and other are small (around 1%).", "The heat of pyrolysis that was studied is the value used in the reactions from the kinetic scheme where the components of wood become char.", "In this chemical scheme [15] these reactions are exothermal, however, they are not suited for studying ignition.", "Therefore, the a-posteriori simulations reduced these values to zero and the results stay the same for all scenarios.", "That confirms the inadequacy of exothermic heat of pyrolysis for this fire application.", "The in-depth radiation for wood depends on the radiation source.", "For wavelengths below 2 μm, corresponding to the wavelengths of tungsten lamps, in-depth absorption is negligible.", "For higher wavelengths, like the cone calorimeter at the temperatures in this study, the wood sample absorbs energy in-depth.", "The varying parameter for the sensitivity analysis is κ (m−1), the absorption coefficient.", "As shown in Fig. 6 for the scenario using a threshold of 50 kW/m2, a lower κ means higher in-depth absorption.", "The temperature value at 3 mm depth is very similar (4.3% increase) to the a-priori case without in-depth radiation, but there is significant variation (76% increase) in the temperature at 19 mm, as more energy is absorbed at a higher depth.", "For a higher κ, the temperature at 3 mm increases (21.8%) compared to the a-priori case, but at 19 mm the temperature does not increase significantly (5.0%).", "For both cases with in-depth absorption, MLR is higher, but for a lower κ, the increase in MLR starts later than for the other two cases, whereas for a higher κ the increase in MLR occurs at a similar time to the a-priori case.", "This shows that in-depth radiation should be taken into account when modelling cone calorimeter experiments.", "The a-priori simulations had first-order reactions, as used in Bellan [15], therefore it is of interest to study the influence of the reaction order.", "Two different reaction orders from the literature are considered.", "The first is for the reactions where the components of wood decompose into char, considering the same value (4.78 [17]) for all 3 components.", "This is marked as (a) in Table 3.", "The second value for the reaction order is taken from Huang et al. [18] and is marked as (b) in Table 3.", "Each reaction of the wood components decomposing into char has a different value: 2.4 for hemicellulose, 0.48 for cellulose, 10.4 for lignin.", "Fig. 7 shows that temperatures at the surface and at 3 mm depth decrease insignificantly (less than 1%) when the reaction order is not 1.", "Changes in MLR are more significant, increasing by 12.5% for case (a) and 7.5% for case (b), but the same shape of the curve is kept for all cases.", "Therefore the reaction order is an influential parameter for MLR, but it does not affect the temperatures in a significant way.", "The same literature values for the reaction orders were used.", "The purpose of this study was to show whether or not the reaction order of the tar reaction is more influential than the char reaction.", "Fig. 8 shows that using a reaction order different from 1 for the tar reaction significantly changes temperature and MLR results.", "The surface temperature is close to the temperature at 3 mm, which are lower and higher respectively compared to the base case where n=1.", "The peak of the MLR curves is significantly higher when using a reaction order different from 1.", "This shows that the tar reaction is more influential than the char reaction.", "Babrauskas [20] reviewed the first studies looking at the ignition criteria for spontaneous ignition of wood that did not involve oven-based experimental setups.", "These include Moran [21], Shoub and Bender [22], Melinek [23], and Abu-Zaid [24].", "A few of the studies have calculated the surface temperature from correlations, thus being excluded from further analysis as well.", "There are a few more recent studies that have been included, namely Shi [25], Boonmee and Quintiere [26] and DiDomizio [3].", "The most studied criterion is the critical ignition temperature, followed by the critical heat flux for ignition.", "Critical mass loss rate appears only in the newer studies and has a wide range of values.", "Table 4 shows the scenarios where ignition occurred, as well as the measured values of MLR, heat flux and predictions of surface temperature at the time of ignition.", "Because the surface temperature was not measured, the model predictions of surface temperature are used in the analysis of ignition criteria.", "For all of the criteria, the values obtained in this work fit within the ranges found in literature.", "However, this is not indicative of the suitability of the criteria; since most of these values were obtained using a cone calorimeter or a fire propagation apparatus with constant irradiation exposures, it is not clear whether these values are applicable to other scenarios.", "Table 5 collects the maximum temperatures, MLR and irradiation reached in the scenarios where ignition did not occur.", "The maximum MLR in these cases is at least as high as the values recorded for the cases where non-piloted ignition occurred, and the heat flux and surface temperature are within the same ranges.", "The most interesting case is the scenario with the threshold at 28 kW/m2, where ignition occurred in four instances, but did not occur in one repeat.", "The heating conditions were the same (irradiation) and the sample behaved similarly (temperature & MLR); from this it must be concluded that neither of these criteria are able to predict the onset of ignition alone.", "The final ignition criteria analyzed is the time-energy squared correlation, developed by Reszka et al. in [7].", "This criterion requires a set of experiments for establishing the correlation between the exposure time and the energy squared.", "Then, the line of best fit of the correlation, which in the case of this set of experiments was a power fit, is compared with the exposure for every scenario and the intersection between the two lines is the predicted time to ignition.", "The predicted times to ignition are shown in Table 6.", "In the case of higher heat flux (50 kW/m2), the time to ignition is overpredicted by 17%, while for the 28 kW/m2 it is underpredicted by 21%.", "The worst performance is for the 30 kW/m2 scenario, where ignition is predicted to not occur.", "Thus, this criteria is not suitable for this set of experiments.", "A one dimensional numerical model was developed and applied to study wood pyrolysis when subjected to transient irradiation.", "The model was validated using cone calorimeter experiments on Canadian white spruce which was exposed to a two-step transient irradiation scenario, the first part consisting of a growing irradiation followed by a constant threshold.", "These thresholds had a range between 25 and 50 kW/m2.", "The experiments measured in-depth temperatures and mass loss rate, which were used to validate the model predictions.", "The model used a chemical scheme with an initial reaction in which the virgin components of wood (cellulose, hemicellulose, lignin) become active, followed by the decomposition of each component into char and volatiles, and tar.", "The kinetic parameters, as well as the thermal properties of the wood were taken from literature for the a-priori model.", "The surface temperature predictions of the a-priori simulations, the measured MLR and the measured heat flux were used in the analysis of the criteria for non-piloted ignition.", "While all the criteria are within the ranges of the values suggested in the literature, none can reliably predict if ignition will occur.", "Thus, the solid-phase criteria for non-piloted ignition are not easily applicable in cases with transient irradiation.", "For the a-posteriori predictions, which were used as a sensitivity analysis, the heat of pyrolysis, reaction order and absorption coefficient were varied, with values taken from the literature.", "It was found that exothermic heats of pyrolysis do not have any influence on the predictions and the reaction order of the char reaction has an impact on the MLR (max.", "12.5%), but it is insignificant on the temperatures (less than 1%).", "However, the reaction order of the tar reaction is more influential, increasing the peak MLR and changing the temperature results when it is different from 1.", "The in-depth absorption is an important factor in cone calorimeter experiments due to the wavelength of the radiation.", "For temperature predictions, the largest difference is a 76% increase in-depth and for MLR the differences are as high as 3 orders of magnitude.", "For the first time in literature, the non-piloted ignition of wood under transient irradiation is studied with an emphasis on evaluating the performance of solid-phase ignition criteria.", "As these criteria have been established using constant irradiation scenarios, they prove to be inconclusive in predicting whether or not ignition will occur, showing similar values in all experiments.", "Therefore, there is a need to look into other indicators of ignition for the more general case of transient irradiation." ]	[]
Anti-stress neuropharmacological mechanisms and targets for addiction treatment: A translational framework	Stress-related substance use is a major challenge for treating substance use disorders. This selective review focuses on emerging pharmacotherapies with potential for reducing stress-potentiated seeking and consumption of nicotine, alcohol, marijuana, cocaine, and opioids (i.e., key phenotypes for the most commonly abused substances). I evaluate neuropharmacological mechanisms in experimental models of drug-maintenance and relapse, which translate more readily to individuals presenting for treatment (who have initiated and progressed). An affective/motivational systems model (three dimensions: valence, arousal, control) is mapped onto a systems biology of addiction approach for addressing this problem. Based on quality of evidence to date, promising first-tier neurochemical receptor targets include: noradrenergic (α1 and β antagonist, α2 agonist), kappa-opioid antagonist, nociceptin antagonist, orexin-1 antagonist, and endocannabinoid modulation (e.g., cannabidiol, FAAH inhibition); second-tier candidates may include corticotropin releasing factor-1 antagonists, serotonergic agents (e.g., 5-HT reuptake inhibitors, 5-HT3 antagonists), glutamatergic agents (e.g., mGluR2/3 agonist/positive allosteric modulator, mGluR5 antagonist/negative allosteric modulator), GABA-promoters (e.g., pregabalin, tiagabine), vasopressin 1b antagonist, NK-1 antagonist, and PPAR-γ agonist (e.g., pioglitazone). To address affective/motivational mechanisms of stress-related substance use, it may be advisable to combine agents with actions at complementary targets for greater efficacy but systematic studies are lacking except for interactions with the noradrenergic system. I note clinically-relevant factors that could mediate/moderate the efficacy of anti-stress therapeutics and identify research gaps that should be pursued. Finally, progress in developing anti-stress medications will depend on use of reliable CNS biomarkers to validate exposure-response relationships.	[ "BIOC", "NEUR" ]	[ "Addiction", "Drug seeking", "Mechanisms", "Medications", "Self-administration", "Stress" ]	[ "1303", "1310", "1312", "1314", "2804", "2807" ]	[ "Stress-related substance use poses a critical challenge for treating all substance use disorders (SUDs), yet this scientific field is at an early investigational stage.", "Candidate therapies for attenuating stress-potentiated drug-seeking/use include medications, neuro-stimulation, cognitive-behavioral, exercise/physical activity, and other approaches.", "Due to that panoramic breadth, this selective review focuses on neuropharmacological strategies grounded in preclinical and clinical evidence that offer translational promise for reducing stress-induced seeking and consumption of nicotine, alcohol, marijuana, cocaine, and opioids.", "This review does not discuss effects of medications on attenuating drug-abstinence signs/symptoms (which are stressful); although withdrawal effects can lead to drug seeking, that outcome is not inevitable, mostly relates to physical dependence on specific substances, and retaining that topic would greatly lengthen this review.", "In short, this restricted scope highlights neurochemical mechanisms for reducing stress-induced seeking/self-administration for the most prevalent abused substances.", "Literature gaps are identified as a means to advance this field of inquiry.", "Mechanisms that link stress reactivity to psychoactive substance use/abuse are complex and incompletely understood.", "Stressors modulate the neurobehavioral effects of abused substances in stressor-, procedure- and drug-class-specific ways, but also activate some common brain pathways (Armario, 2010; Lopez et al., 1999).", "Excellent reviews at a more granular level than this translational review indicate numerous neurochemical systems modulate stress-induced substance-seeking/use including noradrenaline (NA), hypothalamic-pituitary-adrenal (HPA) axis including corticotrophin-releasing factor (CRF) and glucocorticoids, opioid, endocannabinoid (eCB), serotonin (5-HT), orexin/hypocretin, dopamine (DA), glutamate, and γ-amino-butyric acid (GABA) (Briand and Blendy, 2010; Dunn and Swiergiel, 2008; Groeneweg et al., 2011; Herman and Cullinan, 1997; Koob, 2010; Mantsch et al., 2016; Plaza-Zabala et al., 2012; Shalev et al., 2000, 2010; Sinha, 2008; Sinha et al., 2011; Viveros et al., 2005), and other systems that are less well studied (see section 2.10).", "Due to this “neuro-symphony of stress” (Jöels and Baram, 2009), it is only feasible to study a limited number of mechanisms at the same time.", "When relevant, I mention research that has examined pairwise interactions of transmitter systems.", "A motivational systems model (Fig. 1), based on affective neuroscience theories (Alcaro and Panksepp, 2011; Baker et al., 2004; Davidson et al., 2000; Diekhof et al., 2008) suggests that three empirically separable dimensions may underlie stress-induced drug seeking/use, being greatest at the nexus of negative-hedonic or dysphoric (avoidance-punishment), high-arousal (activation), and low-control (disinhibition) states.", "Based on research in the systems biology of addiction (Aston-Jones and Harris, 2004; Briand and Blendy, 2010; Hester and Garavan, 2004; Koob, 2008; Shalev et al., 2000; Sinha, 2008; Spanagel et al., 1992; Waselus et al., 2011; Zhou et al., 2010), this review adopts the approach that stress-related drug-seeking/use is a function of dysregulated neural (particularly limbic) systems underlying these affective/motivational dimensions.", "Throughout this review, I link candidate anti-stress pharmacological approaches to these motivational dimensions (to the extent that current evidence allows).", "Experimental approaches to studying stress-related drug-seeking/use can be classified with regard to: (a) type of stressor, e.g., physical, environmental, and pharmacological, (b) stage in the behavioral cycle of addiction (initiation, progression, maintenance, relapse), and (c) drug-seeking outcome measure (e.g., operant responding for drug, conditioned place preference).", "This literature review focuses on models of maintenance and relapse stages, which more closely translate to individuals presenting for SUD treatment (who have already initiated and progressed).", "Most studies with laboratory animals to date have measured reinstatement of operant responding for drug as the outcome measure; fewer studies have measured conditioned place preference (CPP) but these are included in this review for comprehensiveness.", "The CPP model has limitations, particularly because drug is experimenter-administered and drug exposure is minimal.", "As summarized in Table 1, drug seeking/consumption is enhanced by a wide range of stressors including: social factors (defeat, isolation), physical factors (restraint, cold swim, food deprivation, footshock), and pharmacological probes (e.g., α2-adrenoceptor antagonist yohimbine [section 2.1], kappa-opioid agonist, and neuropeptides [sections 2.2.1, 2.7, 2.10]).", "Each stressor type has strengths and weaknesses, and translation of these models to clinical treatment is limited by several moderating factors, briefly discussed next.", "Experimental models of stress-related drug seeking/use have not routinely included clinically-relevant factors that might mediate/moderate the efficacy of anti-stress therapeutic agents.", "Next, I briefly discuss selected factors worthy of investigation in developing anti-stress pharmacotherapies in these models.", "Detailed analysis of these factors exceeds the scope of this review, but the purpose is to prime the reader to consider these factors in the context of the discussion that follows in section 2.", "The extent to which acute stressors augment the reinforcing effects of psychoactive substances may partly depend on the subject's history of substance exposure (e.g., in utero) or use (i.e., voluntary intake).", "Chronic and/or escalating substance intake has been shown to produce stress-like responses that, in turn, sensitize animals – via CRF, glucocorticoid, and glutamatergic mechanisms – to acute stress-potentiation of the reinforcing effects of cocaine (Ahmed et al., 2000; Graf et al., 2011; Mantsch et al., 2008; Sorge and Stewart, 2005) and alcohol (Liu and Weiss, 2002; Sidhpura et al., 2010).", "These data suggest that agents capable of desensitizing these neurochemical systems could be therapeutically useful, especially for subjects with extensive chronic exposure or high levels of tolerance/dependence.", "Research into effects of stressors on drug-motivated behavior has not adequately investigated the environmental context, particularly non-drug choice alternatives.", "Higher magnitude or probability of non-drug positive reinforcers in the environment during stress-exposure may bias animals away from habitual drug seeking, i.e., serve a stress-protective function.", "This phenomenon has been modeled in rats using environmental enrichment (Chauvet et al., 2009).", "Future studies should examine whether anti-stress pharmacotherapies are more effective in combination with non-drug positive reinforcers.", "In contrast, economic insecurities among humans (i.e., dearth of positive reinforcement in a person's environment) are chronic stressors that contribute to substance use.", "Furthermore, magnitude and/or probability of punishers in the subject's environment may play an important role in modulating stress-induced drug seeking, although this has rarely been investigated.", "For instance, early social isolation of rats enhances cocaine motivation, but isolation did not affect extinction or stress-reinstatement of cocaine seeking (Baarendse et al., 2014).", "Future studies should investigate whether drug intake that is suppressed by punishment (cf. Pelloux et al., 2007) is more easily reinstated by stress than drug intake that is suppressed by non-drug positive reinforcers, and whether other factors such as drug history moderate these effects (see Smith and Laiks, 2017).", "Sleep loss (and/or circadian disruption) is a biological stressor but has rarely been investigated in studies of drug seeking/self-administration.", "This is noteworthy because insomnia is a predictor of relapse to substance abuse (Roehrs and Roth, 2015; Kaplan et al., 2014).", "Also, sleep loss causes hyperalgesia (Alexandre et al., 2017; Roehrs et al., 2006; Vanini, 2016) that, in turn, may increase misuse of opioids and other substances (Edwards et al., 2011; Hipólito et al., 2015; Wachholtz et al., 2015).", "Recognizing the role of insomnia as a stressor leads to the idea of treating insomnia as one cause of substance use.", "Non-addictive sleep-promoting agents might be useful in this regard (see section 2.7).", "Stress can increase nociceptive signaling and contribute to chronic pain states (Ghitza, 2016; Imbe et al., 2006; Jennings et al., 2014; Johnson and Greenwood-Van Meerveld, 2014).", "This overlap is important because it suggests (1) stress-exposure could cross-sensitize subjects – presumably via shared neurobiological pathways – to drug seeking, and (2) therapeutics used to attenuate stress-reactivity could have dual actions on analgesia and drug seeking/use.", "On the other hand, it is well established that stress exposure can sometimes produce analgesia (Butler and Finn, 2009; Parikh et al., 2011).", "Thus, parametric studies are needed to examine basic interactions between (acute and chronic) stressors and (acute and chronic) nociceptive stimulation, and to translate this knowledge toward the development of therapeutic approaches that could potentially address both conditions.", "Early-life or chronic stress as well as psychiatric conditions (e.g., anxiety, depression) are highly prevalent among individuals with SUDs and may increase risk of relapse to substance use especially under stressful conditions (Chida and Hamer, 2008; Lijffijt et al., 2014).", "Experimental animal models have only infrequently created such a behavioral history (e.g., repeatedly exposing animals to anxiety-provoking or helplessness-provoking conditions) or exposed adolescent animals to the biological stressor corticosterone (Al-Hasani et al., 2013a; Bertholomey et al., 2016) to determine its effects on stress-induced drug seeking/use.", "Further systematic research would be valuable for understanding a complex yet important set of clinically relevant influences.", "Impulsivity is an example of one trait variable that may influence stress-induced drug seeking behavior; furthermore, this trait maps onto a key motivational dimension, disinhibition/control, proposed in this review (Fig. 1).", "Specifically, high trait impulsivity (associated with less prefrontal cortical inhibitory control) could, in the presence of a stressor, result in less resistance to drugs and related cues, making the individual more prone to substance use.", "We have found that the relationship between trait impulsivity on current mood state (e.g., Beck Depression Inventory scores) is mediated by effects of cumulative adverse drug-use consequences for both cocaine and heroin users (Lister et al., 2015; Reid et al., 2018).", "Other inter-related trait variables such as anxiety sensitivity (Lejuez et al., 2008), distress tolerance (Brown et al., 2005), emotion regulation (Naragon-Gainey et al., 2017), and resilience or coping ability (Belding et al., 1996; Hyman et al., 2009) are stress-mediator/moderator variables that are not routinely assessed in human studies, but these might potentially predict drug abstinence during addiction treatment (Strong et al., 2012).", "Chronic manipulation of HPA axis (CORT) during adolescence increases sensitivity to YOH-induced alcohol seeking in female more than male adult rats (Bertholomey et al., 2016).", "Neonatal isolation stress increased cocaine seeking behavior in adult rats, however, effects did not differ for males and females (Lynch et al., 2005).", "Some effects of acute stressors on relapse-like behavior are sex-dependent, with females typically more sensitive especially during high-estradiol levels (Anker and Carroll, 2010; Back et al., 2005; Chartoff and Mavrikaki, 2015; Feltenstein et al., 2011; Swalve et al., 2016; Verplaetse et al., 2015) although not all studies observe clear sex differences in stress-induced drug seeking (Buffalari et al., 2012; Zhou et al., 2012; also, see reviews by Becker et al., 2012; Bobzean et al., 2014; Fox and Sinha, 2009; Hudson and Stamp, 2011).", "Consistent with current NIH policy, studies should be adequately powered to detect sex differences in stress-induced drug seeking/use as well as candidate pharmacotherapies that may attenuate these effects.", "Few studies have addressed the role of genetic variation in stress-related substance use and relapse, in contrast to genetic association studies of initial addiction vulnerability.", "We reported that, in heroin-dependent volunteers undergoing buprenorphine dose tapering with an abstinence incentive, variations in genes encoding the kappa-receptor (OPRK1 rs3802281; Greenwald et al., 2012) and glucocorticoid receptor (NR3C1 rs6877893; Greenwald and Burmeister, 2018) predicted opioid relapse potential.", "Variation in OPRK1 rs6989250 is also associated with risk of cocaine relapse (Xu et al., 2013).", "Although CRH-binding protein (CRHBP) genotype variation moderated the association between stress-induced negative affect and negative consequences of alcohol intake in heavy-drinking subjects (Tartter and Ray, 2012), effects of this genotype on stress-induced drinking were not studied.", "Nonetheless, CRF is a promising target, as CRHR1 knockout animals are less sensitive to stress-induced alcohol intake (Hansson et al., 2006; Molander et al., 2012; Pastor et al., 2011).", "CRF-R1 knockdown mice are also less sensitive to stress-reinstatement of cocaine seeking (Chen et al., 2014).", "This section reviews evidence from studies related to various neurochemical systems that offer anti-stress therapeutic potential.", "To promote translational studies, each section indicates positron emission tomography (PET) imaging radiotracers that could be used to investigate proof-of-targeting in future prospective studies.", "The NA system has been the most commonly studied neurochemical domain for stress-related substance use, alone or in combination with other systems (see below).", "Discontinuation of chronic exposure to nicotine (Bruijnzeel et al., 2010; Sofuoglu et al., 2003), alcohol (Muzyk et al., 2011), cocaine (McDougle et al., 1994; Sofuoglu and Sewell, 2009), and opioids (Maldonado, 1997; Van Bockstaele et al., 2001) is a functional stressor associated with increased NA neurotransmission.", "It has been hypothesized that elevated NA release in the extended amygdala, and altered DA-mediated plasticity in the ventral tegmental area (VTA), alter hedonic processing of drug-related stimuli and are common substrates in withdrawal-associated relapse to drug seeking (Aston-Jones and Harris, 2004; Espana et al., 2016; Fitzgerald, 2013; Smith and Aston-Jones, 2008; Weinshenker and Schroeder, 2007).", "Yohimbine (YOH) is an α2-adrenoceptor antagonist that increases NA neurotransmission by blocking feedback at presynaptic autoreceptors (Doxey et al., 1984; Goldberg and Robertson, 1983) and has become an important tool for investigating stress-related drug seeking/use.", "YOH-mediated increases in NA release and synaptic levels regulate HPA axis activity (Armario, 2010; Banihashemi and Rinaman, 2006; Grunhaus et al., 1989; Leri et al., 2002; Smythe et al., 1983), as well as 5-HT and DA neurotransmission (Brannan et al., 1991; Cheng et al., 1993; Hopwood and Stamford, 2001; Maura et al., 1982; McCall et al., 1991; Millan et al., 2000; Mongeau et al., 1993; Raiteri et al., 1990; Söderpalm et al., 1995a, b; Winter and Rabin, 1992).", "In a PET neuroimaging study of rhesus monkeys, YOH increased [11C]-flumazenil binding potential (Matsunaga et al., 2001) indicating YOH actions at GABA-A receptors that might correlate with its anxiogenic (negative-hedonic, arousing) and/or disinhibiting motivational effects (Fig. 1).", "YOH has been used extensively as an experimental stressor in animal and human laboratory models.", "It produces anxiogenic effects in animals, healthy subjects, patients with panic disorder and opioid use disorder, which can be blocked by the α2-adrenoceptor agonist clonidine (Albus et al., 1992; Bremner et al., 1996; Cameron et al., 2000; Charney et al., 1983, 1992; Gurguis et al., 1997; Mattila et al., 1988; Pellow et al., 1987; Stine et al., 2002).", "These anxiogenic effects are presumed to mediate the effects of YOH on the reinforcing effects of drugs and drug-related stimuli.", "Reviews have concluded that YOH is a reliable and potent inducer of drug seeking with translational value (Bossert et al., 2005; Figlewicz et al., 2014; See and Waters, 2011; Shaham et al., 2000a).", "On the other hand, Chen et al. (2015) found that the effect of YOH on food-reinforced operant behavior may partly depend on factors unrelated to stress-induction; namely, YOH did not induce conditioned place aversion, YOH increased responding independent of the rat's history of contingent self-administration of food reward, and YOH increased medial prefrontal cortex (but not nucleus accumbens) dopamine levels.", "As a result, Chen et al. proposed that YOH may invigorate responding for cues that possess weaker rewarding effects in rodents (also see Box 2 in Mantsch et al., 2016).", "Given the extensive use of YOH in experimental stress studies, such findings warrant caution in relying only on YOH as an experimental model, yet encourage further investigation to determine the precise conditions under which YOH exerts its response-enhancing effects.", "For instance, YOH effects could differ for food vs. drug reinforcers, for paradigms involving drug-paired cues, for single-operant vs. choice scenarios, the subject's behavioral history, or other methodological factors.", "In animal studies, YOH administration delays extinction of cocaine seeking (Kupferschmidt et al., 2009) and – as summarized in Table 1 – YOH robustly reinstates previously extinguished responding for nicotine, alcohol, cocaine, and opioids.", "Reinstatement of alcohol seeking by YOH (previously established to be a 5-HT1A partial agonist; Millan et al., 2000; Winter and Rabin, 1992) was blocked not only by the α2 agonist clonidine but also by a selective 5-HT-1A antagonist (WAY-100,635), suggesting an important role for 5-HT in mediating YOH-induced alcohol seeking (Lê et al., 2009; see section 2.3).", "Although YOH did not increase heroin seeking in opioid-dependent rats (Minhas and Leri, 2014), our group was the first to find that YOH increased drug seeking in humans, i.e., the stressor increased opioid-maintained responding in a sample of buprenorphine-maintained heroin-dependent human subjects (Greenwald et al., 2013) and we recently extended this to cigarette puff-maintained responding in tobacco users (Woodcock et al., under review).", "Tests of YOH to reinstate Δ9−THC- responding have not been reported (due to limitations of animal models in demonstrating reinforcing effects of Δ9-THC), although chronic Δ9−THC exposure during adolescence augmented YOH reinstatement of heroin seeking (Stopponi et al., 2014).", "Furthermore, YOH augments cue-induced reinstatement for nicotine (Costello et al., 2014), heroin (Banna et al., 2010) and cocaine (Buffalari and See, 2011; Feltenstein and See, 2006; Fletcher et al., 2008) in animals, and enhances cocaine cue-reactivity in cocaine users (Moran-Santa Maria et al., 2014), suggesting this primarily NA stressor can enhance the conditioned reinforcing properties of drug cues to elevate relapse risk.", "Moreover, across animal and human studies (Adams et al., 2017; Bari and Robbins, 2013; Ma et al., 2003; Sanger, 1988; Schippers et al., 2016; Sun et al., 2010; Swann et al., 2005), YOH and other α2-antagonists increase impulsive responding (i.e., decrease self-control; see Fig. 1), suggesting another mechanism by which YOH could increase substance use (see Table 2).", "Interestingly, NA binding to α2 receptors in prefrontal cortex regulates attentional set, response inhibition and behavioral flexibility (Arnsten, 1998; Aston-Jones and Cohen, 2005; De Martino et al., 2008; Ramos and Arnsten, 2007; Robbins and Roberts, 2007).", "YOH also causes perseverative responding (Caetano et al., 2013), which predicts that YOH should promote drug-maintained responding particularly habitual substance use.", "The foregoing data suggest agents that reduce NA neurotransmission are first-line candidates as anti-stress medications for at least some SUDs; this proposition has been tested in animal studies.", "As indicated in Table 2, the α2-agonists clonidine, lofexidine and guanfacine block stress-reinstated responding for nicotine, alcohol, heroin, cocaine, and heroin/cocaine “speedball” (Highfield et al., 2001) at doses that blocked footshock-induced NA release in prefrontal cortex and amygdala.", "We recently proposed (Woodcock et al., under review) that the occupancy ratio of NA receptors (higher-affinity α2-receptors vs. lower-affinity α1-and β-receptors) may underlie stress-induced drug use.", "Specifically, we hypothesized that during acute stress-exposure, NA released into the synapse may exceed capacity of higher-affinity α2 receptors, possibly leading to spillover binding at lower-affinity α1-and β-receptors; and, in turn, this altered receptor occupancy ratio could potentiate drug seeking.", "If this theory were confirmed, it could suggest that α1-and β-receptor antagonists may also attenuate drug seeking, especially at higher stress intensities (when NA transmission is greatest).", "In fact, the α1 antagonists, prazosin and doxazosin, and the α2 agonist guanfacine blocked several types of stress-reinstatement of alcohol-seeking in rodents (Funk et al., 2016; Lê et al., 2011; Riga et al., 2014), as well as YOH-induced motoric impulsivity (Adams et al., 2017).", "Stress-reinstatement of cocaine seeking is attenuated by the β-receptor antagonist propranolol (Mantsch et al., 2010) and the NA synthesis inhibitor nepicastat (Schroeder et al., 2013).", "However, when injected directly into the amygdala central nucleus, the α1-antagonist prazosin and the β1/2-antagonist propanolol did not attenuate footshock-reinstatement of nicotine seeking, whereas the α2 agonist clonidine did (Yamada and Bruijnzeel, 2011), highlighting different effects of site-specific versus systemic drug administration across studies.", "One study found a double dissociation in NA receptor subtypes mediating rewarding and somatic withdrawal effects during mecamylamine-precipitated nicotine withdrawal: only prazosin attenuated brain reward threshold deficits during nicotine withdrawal, whereas only clonidine and propanolol attenuated somatic signs (Bruijnzeel et al., 2010), suggesting that these NA agents address different motivational mechanisms.", "In clinical practice, α2-receptor agonists are typically used as antihypertensive agents and for improving cognitive control in attention-deficit/hyperactivity disorder.", "Clonidine is a second-line treatment for smoking cessation (Gómez-Coronado et al., 2018) and guanfacine exhibited a promising signal in a pilot-test as a anti-stress/cognitive-control agent in tobacco smokers (McKee et al., 2015).", "Regarding cannabis use disorder, one study found that lofexidine reduced marijuana relapse-like behavior in a human laboratory model (Haney et al., 2008) but was not generally effective, when combined with oral THC, for reducing outpatient cannabis use (Levin et al., 2016).", "For cocaine use disorder, during early cocaine abstinence, guanfacine attenuated stress reactivity and craving (Fox et al., 2012) and enhanced inhibitory control (Fox et al., 2015), which might treat dysphoric and impaired self-control dimensions in the drug-motivational space (Fig. 1).", "Another study found guanfacine did not attenuate stress responses, prompting the authors to question its value for cocaine use disorder (Moran-Santa Moria et al., 2015).", "Furthermore, these human studies focused on drug craving rather than drug seeking/use, which may limit their clinical utility.", "Another caveat is that a study in rhesus monkeys found that, in the absence of stress, chronic lofexidine treatment produced a leftward shift in the cocaine self-administration dose-response curve; accordingly, the authors suggested α2 agonist maintenance may not be generally useful for cocaine use disorder (Kohut et al., 2013).", "In opioid-dependent humans, where YOH produced anxiety and opioid withdrawal-like effects (Greenwald et al., 2013; Oliveto et al., 2003; Stine et al., 2002), α2 agonists suppress opioid withdrawal symptoms (Gowing et al., 2014) and stress-induced craving (Jobes et al., 2011; Sinha et al., 2007; but see Moran-Santa Maria et al., 2015).", "Although maintenance on clonidine (vs. placebo) significantly lengthened duration of opioid abstinence among methadone-maintained patients, survival curve analysis revealed no significant clonidine vs. placebo group difference in time to opioid relapse, suggesting circumscribed efficacy (Kowalczyk et al., 2015).", "Some data suggest α-2A receptors primarily mediate anti-stress therapeutic effects of α2 agonists (e.g., guanfacine is a α-2A-specific partial agonist), whereas uncertainty remains regarding the roles of α-2B and α-2C subtypes, as well as whether these effects occur via pre-synaptic autoreceptors or heteromers with non-NA neurons such as glutamate (Shields et al., 2009).", "α1 antagonists have been investigated for efficacy in alcohol use disorder, with mixed results.", "In a recent, placebo-controlled, phase 2 trial, six-week treatment with prazosin (up to 16 mg/day) did not exhibit overall efficacy (Wilcox et al., 2018).", "Interestingly, pretreatment blood pressure – possibly reflecting chronic stress levels – has been found to moderate the efficacy of prazosin and doxazosin in reducing alcohol intake in two clinical trials (Haass-Koffler et al., 2017; Wilcox et al., 2018), such that these medications reduced alcohol intake to a greater degree among patients with higher pretreatment blood pressure.", "This review emphasizes a system biology approach to the influence of stress on substance use.", "Notably, the NA system has been the most extensively studied on its own and interacting with other neurochemical systems.", "Accordingly, Table 3 summarizes studies that have experimentally analyzed effects of manipulating NA signaling in combination with other systems, specifically for an effect on stress-related substance use.", "This table is intended to advance the idea that multi-modal medication (polypharmacy or “cocktail”) approaches may be useful, and perhaps preferable (i.e., safer and more effective than unimodal intervention), for treating stress-related substance use.", "Table 3 also illustrates the current research landscape: alcohol and cocaine have most often been studied using this systems biology approach, but there are substantial research gaps for other abused drugs.", "From a clinical perspective, it is notable that the use of other (non-NA) medications in combination with NA agents might minimize incidence of hypotension (i.e., produce a dose-sparing effect for the NA medication), which is the primary side effect of the NA medication class.", "Finally, prescribing NA medications for substance abusers who have hypertension could help treat this common comorbid condition.", "For proof-of-target studies, PET radiotracers exist for measuring occupancy of α2C receptors ([11C]-ORM-13070; Arponen et al., 2014) and noradrenaline transporter ([11C]-MENET; Adhikarla et al., 2016), but none are available for α1-or β-adrenergic receptors.", "For completeness of coverage, this section briefly notes promising alternative candidates with relatively less empirical evidence.", "Oxytocin is synthesized in the hypothalamus and binds to oxytocin and vasopressin receptors in brain regions implicated in regulating stress-reactivity, emotional and social behaviors (Heinrichs and Domes, 2008; Neumann and Landgraf, 2012; Windle et al., 2004).", "Studies of oxytocin suggest its clinical potential for several psychiatric conditions (Keech et al., 2018; Meyer-Lindenberg et al., 2011; Naja and Aoun, 2017; Quintana et al., 2017) although some therapeutic effects are likely to be sex-dependent (Bisagno and Cadet, 2014).", "Overall, few studies relate to substance abuse, and oxytocin might have greater promise for treating stress-related psychiatric conditions comorbid with SUDs (Zanos et al., 2017).", "An oxytocin analogue, carbetocin, attenuated forced-swim stress-reinstatement of morphine CPP, while attenuating stress-induced plasma CORT response (Zanos et al., 2014).", "Amygdala arginine vasopressin (AVP)1b receptors have been implicated in footshock-reinstatement of heroin seeking (Zhou et al., 2008, 2015), suggesting V1b receptor antagonism might be a viable target for blocking stress-related relapse.", "One V1b antagonist (ABT-436) is in clinical trials for treatment of alcohol use disorder (Ryan et al., 2017).", "Neuropeptide S (NPS) acts at the NPS receptor to increase arousal and locomotion while decreasing anxiety-like behavior (Leonard et al., 2008; Rizzi et al., 2008; Xu et al., 2004).", "This paradoxical profile – anxiolysis but increased locomotion (often associated with reinforcing effects) – resembles that of nicotine, creating uncertainty as to whether agonists or antagonists of this system would be optimal anti-stress candidates.", "Schmoutz et al. (2012) adopted an NPS receptor antagonist approach, based on the idea that NPS may promote addictive behaviors.", "Using RTI-118, they found it decreased YOH- as well as cue- and cocaine priming-induced reinstatement of cocaine seeking, indicating a broad efficacy profile (Schmoutz et al., 2012).", "NPS receptors are co-localized with other hypothalamic-originating neuropeptides discussed in this review, including orexin and CRF receptors.", "Hypothalamic NPS infusion has been shown to reinstate alcohol and cocaine seeking, effects that are reversed by the orexin-1 antagonist SB-334867 (Cannella et al., 2009; Kallupi et al., 2010; Ubaldi et al., 2016) and the CRF-1 antagonist antalarmin (Paneda et al., 2009).", "Neuropeptide Y binds multiple receptor subtypes; however, most studies have investigated Y1 (mostly postsynaptic) and Y2 (mostly presynaptic) receptors with the latter negatively modulating NPY release (Colmers et al., 1991; Eva et al., 2006; King et al., 1999) to produce numerous biobehavioral effects including anxiolysis (Heilig, 2004).", "The actions of NPY counteract CRF-induced anxiogenesis (Heilig et al., 1994; Sajdyk et al., 2004; Valdez and Koob, 2004).", "Central NPY infusion blocked YOH-reinstatement of alcohol seeking (Cippitelli et al., 2010).", "Systemic pretreatment with the Y2-selective antagonist, JNJ-31020028, failed to block footshock-reinstatement of alcohol responding (Cippitelli et al., 2011).", "A similar stress-reinstatement blocking test has not been reported for a Y1-selective agonist.", "Experimental results from studies of this system could be complicated by findings that Y1 and Y2 receptors (which have opposite functions regarding anxiety-like behavior) can form homo- and hetero-dimers whose net effect might negate the effects of receptor-selective stimulation (Dinger et al., 2003; Silva et al., 2003).", "Substance P is a tachykinin-family neuropeptide selective for the neurokinin-1 (NK-1) receptor.", "The NK-1 receptor antagonist aprepitant is FDA-approved for treating chemotherapy-induced nausea and biosimilars are in the pipeline.", "Clinical development of NK-1 antagonists has met with difficulties perhaps, not surprisingly, because these agents share functional features with CRF-1 antagonists that have associated negative findings (see section 2.2.1; Schank and Heilig, 2017).", "Preclinical studies have shown that the NK-1 antagonist L822429 blocks YOH- or footshock-reinstatement of alcohol and cocaine seeking (Schank et al., 2011, 2014, 2015).", "Although a phase 2 clinical study found that the NK-1 antagonist LY686017 decreased spontaneous and psychological stress-induced alcohol craving and cortisol responses in high trait-anxious, alcohol-dependent volunteers (George et al., 2008), a subsequent placebo-controlled study of aprepitant administered to alcohol-dependent patients with co-occurring PTSD failed to replicate these findings (Kwako et al., 2015a).", "Although it remains unclear whether NK-1 antagonists could reduce acute stress-induced effects among individuals who are not highly anxious, this would considerably restrict the clinical utility of this class of medication.", "The PET ligand [11C]-GR205171 is available for measuring NK-1 binding potential in humans (Frick et al., 2015; Ridler et al., 2014; Spinelli et al., 2014).", "Relaxin-3 is a peptide acting at its cognate receptor RXFP3 and, similar to orexin, relaxin-3 agonists increase stress-reactivity, food intake and arousal.", "In contrast, relaxin-3 antagonism, at least partly mediated via the BNST, blocked YOH-reinstatement of alcohol- but not sucrose-seeking (Ryan et al., 2013); furthermore, RXFP3 deletion blocked physical (repeated restraint followed by swim) stress-reinstatement of alcohol seeking without disrupting baseline alcohol or saccharin intake, or hepatic metabolism of alcohol (Walker et al., 2015).", "Thus, relaxin-3 antagonists may be reasonable anti-stress agents, although efficacy with other abused drugs has not been tested to date.", "FDA-approved nicotine replacement treatments (NRTs) do not attenuate stress-induced drug seeking or use (Kotlyar et al., 2006; Ray et al., 2013).", "Interestingly, the α3β4 nAChR partial agonist AT-1001 dose-dependently attenuated YOH-reinstatement of nicotine seeking (Yuan et al., 2017).", "Furthermore, AT-1001 selectively blocked YOH- but not cue-reinstatement of alcohol seeking without affecting baseline alcohol or food intake (Cippitelli et al., 2015).", "This novel approach could potentially address some limitation of current NRTs and assist the many individuals who concurrently use tobacco and alcohol.", "Pioglitazone, agonist at the peroxisome proliferator-activated receptor-gamma (PPARγ) subtype, was shown to attenuate YOH-reinstatement of alcohol seeking (Stopponi et al., 2011, 2013) and heroin seeking (de Guglielmo et al., 2017).", "As noted in section 1.3, infusion of CRF (which mimics supra-physiologic endogenous release from the hypothalamus) reinstates previously extinguished drug seeking in animal models.", "Notably, CRF-1 receptor knockout animals are less sensitive to stress-induced cocaine seeking (Chen et al., 2014).", "Thus, CRF-1 antagonists have been explored for blocking stress-induced reinstatement.", "Early studies found that ICV infusion of non-selective CRF-1/2 receptor antagonists decreased footshock reinstatement of responding for heroin (Shaham et al., 1997), cocaine (Erb et al., 1998), alcohol (Lê et al., 2000; Liu and Weiss, 2002) and nicotine (Zislis et al., 2007).", "As summarized in Table 2, selective CRF-1 antagonism attenuated stress-reinstatement of responding for nicotine, alcohol, cocaine and heroin.", "Although these preclinical studies suggest CRF-1 antagonism might be a useful approach for reducing stress-induced drug seeking, this apparent promise must be tempered by contradictory results from recent clinical studies.", "Two CRF-1 antagonists, pexacerfont and verucerfont, failed to demonstrate anti-craving effects in patients with alcohol use disorder (Kwako et al., 2015b; Schwandt et al., 2016).", "A third CRF-1 antagonist, GSK561679, failed to show anxiolytic effects in a human laboratory model (Grillon et al., 2015) and lacked efficacy in a clinical trial with posttraumatic stress disorder patients (Dunlop et al., 2017).", "Given this lack of translational efficacy, considerable caution is warranted for similar congeners in this pharmacological class.", "Several methodological factors could have led to poor translation (Pomrenze et al., 2017; Shaham and de Wit, 2016); if these are properly addressed, there could still be a path forward for these agents but, in all likelihood, therapeutic application may be narrower than originally hoped (Spierling and Zorrilla, 2017).", "Alternative study designs may be needed that measure not only direct effects of the medication on substance use, but also intermediate stress-related phenotypes, e.g., coping or resilience (Contoreggi et al., 2013).", "One potential moderating factor is that some clinical studies were conducted exclusively with female volunteers, which could influence the generalizability of findings.", "Contribution of CRF-2 receptors to stress-induced drug seeking is unclear, due to mixed methods and findings in the literature (Blacktop et al., 2011; Bruijnzeel et al., 2009; Wang et al., 2007; Zorrilla et al., 2014).", "CRF-2 receptors mediate anxiolysis (Bale et al., 2000; Kishimoto et al., 2000; Risbrough et al., 2004), suggesting CRF-2 agonists (not antagonists) are more appropriate anti-stress medication candidates but, presently, there are no published studies on this topic.", "Currently, there are no viable CRF-1 or CRF-2 receptor PET ligands for use in studies.", "Brown et al. (2009) found that ICV infusion of NA reinstated cocaine seeking, which was blocked by pretreatment with the non-selective CRF antagonist D-Phe CRF12-41.", "In contrast, ICV CRF reinstated cocaine seeking but this was not blocked by clonidine pretreatment.", "Pretreating with the CRF antagonist or clonidine failed to block YOH-induced reinstatement.", "These findings suggest a functional interaction between NA and CRF systems in mediating stress-reinstatement of cocaine seeking, such that CRF receptor activation occurs downstream from NA sites of action (i.e., at extra-hypothalamic sites, independent of the HPA axis).", "McReynolds et al. (2014) demonstrated that reinstatement of cocaine CPP using forced swim or systemic injection of the β2-receptor agonist clenbuterol was blocked by antalarmin or the β2-antagonist ICI-118,551; whereas clenbuterol reinstatement of cocaine CPP was only blocked by antalarmin but not the β2-antagonist.", "The authors hypothesized that stress-induced liberation of NA activates, via β2 receptors, CRF neurons that lead to drug-motivated responding.", "Several early studies demonstrated that footshock stress, while increasing corticosterone (CORT) levels in rats, did not reinstate responding for heroin (Shaham et al., 1997), cocaine (Erb et al., 1998), or alcohol (Lê et al., 2000).", "Although acute pretreatment with the corticosteroid synthesis inhibitor, metyrapone, itself reinstated heroin-seeking behavior, neither acute nor chronic metyrapone exposure, nor adrenalectomy, attenuated footshock-reinstatement of heroin seeking (Shaham et al., 1997).", "These data implied that CORT does not specifically mediate reinstatement.", "Although one study found that acute pretreatment with the corticosteroid synthesis inhibitor, ketoconazole, blocked footshock reinstatement of cocaine seeking (Mantsch and Goeders, 1999), ketoconazole has other pharmacological actions, making it unclear whether this effect was HPA axis-specific.", "Furthermore, sustained ketoconazole treatment in a clinical study led to increased use of cocaine and heroin among methadone-maintained patients (Kosten et al., 2002).", "Despite the important role of the HPA axis in addiction (Goeders, 2003), preclinical studies have found that glucocorticoid manipulations, rather than directly influencing drug seeking (given the negative findings above), may instead sensitize the animal to respond more in the presence of drug-related stimuli.", "Graf et al. (2013) found that exposing rats to footshock failed to reinstate cocaine seeking, but footshock did facilitate reinstatement to a subthreshold cocaine priming dose.", "Further, CORT administered to adrenalectomized rats also reinstated cocaine seeking with a subthreshold cocaine priming dose.", "Interestingly, CORT infused into the nucleus accumbens (NAc) was found to decrease DA clearance, suggesting CORT potentiation of NAc DA signaling may partly underlie stress-induced reinstatement.", "This research group also found that acute CORT alone did not reinstate extinguished cocaine CPP (McReynolds et al., 2017b) or self-administration (McReynolds et al., 2017a); however, CORT enhanced efficacy of a sub-threshold cocaine priming dose to reinstate cocaine CPP and self-administration (McReynolds et al., 2017a, b); thus, the stressor (CORT) “set the stage” for acute cocaine exposure to reinvigorate drug- or drug-paired responding.", "Also, these investigators found CORT and footshock-stress effects on cocaine-primed reinstatement of self-administration that were mediated by eCB signaling via CB1 receptors and the enzyme monoacylglycerol (McReynolds et al., 2016, 2017a).", "This research illustrates an interaction between the HPA axis and eCB system in the control of stress-occasioned relapse-like behaviors and suggests a role for using eCB modulators to attenuate the direct and indirect effects of stressors on drug-maintained behaviors (see section 2.6).", "Given the complexities of the HPA axis (e.g., negative feedback loops within the HPA axis; see Fig. 2), and the findings above that adrenalectomy or inhibition of corticosteroid synthesis did not block drug seeking, the potential utility of glucocorticoid receptor (GR) antagonists as anti-stress therapeutics must be seriously questioned.", "Also, nicotine abstinence is associated with HPA axis hypo-sensitivity (al’Absi et al., 2005; Semba et al., 2004), so it is unclear whether GR antagonists would be useful.", "On the other hand, a recent study found that the dual GR/progesterone antagonist mifepristone and the GR-specific antagonist CORT113176 each dose-dependently reduced escalated alcohol intake in rats and, in a companion clinical study, maintenance on mifepristone 600 mg/day (vs. placebo) reduced alcohol drinking among individuals with alcohol use disorder (Vendruscolo et al., 2015).", "Further investigation of these agents is warranted to shed light on the specific mechanisms through which this effect may occur, and whether it may be unique to alcohol or apply to other abused drugs.", "Consistent with the systems biology focus in this review, NA and glucocorticoid systems produce co-operative effects in multiple paradigms (Dunn and Swiergiel, 2008; Forray and Gysling, 2004; Koob, 2008; Schwabe et al., 2010, 2012; Torregrossa et al., 2011; Valentino and Van Bockstaele, 2008; van Stegeren et al., 2010; Vasa et al., 2009).", "This prompted our recent study to co-activate these systems using separate and combined challenges with YOH and hydrocortisone (synthetic CORT) (Greenwald et al., in preparation).", "In a within-subject randomized crossover design, heroin-dependent buprenorphine-maintained volunteers were pretreated with oral YOH (0-, 27- and 54-mg) and CORT (0-, 20- and 40-mg) alone and combined.", "YOH/CORT increased opioid (hydromorphone) choices, relative to placebo pretreatment, while reducing money choices (i.e., stress increased opioid preference) but this effect was modulated by individual's pre-experimental variation in daily nicotine use.", "This pharmacological stress model was recently applied to cigarette smokers.", "Relative to placebo, YOH-54mg/CORT-10mg pretreatment induced similar physiological stress responses as above.", "Controlling for nicotine-dependence level, acute stress increased nicotine seeking (Woodcock et al., under review).", "Several studies implicate 5-HT neurotransmission in stress-induced reinstatement of alcohol seeking (whereas similar studies with abused drugs are lacking).", "In an initial study, alcohol seeking was reinstated by footshock and reversed by the 5-HT reuptake inhibitor fluoxetine (Lê et al., 1999).", "Subsequently, the 5-HT-1A agonist 8-OH-DPAT (which decreases 5-HT cell firing and transmitter release) infused in the median raphe nucleus (MRN) reinstated alcohol seeking; furthermore, intra-MRN infusion of CRF reinstated alcohol seeking (Lê et al., 2002).", "In a follow-up study, CRF antagonist infusion in the MRN attenuated YOH-reinstatement of alcohol seeking (Lê et al., 2013).", "Together, these findings point to 5-HT/CRF interaction within the MRN as mediating this stress effect.", "In addition to its primary α2-antagonist action, YOH also acts as a 5HT-1A partial agonist, which could partly explain its stress-reinstating efficacy.", "Furthermore, intra-MRN infusion of muscimol (GABA-A receptor agonist) reinstated alcohol seeking, implicating a 5-HT/GABA-A interaction in the MRN (Lê et al., 2008).", "The 5-HT3 receptor antagonists, ondansetron and tropisetron (FDA-approved for treating nausea and vomiting), have been demonstrated to attenuate footshock-reinstatement of alcohol seeking in rats (Lê et al., 2006).", "However, these anti-stress effects have not yet been explored for other abused substances.", "In summary, limited data suggest agents that promote 5-HT release, prevent reuptake, or block 5-HT-3 receptors could be useful therapeutics, although such effects may depend on raphe-mediated interactions; thus, systemic delivery could produce mixed effects.", "Given widespread clinical use of SSRIs and 5-HT-3 antagonists, systematic research across classes of abused drugs is warranted.", "Target-selective PET tracers are available that measure 5-HT transporter occupancy using [11C]DASB (for review, Spies et al., 2015); 5-HT synthesis using α-[11C]-methyl-L-tryptophan (Kumar et al., 2011); and 5-HT1A receptor binding using [carbonyl-11C]-WAY100635 or possibly [11C]-CUMI-101, but none for 5-HT-3 receptors (for review, Kumar and Mann, 2014).", "Although the non-selective opioid receptor antagonist naltrexone (NTX) is used to treat alcohol and opioid use disorders, NTX does not block stress-induced alcohol or opioid use (e.g., Hyman et al., 2007; Lê et al., 1999).", "Notably, NTX can increase YOH reactivity (Rosen et al., 1999), and a clinical trial found that adding the α2-agonist guanfacine to NTX did not improve opioid abstinence over NTX alone (Krupitsky et al., 2013).", "It is presently unclear whether opioid agonist medications might (in addition to their withdrawal-suppressing and opioid blockade properties; see Greenwald et al., 2014) attenuate stress-reactivity among individuals with opioid use disorder.", "One animal study found that maintenance on the mu-agonist methadone (MTD) did not block stress-reinstatement of heroin seeking (Leri et al., 2004).", "Results from cross-sectional clinical studies suggest MTD might blunt stress-reactivity (Kakko et al., 2008; Kreek et al., 1984; Schluger et al., 2001, 2003) but those studies have methodological flaws, e.g., variable maintenance doses, potential pre-morbid differences between patients and controls, and failure to match controls on other substance use.", "Buprenorphine (BUP) is a partial mu-receptor agonist/kappa-receptor antagonist with rapid-onset antidepressant and anxiolytic effects in animal models (Falcon et al., 2015) and human studies (Bershad et al., 2015; Bodkin et al., 1995; Karp et al., 2014; Nyhuis et al., 2008).", "In the clinical dosing range for opioid use disorder, BUP may be stress-protective via one or both of these mechanisms; our lab is presently investigating this issue.", "Stressors have been shown to release dynorphin and activate KORs located on DA and 5-HT neurons, thereby reducing DA and 5-HT levels (Carlezon et al., 2006; Ebner et al., 2010; Gehrke et al., 2008; Land et al., 2008; Zhang et al., 2007) that, in turn, are associated with negative-hedonic effects of stressors (Fig. 1).", "Furthermore, KOR agonist administration reliably reinstates responding for cocaine (see Table 1).", "Accordingly, KOR antagonists are being evaluated as potential anti-depressants (Carr et al., 2010; Filho et al., 2013; Grimwood et al., 2011; Zhang et al., 2007) and anti-stress/drug-relapse prevention agents.", "As summarized in Table 2, KOR antagonists reliably attenuate stress-reinstated responding for nicotine, alcohol (but see Schank et al., 2012), cocaine, and heroin.", "Although no tests of KOR antagonism of stress-related substance abuse have been conducted in humans to date, PET ligands for measuring KOR occupancy are available including [11C]-LY2795050 (Naganawa et al., 2014) and [18F]-LY2459989 (Li et al., 2018).", "Evidence also suggests dynorphin projections to NA and GABA neurons in the locus coeruleus (Kreibich et al., 2008; Reyes et al., 2007) modulate stress-related drug reinforcement.", "Studies in rats have identified KOR/noradrenergic interactions in the locus coeruleus that mediated reinstatement of cocaine lever-responding (Al-Hasani et al., 2013b), and KOR/serotonergic projections from the dorsal raphe to the NAc that mediated reinstatement of cocaine conditioned place preference (CPP) (Land et al., 2009).", "Finally, interactions between dynorphin/KORs and CRF have been proposed as one mechanism by which stressors lead to changes in allostatic load, altering the motivational valence of the abused substance (Bruchas et al., 2010; Koob, 2015).", "DORs are a potential anti-stress target, given their established role in anxiety-like behaviors in animal models; however, their pharmacology is incompletely understood, including differing functions of DOR subtypes, stress-induced translocation of DORs from cytoplasm to cell surface, and potential for DORs to form heteromers with mu-receptors (van Rijn et al., 2013).", "In the only relevant study to date, YOH-reinstatement of alcohol seeking was attenuated by a DOR-1 antagonist, SoRI-9409 (Nielsen et al., 2012).", "Further research with DOR subtype-specific agonist and antagonists is needed to determine whether such agents could be useful anti-stress therapeutics.", "The PET ligand [11C]methyl-naltrindole is clinically available (Madar et al., 1996; Smith et al., 1999) and has been used to measure DOR binding potential in alcohol-dependent individuals (Wand et al., 2013; Weerts et al., 2008, 2011).", "Although the NOP system is involved in stress-reactivity and addictive behaviors, historically less clear is whether NOP agonists or antagonists should be effective anti-stress agents.", "On the one hand, NOP agonists have shown therapeutic promise against stress-reinstatement of alcohol seeking.", "Footshock-reinstatement of alcohol seeking was attenuated by ICV administration of nociceptin (Martin-Fardon et al., 2000) and systemic administration of the non-peptide, brain-penetrant NOP agonist MT-7716 in post-dependent rats but not alcohol-naïve rats (de Guglielmo et al., 2014).", "The latter findings suggest a history of alcohol dependence dysregulates the nociceptin system, which can be reversed with NOP agonist treatment.", "A different small-molecule NOP agonist, SR-8993, blocked YOH-reinstatement of alcohol seeking (Aziz et al., 2016).", "Conversely, other studies have found that NOP antagonists and NOP knockout mice produce anti-obesity and anti-depressant effects (review by Witkin et al., 2014), and the orally bioavailable, brain-penetrant, NOP antagonist LY2940094 blocked YOH-reinstatement of alcohol seeking in alcohol-preferring rats (Rorick-Kehn et al., 2016).", "Witkin et al. (2014) proposed a hypothesis to reconcile these differences; specifically, nociceptin serves as part of a stress-coping negative-feedback mechanism, eliciting a stress-like response within the HPA axis but anti-stress effects at extra-hypothalamic sites.", "For future studies, the PET ligand [11C]NOP-1A (Lohith et al., 2012) is available to measure NOP binding potential in human subjects (Narendran et al., 2017).", "Initial studies of the eCB system as an anti-stress target focused on CB1 receptor antagonists.", "Administration of the CB1 antagonist rimonabant to rats failed to attenuate footshock-reinstatement of alcohol seeking (Economidou et al., 2006; 2007) and cocaine seeking (De Vries et al., 2001).", "Moreover, side effects of rimonabant in clinical trials led to discontinuation of its therapeutic development.", "The neutral CB1 antagonist AM4113 dose-dependently reduced YOH-reinstatement of nicotine seeking without altering inactive-lever or food-maintained responding acutely, although chronic treatment led to weight loss (Gueye et al., 2016).", "In contrast, the CB1 antagonist AM251 blocked forced swim-induced cocaine reinstatement (Vaughn et al., 2010) and AM251 blocked CRF- but not footshock-reinstatement of cocaine seeking (Kupferschmidt et al., 2012).", "Given these mixed effects of CB1 antagonists on stress-reinstatement, it is unclear whether findings may depend on interactions between the CB1 antagonist, stressor, and abused substance.", "An alternative strategy is to modulate signaling indirectly within the eCB system.", "One promising approach is to promote eCB transmission by facilitating actions of the endogenous ligand anandamide (AEA).", "Although acute administration of the FAAH inhibitor URB597 (which increases AEA levels by inhibiting its catabolism) did not attenuate footshock- or YOH-reinstatement of alcohol seeking (Cippitelli et al., 2008), Chauvet et al. (2014) argued that chronic administration of anti-stress medication is a preferable model, because (1) this directly pertains to the human condition, (2) chronic exposure controls for possible tolerance development, and (3) medication-induced neuroadaptations may be required for a therapeutic effect.", "Using this approach, they demonstrated that URB597 significantly attenuated YOH-reinstatement of cocaine seeking (Chauvet et al., 2014).", "Targeting fatty acid binding proteins, which transport AEA intracellularly to FAAH for degradation, is a related strategy to interfere with stress-induced drug seeking (Hamilton et al., 2018).", "The eCB cannabidiol (CBD) has diverse neuropharmacological actions but salient among these includes enhancing AEA levels with weak antagonist actions at CB1 and CB2 receptors (Campos et al., 2012; Izzo et al., 2009; Kathman et al., 2006; McPartland et al., 2015; Mijangos-Moreno et al., 2014; Pertwee, 2008; Petitet et al., 1998; Russo et al., 2005; Thomas et al., 2007).", "Transdermal CBD administration was recently found to attenuate acute and repeated YOH-reinstatement of cocaine and alcohol seeking and produced anxiolytic and anti-impulsive effects without disrupting other reward behavior or locomotion, and this profile of effects endured long after CBD was biologically detectable (Gonzalez-Cuevas et al., 2018).", "Further studies are needed, but CBD represents a novel anti-stress therapeutic approach.", "PET radiotracers are clinically available that measure binding potential in the eCB system including [11C]-CURB (Boileau et al., 2015; Rusjan et al., 2013) and [11C]-MK3168 (Postnov et al., 2018) for FAAH; [11C]-OMAR (Normandin et al., 2015) and [11C]-MePPEP and [18F]-FMPEP-d (Terry et al., 2010) for CB1 receptors; and [11C]-NE40 for CB2 receptors (Ahmad et al., 2013).", "Measurement of eCB levels in biological matrices is promising and undergoing refinement (Dlugos et al., 2012; Feuerecker et al., 2012; Vaughn et al., 2010; Walter et al., 2013; Witkamp and Balvers, 2016).", "Orexin fibers (originating mostly in the lateral hypothalamus) project widely in the brain, including to VTA, NAc and extended amygdala and, thus, are well-positioned to influence motivated behavior.", "The orexin system has been demonstrated to influence stress-induced seeking for several abused substances.", "ICV administration of orexin-1 but not orexin-2 peptide reinstates drug seeking (Boutrel et al., 2005; Harris et al., 2005; Plaza-Zabala et al., 2010; Wang et al., 2009), consistent with a broader role for orexin-1 in compulsive behavior (Pich and Melotto, 2014).", "Notably, the orexin-1 receptor-selective antagonist SB-334867 dose-dependently reduced footshock- and YOH-reinstatement of cocaine responding, particularly in rats given long-access to cocaine, a model of escalated drug use (Boutrel et al., 2005; Schmeichel et al., 2017; Wang et al., 2009).", "A set of studies found that restraint stress initially activated hypothalamic orexin neurons to release orexins into the VTA, activating postsynaptic VTA DA neurons, causing retrograde eCB inhibition of GABA release, leading to VTA DA disinhibition that resulted in reinstatement of cocaine CPP (Tung et al., 2016).", "Systemic pretreatment with SB-334867 attenuated YOH-reinstatement of alcohol seeking, without altering locomotion (Richards et al., 2008).", "However, a later study found that only micro-injection of an orexin-2 receptor antagonist (but not SB-334867) into the pontine nucleus incertus attenuated YOH-reinstatement of alcohol seeking (Kastman et al., 2016).", "These mixed results could be due to different methods.", "A human study found that plasma concentrations of orexin – which may reflect diffusion of the peptide between CNS and peripheral compartments (Kastin and Akerstrom, 1999) – during alcohol abstinence positively correlated with psychological distress levels (von der Goltz et al., 2011); thus, it is plausible that orexin modulates stress- or abstinence-related alcohol use.", "However, there are also contrary data in cigarette smokers, showing a negative relationship between peripheral orexin-1 levels and nicotine craving (von der Goltz et al., 2010).", "For nicotine, SB-334867 blocked reinstating effects of ICV orexin and footshock on nicotine seeking; in contrast, SB-334867 did not block CRF-induced nicotine reinstatement, and the CRF-1 antagonist antalarmin did not block ICV orexin-induced nicotine reinstatement, indicating autonomous control of stress-related drug-seeking across these two systems (Plaza-Zabala et al., 2010).", "For opioids, pretreatment with either orexin-1 or orexin-2 receptor antagonists in the NAc shell (which processes positive and negative affectively-valenced behaviors) attenuated reinstatement of morphine CPP (Qi et al., 2013).", "Suvorexant is a non-selective orexin-1/2 receptor antagonist approved for treating insomnia.", "Evidence indicates that orexin-2 antagonism is responsible for the sleep-inducing effect (Andrews et al., 2016).", "Consistent with findings above, orexin-1 antagonists such as GSK1059865 (Gozzi et al., 2011) are being developed as anti-stress and anti-addiction pharmacological tools, although none are clinically available at this time.", "Although there are emerging orexin-2 ligands for PET imaging such as [11C]-MK-1064 (Gao et al., 2016) and [11C]-CW4 (Wang et al., 2013), at this time there are no viable orexin-1 radiotracers.", "Multiple signals converge on glutamate signaling to alter stress-induced drug seeking.", "The effect of footshock stress on cocaine responding was shown to depend on glutamate via dorsal PFC/NAc core interaction (McFarland et al., 2004) and VTA CRF signaling (Wang et al., 2005; Williams et al., 2014), ultimately leading to increase NAc DA transmission (Wise, 2009).", "Cold-water forced-swim stress was shown to alter AMPA/NMDA receptor ratio in the NAc shell, an effect that was reversed with the glucocorticoid receptor antagonist RU486 (Campioni et al., 2009).", "NA signaling through α2 and β2 receptors in the dorsal BNST, can bidirectionally modulate glutamate transmission (Egli et al., 2005).", "Although these examples are not exhaustive, they illustrate that glutamate signaling in stress-related behavior is mostly secondary to other systems.", "This implies that a direct pharmacological approach to modulating glutamate function may be challenging for treating stress-related substance use.", "Two agents with glutamate transmission-dampening properties, a mGluR2/3 agonist (LY379268) and mGluR5 antagonist (MTEP), each dose-dependently reduced footshock-reinstatement of cocaine seeking (Martin-Fardon and Weiss, 2012) and alcohol seeking (Sidhpura et al., 2010; Zhao et al., 2006).", "These findings suggest mGluR2/3 and mGluR5 targets (primarily located in pre-/peri- and post-synaptic spaces, respectively) should be studied further, given the established anxiolytic effects of LY379268 and MTEP in animal models (e.g., Kenny and Markou, 2004).", "On the other hand, a review of medications for treating cocaine addiction (not specific to stress-induced drug seeking) suggested that allosteric modulators could be pursued (Kalivas and Volkow, 2011).", "Given the evidence above, reasonable candidates to be considered would be positive allosteric modulators of mGluR2/3 receptors and negative allosteric modulators of mGluR5 receptors.", "Given similar neuroadaptations from chronic alcohol (relative to cocaine) use, including a hypersensitive glutamate system (Vengeliene et al., 2008), it may be fruitful to investigate these allosteric modulators for stress-induced alcohol use.", "Yet, an initial test of this hypothesis found that the mGluR2-selective positive allosteric modulator AZD8529 attenuated cue- but not footshock-reinstatement of alcohol seeking in rats (Augier et al., 2016).", "Those authors note that this difference in findings across studies could imply that mGluR3 rather than mGluR2 receptors are distinctly involved in stress-related drug responding, or that orthosteric agonists and positive allosteric modulators could modulate glutamate function in distinct ways.", "Several biomarkers are available to measure glutamate function in human subjects.", "Emerging PET radiotracers for measurement of brain glutamate targets include [18F]-FIMX for mGluR1 receptors (Zanotti-Fregonara et al., 2016), and [11C]-ABP688 (Kågedal et al., 2013) and [18F]-FPEB (Sullivan et al., 2013) for mGluR5 receptors.", "High-field proton magnetic resonance spectroscopy (1H-MRS) has proved sensitive to pharmacotherapy-related repeated-measures change in brain-regional glutamate concentrations in substance abusers (Greenwald et al., 2015; Umhau et al., 2010).", "The GABA-A agonist muscimol, when injected into the median raphe nucleus, reinstated alcohol seeking (Lê et al., 2008).", "However, intra-VTA modulation of GABA-A receptors using the GABA-A antagonist bicuculline did not block footshock- or intra-VTA CRF-induced reinstatement of cocaine seeking (Blacktop et al., 2016).", "However, it is possible that a GABA-A positive allosteric modulator could offer a useful alternative; one such compound is in development (Martinez Botella et al., 2017).", "In contrast, the GABA-B agonist baclofen was found to block YOH-induced alcohol seeking (Williams et al., 2016) and to attenuate forced swim stress-reinstatement of morphine CPP (Meng et al., 2014).", "However, these encouraging results must be reconciled with conflicting findings that intra-VTA infusion of a GABA-B antagonist, 2-hydroxysaclofen, blocked reinstatement of cocaine seeking by footshock and intra-VTA CRF administration (Blacktop et al., 2016).", "Thus, it is unclear whether using a GABA-B agonist or antagonist would be preferable against stress-induced drug seeking, or whether this might vary by the abused substance or route of delivery.", "GABA modulation with pregabalin (a clinically available medication) was found to block YOH-reinstatement of alcohol seeking (Stopponi et al., 2012) and cocaine seeking (de Guglielmo et al., 2013).", "The GAT-1 inhibitor tiagabine has been demonstrated to reduce anxiety- and depressive-like behaviors in animal models (Thoeringer et al., 2010) but has not yet been tested in experimental models of stress-induced drug seeking/use.", "Biomarkers for GABA system targets include the PET radiotracer [11C]-flumazenil for GABA-A receptors (Jucaite et al., 2017; Lingford-Hughes et al., 2005), and 1H MRS imaging for cortical GABA concentrations (Mason et al., 2006; Streeter et al., 2005).", "The development of anti-stress medications is critical for the advancement of treating all SUDs.", "The impact of stressors extends across abused substances (which contrasts with the effects of drug-priming and drug-cue exposure, which tend to be specific to drug classes).", "Therefore, progress in this field has significant potential to apply scientifically across all drug classes and to improve treatment of all patients with SUDs.", "This review describes available evidence on promising neuropharmacological approaches, using mechanistic studies based on chemical and non-chemical probes (Table 1).", "Although studies of Δ9-THC/marijuana were surveyed for this review, there is a paucity of studies in this area.", "Based on the quality of evidence to date, promising first-tier neurochemical targets include: NA (α1-and β-antagonist, α2 agonist), kappa-opioid antagonist, NOP antagonist, orexin-1 antagonist, and eCB modulation (e.g., cannabidiol, FAAH inhibition); second-tier candidates may include CRF-1 antagonists, serotonergic agents (reuptake inhibitors, 5-HT-3 antagonists), glutamatergic agents (mGluR2/3 agonist/positive allosteric modulator, mGluR5 antagonist/negative allosteric modulator), GABA-signaling promoters (e.g., pregabalin, tiagabine), vasopressin 1b antagonist, NK-1 antagonist, and PPAR-γ agonist (e.g., pioglitazone).", "DA antagonists were excluded from this review because, although brain site-specific studies in animals support a role for DA in mediating stress-induced drug seeking (McFarland et al., 2004), when DA antagonists are administered systemically (and especially chronically) they produce side effects that do not translate well into clinical practice.", "Table 2 complements this review by adding a layer of theoretical analysis pertaining to affective/motivational mechanisms that may be modulated while intervening in these neurochemical systems.", "I propose the overarching hypothesis that, to be effective, anti-stress medications for substance use disorders must alleviate the multidimensional burden of stressors that can lead to behavioral deficits including avoidance (valence dimension), hyperactivation (arousal dimension), and impulsivity (control dimension), that can perpetuate substance use and its adverse consequences.", "Table 2 illustrates that few candidate pharmacotherapeutic approaches are likely to tackle all these problems.", "Accordingly, to address these affective/motivational mechanisms of stress-related substance use, it seems advisable to combine agents with actions at complementary targets for greater efficacy; however, it should be noted that systematic studies are lacking except for interactions with the NA system (Table 3).", "Future research could be directed at whether some agents may function to desensitize subjects from chronic stress-induced effects (e.g., glucocorticoid receptor antagonist), whereas other agents might help to block acute stress-induced effects (e.g., α2 agonist or α1 antagonist).", "It is plausible that both types of agents together may be more effective than either alone.", "Some agents may also have additional effects beyond anti-stress, such as to block cue-induced drug seeking (e.g., eCB modulators, orexin-1 antagonist).", "A key methodological factor concerns whether medication development studies employ acute vs. chronic administration.", "Although acute medication administration is the dominant paradigm to date, partly due to its efficiency, Chauvet et al. (2014) effectively argued that chronic exposure is desirable for applied clinical relevance, to avoid false negative results attributable to development of tolerance, and to enable within- and/or between-system neuroadaptations (including epigenetic changes) to occur.", "Future studies should carefully weigh these issues, e.g., whether to screen medications acutely (but perhaps also chronically) during early-stage development but move to chronic dosing in phase 2 studies.", "Programmatic studies should include biomarkers of stress-reactivity and medication targeting to confirm mediation of effects on drug-seeking behavior.", "Section 2 mentioned clinically available PET ligands for measuring occupancy of molecular targets and use of 1H-MRS for measuring glutamate and GABA brain-regional concentrations.", "These CNS biomarkers should be paired with measurement of medication plasma concentrations, and target occupation/stimulation and plasma pharmacokinetic data should be correlated with one another and with pharmacodynamic effects (clinical endpoints) to generate exposure-response functions.", "We successfully applied a similar PK/PD modeling strategy for optimizing sublingual and depot buprenorphine treatment of opioid use disorder (Greenwald et al., 2014; Nasser et al., 2014, 2016) and this approach was recently applied in the development of the NOP antagonist LY2940094 (Rorick-Kehn et al., 2014, 2016).", "As indicated in Table 2, which maps neurobiological and motivational foundations of anti-stress medications, it seems unlikely that any single medication will be highly effective for attenuating stress-mediated negative-hedonic, arousal/activating, and disinhibiting/loss-of-control effects on drug-maintained behaviors.", "Furthermore, chronic dosing may lead to between-system adaptations resulting in loss of efficacy over time, so it may be advantageous to employ a redundant approach (e.g., use two medications that each target one motivational dimension, but via different pharmacological stimulation).", "This approach emphasizes using one or more anti-stress medications that exert actions that address multiple motivational features of stress-reactive drug use.", "Using the model proposed here, a predictable consequence of deploying highly targeted compounds would be restricted efficacy.", "Individual difference factors need to be considered in the context of medication development; inattention to these variables will also limit efficacy (section 1.4).", "This review has identified promising, evidence-based neurochemical mechanisms and potential pharmacotherapeutic leads to combat stress-induced substance seeking/use, a major problem for all SUDs.", "Medications that prove safe and effective could be added to existing SUD pharmacotherapies (e.g., agonist therapies for opioid or nicotine use disorder) to augment treatment efficacy, as most FDA-approved agonist treatments were not designed as anti-stress agents.", "To address motivational dimensions of stress-related substance use, it will be theoretically and pragmatically valuable to test agents with complementary actions alone and in combination to improve efficacy.", "As these agents are developed, it will be important to recognize numerous clinically-relevant factors (some of which could be modeled in animal studies) that could mediate/moderate the efficacy of anti-stress agents.", "Finally, progress in developing anti-stress medications will also depend on use of reliable CNS biomarkers to validate exposure-response relationships.", "NIH 2 R01 DA015462 from the National Institute on Drug Abuse, Helene Lycaki/Joe Young Sr. funds (State of Michigan), and the Detroit Wayne Mental Health Authority supported preparation of this manuscript.", "The author is responsible for the content of this manuscript, but gratefully acknowledges the many valuable contributions offered by two anonymous reviewers.", "The author has received compensation as a scientific consultant to Indivior, Inc., which manufactures and markets buprenorphine products, and he has co-authored publications without compensation regarding RBP-6000 (Sublocade™)." ]	[ "Selective review of stress-potentiated seeking/use of nicotine, alcohol, opioids, cocaine, and cannabis.", "Affective/motivational theoretical model is mapped onto a systems biology approach.", "Neurochemical targets are discussed, and potential combination approaches are identified.", "Clinically-relevant factors that could influence efficacy of anti-stress therapeutics are discussed.", "Research gaps are identified and future avenues of investigation are suggested." ]
Do greenhouse gas emission calculations from energy crop cultivation reflect actual agricultural management practices? – A review of carbon footprint calculators	A wide range of calculators have been developed to assess the greenhouse gas (GHG) emissions of agricultural products, including biomass for bioenergy production. However, these calculators often fail in their ability to take into account the differences in pedoclimatic conditions, agricultural management practices and characteristics of perennial crops and crop rotations. As a result, the predictions of GHG emissions by these calculators are characterized by a high level of uncertainty, and calculators may fail in their ability to detect mitigation options along the production chain. The aim of this study was to analyze the available calculators for calculating GHG emissions from energy crop cultivation based on Carbon Footprint (CFP) approaches according to the goal and scope of the calculator, the methodology used to account for GHG emissions from energy crop cultivation, energy crop cultivation management practices and the ability to model crop rotation. Out of 44 environmental assessment calculators for agricultural products, we identified 18 calculators which are capable of assessing GHG emissions from energy crop cultivation. These calculators differ in their goal and scope and which farming operations related to crop management are taken into account; this makes it difficult to compare and interpret the results from these CFP assessments. Only seven calculators out of 18 can calculate GHG emissions from energy crop rotations. At the moment, none of these calculators are able to consider actual effects from energy crops in rotation in the context of nutrient shifts, reductions in the use of agricultural operating needs, or the sequence and composition of crop rotations. However, by expanding the system boundaries of the CFP study, by taking the whole energy crop rotation and local agricultural management practices into account, the opportunity to identify more GHG mitigation options increases.	[ "ENER" ]	[ "Carbon Footprint", "Crop management", "Crop rotation", "Energy crops", "Greenhouse gas emissions", "Life Cycle Assessment" ]	[ "2105" ]	[ "Human influence on climate change was again confirmed by the latest report from the Intergovernmental Panel on Climate Change (IPCC) [1].", "Anthropogenic greenhouse gas (GHG) emissions from fossil fuel combustion and industrial processes contributed about 78% to the total increase of GHGs in the atmosphere over the last 40 years [1].", "Furthermore, the Agriculture, Forestry and Other Land Use sector (AFOLU) accounted for about a quarter of anthropogenic GHG emissions [1].", "In response to this, a growing number of governments have begun introducing renewable energy policies in an effort to reduce GHG emissions by replacing non-renewable fossil fuels with renewable energy sources.", "The European Commission has committed itself to increase the proportion of renewable energy to 20% of the overall share of the energy consumption and to 10% of transportation-related energy consumption by 2020 [2].", "In 2008, 12.9% of the total global primary energy supply had already originated from renewable energy sources, of which bioenergy contributed the dominant share (80%) [3].", "This implies that the production and use of biomass to generate power, heat and fuel has significantly increased in recent years [4].", "Biomass for the supply of energy is traditionally obtained from fuelwood.", "However, in the last decade, the use of crop residues and dedicated energy crops delivering the demanded biomass increased.", "Energy crops are agricultural crops solely cultivated for energy-related use.", "Several food crops (e.g. maize or sugar beet) can also be grown as energy crops if they have high yields and, preferably, a low demand for agrochemical inputs [5].", "Energy generation from energy crops has an almost-closed CO2 cycle (in which the combustion of biomass releases the same amount of CO2 as was captured by the crop during growth).", "However, it is not carbon neutral over its whole production chain, since GHG emission occurs during the production stage, e.g. through production of fertilizer, pesticides, farming machinery or fuel combustion from machinery used [5].", "Agricultural management practices have a considerable effect on the amount of GHG emissions from energy crop production and, correspondingly, on the entire biomass energy production chain [6].", "Consequently, agriculture, including energy crop cultivation, holds significant potential for reducing GHG emissions [7].", "However, appropriate assessment tools are required to identify the GHG emission benefit of bioenergy compared to its fossil alternatives.", "The most widely used approach is the Life Cycle Assessment (LCA) defined by ISO Standards 14040 [8] and 14044 [9,10].", "LCA is defined as a method for compiling and evaluating all inputs, outputs and the potential environmental impact of a production system throughout its life cycle.", "It enables the user to measure and quantify the environmental impacts of a product.", "Furthermore, it helps to identify hot spots where the most significant impacts occur, giving the user the opportunity to develop strategies for improving the product's environmental performance [8].", "In addition to the LCA guidelines, the Carbon Footprint (CFP) defined by ISO Standard 14067 [11] provides requirements and guidelines for the quantification and communication of GHG emissions in a production chain.", "The CFP is a specific method within the LCA approach and summarizes all GHG emissions and removals occurring within the established product system boundaries, expressed as CO2 equivalents.", "There are a considerable number of tools working with the CFP approach for calculating the GHG emissions from agricultural products [12,13].", "An overview of currently available tools for quantifying GHG emissions at landscape scale from AFOLU was provided by Denef et al. [13].", "They divided those tools into three categories: (1) calculators, (2) protocols and guidelines, and (3) process-based models.", "Based on these results a review of these tools was conducted by Colomb et al. [14,12] to evaluate the methodological differences between these tools, to promote transparency and to provide guidance for the user to choose the most appropriate tool.", "As distinct from Colomb et al. [14], our review focuses only on calculators, including web-based and software-based calculation tools, which are able to quantify GHG emissions from energy crop cultivation at farm scale.", "For this subset we provide an extended analysis of the complex crop cultivation system, including an evaluation of the calculators for their ability to take energy crop production specific characteristics, crop rotation effects and farm specific management practices into account.", "CFP calculators are used by farmers, agricultural suppliers and scientists to identify the potential for GHG mitigation in their local agricultural production chains [15].", "In order to be able to detect these GHG emission mitigation potentials, however, calculators should account for local agricultural management practices on the farm and especially for energy crop specifications by taking into account differences in pedoclimatic conditions, farming practices, farming technologies [16], the characteristics of perennial crops [17], and crop rotations (sequence and composition of crops) [18].", "Diversification of crop rotation patterns is one option for GHG emission reduction in cropping systems [19], but CFP studies from crop cultivation typically only take into account one vegetation period of one single crop [18].", "Accordingly, as agriculture systems are highly complex, not all underlying material flows can be quantified when the assessment is limited to such a short time period.", "As result, calculation systems leave out crop rotation effects, including all interactions between the previous crop and the assessed crop, such as nutrient shifts, reduction in the use of agricultural operating needs, different intensity and the timing of farming activities [18].", "Furthermore, CFP studies frequently fail to adequately consider the specifics of energy crop cultivation, such as differences in the timing of sowing and harvesting dates, the allocation of byproducts (e.g. the production of digestate and its reuse as fertilizer), and cultivation management (e.g. increased fuel use for the whole plant harvest, tillage frequency, and fertilizer quantities) [5,20].", "There are various case studies that use the CFP approach to assess the GHG emissions of biomass energy production.", "Cherubini and Strømman [20] presented an overview of these case studies and an assessment of the key methodological issues.", "They pointed out that there are wide ranges and uncertainties in bioenergy CFP case studies due to differences in methodological assumptions (e.g. different reference systems, the database used, functional units, and allocation procedures) and the many variables involved in this calculation (e.g. selection of system boundaries, including land use change and accounting for field emissions from different fertilizer types and crop residues).", "Furthermore, some of these key parameters regarding agricultural processes are still not well understood and depend heavily on local and climate conditions [21].", "The aim of this paper is to review currently available calculators for their ability to quantify GHG emissions from energy crop cultivation by taking into account the specific features of energy crop production and local management practices (as explained above).", "Following Buytaert et al. [22], who note that LCA is the most suitable assessment tool to assess emissions from bioenergy production systems, we focused our review on calculators that are based on the specifications of the LCA approach for GHG emission assessment, the CFP.", "Additionally, for CFP studies focusing on agricultural processes, the system boundary can be restricted to “cradle to farm gate” instead of “cradle to grave” to avoid complications of a full CFP study [23].", "Following the recommendation of Audsley et al. [23], we set the system boundaries of our study at the farm gate ending with crop harvest, but including byproducts such as organic fertilizers (e.g. digestate, manure, slurry).", "Our analysis of the calculators is based on four criteria: (1) the goal and scope of the calculator, (2) the methodology used to account for GHG emissions from energy crop cultivation, (3) energy crop cultivation management and (4) the ability to model crop rotation.", "The review process was performed in three steps: first, we identified calculators which account for GHG emissions of agricultural products.", "Out of these, in the second step, we identified the calculators which could account for GHG emissions from energy crop cultivation.", "In the third step, we analyzed and compared the resulting calculators regarding to the four criteria described in Table 2.", "Our search for calculators was carried out in English and German and covered only published information, which includes peer-reviewed literature, reports, calculator descriptions and websites.", "A systematic database search of peer-reviewed articles was conducted using the electronic Web of Science.", "All analyses were conducted between January and November 2014.", "The following thematic search terms were used: energy crops AND review, LCA AND agriculture, environmental impact AND bioenergy, Carbon Footprint AND energy crops, LCA AND modeling AND bioenergy.", "The composite terms were placed inside quotation marks, and an asterisk was used at the end of each term to capture all possible extensions and variations of a particular word.", "The documents were considered relevant if they matched at least one of the topical search terms in their titles, abstracts or keywords and were published in the last 25 years.", "After identifying the relevant papers, we used references and citations from these papers to search for cited reports, websites and models.", "In two cases we consulted the software developer directly for further information.", "Calculators that were developed exclusively for internal use by companies, consultancies or scientists for a very specific product were not included in our review.", "In the end, we identified 44 environmental assessment calculators for agricultural products.", "An overview of these 44 calculators, including their specific properties (user interface, method, GHG indicator, availability, target user group and literature source) is provided in Table S1 (supplementary material).", "Fig. 1 depicts our selection process.", "Methodologies for governmental certifications of bioenergy sustainability often use CFP methodology [24,25] and the guidelines of the IPCC for AFOLU [26] to assess GHG emissions from biomass production [24,25].", "Therefore, we selected all calculators out of the 44 earlier identified calculators which were able to calculate GHG emissions from crop cultivation (focusing on all processes occurring from “cradle to farm gate”) following this methodology and these guidelines.", "The result is that all GHG emissions occurring during the production process are aggregated into one single impact category of “climate change” by using the category indicator the Global Warming Potential (GWP) or CML 2001 [7,27].", "Calculators following other environmental assessment methods as described by Payraudeau and van der Werf [28] were excluded, as well as calculators designed to model detailed soil-plant-atmosphere processes on farms.", "Adopting these criteria in our selecting process, we identified 34 calculators matching these criteria.", "From the remaining 34, we selected all calculators that were able to model GHG emissions from arable crop cultivation.", "Calculators modeling only horticultural crops and calculators working only with fixed datasets for crop cultivation, without the possibility to modify the inputs and outputs or cultivation processes, were excluded.", "This resulted in 31 suitable calculators.", "Finally, from the remaining 31 we selected all calculators that were capable of calculating GHG emissions from energy crop cultivation.", "Since several arable crops for food, feed or fiber production can also be grown as energy crops, our analysis included calculators designed for GHG emission assessment from arable crop cultivation; while these are not exclusively designed for energy crops, they nevertheless are capable of assessing GHG emissions from energy crop cultivation as well.", "Calculators modeling crops without any specification of crop type or only with broad crop categories (e.g. general cropping system, rice fields) without a category for energy crops were excluded.", "Eighteen calculators were identified that fulfill the requirements (Table 1).", "Thirteen of the calculators are freely available and can be downloaded directly from their website or by contacting the developer.", "We tested these calculators to determine their features, inputs and outputs, functionality and operability.", "For all 18 calculators, their methodological and practical aspects were obtained from published information, including peer-reviewed literature, reports and calculator descriptions on the websites, plus the results from our calculator tests.", "Before analyzing them in the third step of our review, we screened them for their ability to assess GHG emissions from energy crop rotations and their effects.", "Seven of these 18 calculators were capable of modeling energy crops in rotation, but none of these calculators could assess energy-crop rotation effects like interactions between crops such as nutrient management or green manuring.", "The CFP methodology defines four phases to assess GHG emissions along the production chain: (1) the goal and scope definition phase, (2) the inventory analysis phase, (3) the impact assessment phase and (4) the interpretation phase [11].", "The first phase defines the general framework of the CFP study.", "The data collection for each process is carried out in the second phase, and this data is summarized into one CFP result in the third phase.", "In the final CFP phase, the results from the first three phases are evaluated in light of their completeness and sensitivity; on this basis, researchers form their conclusions, including any limitations of the study and finally give recommendations.", "The adoption of these phases in GHG emission calculators is essential for the calculated result and therefore for the applicability and utilization of the results.", "As the first three CFP phases are particularly relevant for the design and development of the calculator and for the results of the CFP study, we focused on the first three phases in our study.", "We chose the following four criteria for the comparison of the selected calculators: (1) the goal and scope of the calculator, (2) the methodology used to account for GHG emissions from energy crop cultivation, (3) energy crop cultivation management and (4) the ability to model crop rotation.", "These criteria were assigned to the first three CFP phases, and indicators and variables related to these CFP phases were identified (Table 2).", "The importance and relevance of each criterion (including CFP phases, indicators and variables) are described in detail in the following paragraphs.", "The following indicators should be considered and clearly described in the first CFP phase: the goal of the study, the system boundary, the allocation method and the functional unit [11].", "By defining these indicators, the limits of the processes included in each calculator as well as the working plan of the entire CFP study can be defined.", "When characterizing the goal of CFP studies, the intended application and audience as well as the reason for the study has to be defined [8].", "GHG emission assessment of crop production can be undertaken for various reasons.", "Colomb et al. [14] divided these reasons into four categories: raising awareness, reporting, project evaluation and product assessment.", "Calculators whose goal is raising awareness often have an educational purpose by giving information about climate change in crop cultivation and are often used by farmers and farming consultants.", "Calculators in the second category, reporting, assess GHG emissions at the farm level (used by farmers) or landscape level (used by policy-makers) to compare results with other farms or countries and to help propose GHG mitigation options.", "The third category, project evaluation, includes calculators assessing the GHG emissions of a project or a policy, often used by policy-makers, NGOs, technicians or consultants comparing different projects (e.g. different management systems, agricultural innovations).", "The fourth category, product assessment, covers calculators used by private businesses for assessing GHG emissions from agricultural production chains to compare different production systems and to provide GHG reduction plans.", "The system boundary defines processes, inputs and outputs of the production system to be included in the inventory analysis [8].", "The CFP study may be performed for the complete production chain “from cradle to grave” to the end product – e.g. biodiesel from oilseed rape, or just for the first product in the production chain, “from cradle to farm gate” – e.g. rapeseed cultivation.", "If the objective of the study is to evaluate GHG emissions from the cultivation process, the post farm gate processes can be neglected in the assessment.", "However, for determining the global impact up to consumption, all processes “from cradle to grave” (including post-farm gate processes) should be considered in the assessment [17].", "Depending on the goal of the calculator, the scale of the assessment can range from the global level, to the national, regional or individual farm level, or even down to individual farming processes.", "The time scale is another important factor in the system boundary consideration.", "CFP can be carried out for the whole life cycle of one crop, which could be less than one year for annual crops or more than one year for perennial crops.", "It is essential to define the time scale for each CFP and describe the findings in the CFP report in order to make it comparable to other CFP studies.", "Annual crops are typically assessed for one vegetation period from seedbed preparation to harvesting.", "The influence of the previous crop on the assessed crop is often outside the system boundary of typical CFP studies.", "For perennial crops, the system boundaries can be set either to one single production year or to the entire life cycle, from crop establishment to the final harvesting period.", "Further reflections on this complex issue of modeling crop rotations and the effects it has on single crops, will be discussed in Section 2.2.3.", "Allocation issues occur when a single process delivers more than one product or service (multifunctional process).", "Energy crop cultivation and processing of biomass can lead to multiple outputs, e.g. oil and oilseed meal from oilseed crushing, or biogas and digestate from anaerobic biomass digestion.", "There are three different methods available to allocate the processes emissions to different products [29].", "The first allocation method expands the system boundary (until the use of the byproduct is included) and then applies the substitution method.", "The second method divides the emissions of the entire system among the different byproducts by using a specific indicator (either a physical indicator, e.g. weight or energy content, or a socioeconomic indicator, e.g. market value).", "The third allocation method ignores the allocation process and allocates all emissions to the main product or avoids allocations by using a suitable fictional unit.", "The chosen allocation method is extremely important for bioenergy systems, due to its large impact on the final CFP result [21].", "The functional unit should be consistent with the goal and scope of the CFP study and provide a reference unit for all life cycle flows and indicators, allowing the comparison between systems [9].", "The results from CFP studies from energy crop production can be expressed as kg of CO2 equivalent per unit area; per unit product; per emission category or per farm.", "Defining the data requirements, assumptions and quality requirements of the data is part of the first CFP phase and is influenced by the goal of the calculator and the goal of the CFP study correspondingly.", "The IPCC provides three calculation pathways, called Tiers, in the AFOLU guidelines to account for land-based GHG emissions [26].", "The Tiers differ in their degree of complexity: Tier 1 is the least accurate methodology, though the simplest to use, as it provides equations and global default values; Tier 2 may use the same methodological approaches as Tier 1, but requires specific regional data and emission factors, while Tier 3 level methodologies are based on actual measurements or model simulations.", "Using a higher Tier generally improves the accuracy of the inventory analysis and reduces uncertainty, but requires a higher amount and quality of input data.", "Making sure that the chosen GHG emissions calculation pathway (Tier) corresponds to the geographical coverage of the calculator is very important [14].", "Global or national calculators use the Tier 1 approach, in which only a small amount of input data is required and global or country average emission factors are used.", "Calculators using the Tier 2 approach often focus on a regional application, and pedoclimatic and management data is needed.", "Using the Tier 3 approach for assessing the GHG emissions enables the calculator to obtain farm-specific results in different timeframes (day, month and/or year).", "However, this requires specific measurements or complex pedoclimatic and management input data, which is often too time-consuming to obtain.", "Furthermore, calculators using the Tier 3 approach are locally restricted or focus on a specific product or emission processes; this could be unfeasible for most CFP studies.", "The results of the GHG calculator and the integrated calculation pathway can only be as precise and reliable as the input data used to compute these results.", "Various LCA databases are available, providing datasets from agriculture, energy supply, transportation, biofuels and biomaterials, bulk and special chemicals, construction and packaging materials, basic and precious metals, and metal processing, as well as waste treatment.", "These datasets integrated in the calculators enable users to calculate their production chain by simply combining the single production steps which are provided in a kit of modules from the chosen database.", "All datasets are representative of previously completed LCA study results.", "The result of a CFP study largely depends on which database is used.", "In the third CFP phase, the impact categories and category indicators are selected consistent with the goal of the study.", "The collected emission data from the inventory analysis are assigned to the selected impact category [9].", "In our review, we focused on calculators following the IPCC guidelines [26] and using the impact category “climate change” with the category indicator GWP [7,27].", "The GWP can be calculated over a specific time interval: 20, 100 or 500 years, and aggregates all emitted GHG into one unit (kg of CO2 equivalent per functional unit), which makes it easier to compare GHG emissions from different products.", "The production of biomass from energy crops requires multiple steps: tillage, sowing, fertilization, use of pesticides, and harvest.", "GHGs are emitted from each farming operation.", "However, agricultural croplands that are intensively managed offer many opportunities for reducing GHG emissions through changes in agronomic practices [7].", "The IPCC [26] recommends taking into account all indirect and direct emissions caused by farming operations when calculating the CFP of crop cultivation.", "Indirect emissions occurring during the production of all inputs (agricultural operating needs such as seeds, fertilizer, pesticides, agricultural machinery, fuel, building materials and energy) and have often been considered by using combined emission factors expressed in CO2 equivalent from available databases.", "Indirect emissions from the production of agricultural operating needs can have a significant impact on the CFP results [23]; fertilizer production in particular is responsible for high GHG emissions [30].", "Therefore, distinguishing between fertilizer types used in agricultural production can have a great impact on the CFP results [30].", "The production of pesticides is less GHG emission intensive, but the distinction between different types can affect the CFP result as well.", "Direct GHG emissions occur on the field through the application of crop residues and fertilizer (organic and mineral).", "According to the IPCC guidelines for AFOLU [26], CO2, N2O and CH4 should be considered for direct emissions and NH3 and NOx for indirect emissions when estimating anthropogenic GHG released during crop cultivation.", "CO2 emissions result from liming and urea application.", "Nitrous oxide (N2O) emissions from managed soils arise from anthropogenic nitrogen input, such as mineral and organic fertilizer and crop residues, including N2O through two indirect emissions pathways.", "The first indirect N2O pathway is nitrogen volatilization, which occurs for example when NH3 and NOx are deposited onto soil and water.", "Leaching and runoff of nitrogen from fertilizer application is the second pathway for indirect N2O emissions.", "Both indirect emissions pathways lead to further processes in which N2O emissions occur.", "The type of fertilizer applied on the field affects the direct and indirect GHG emissions caused by the fertilizer [31].", "This can be seen in a simple form, by distinguishing between mineral and organic fertilizer in general, but also in a more advanced distinction as seen between mineral and organic fertilizer types.", "The use of digestate as an organic fertilizer to substitute for mineral fertilizer is one option for reducing GHG emissions [32] by eliminating the GHG emissions from mineral fertilizer production.", "However, digestate application increases diesel consumption and correspondingly GHG emissions [33].", "Consequently, the possibility of distinguishing between fertilizer types could be beneficial for the CFP of energy crop cultivation.", "From crop residues remaining on the field, GHG emissions occur through the process of nitrification and denitrification, and should be included in the CFP calculations.", "The amount of nitrogen added to the field annually through crop residues (above-ground and below-ground) including nitrogen-fixing crops, is related to crop type, yield, residual nitrogen content, ratio of below-ground and above-ground biomass and the crop management system (what is left on the field, e.g. straw, stubble) [26].", "CO2, CH4 and N2O and other air pollutants (e.g. CO, NOx) are emitted during fuel combustion [34].", "The amount of fuel used for each cropping system as a function of machinery operation for tilling, drilling, seeding and harvest is related to machinery performance (technical standard) and the type of machinery used, soil type, and harvest yield, as well as crop management (e.g. the tillage system, the amount and type of fertilizer and pesticides applied) [35].", "CO2 emissions can occur through changes in soil organic carbon (SOC) stock changes caused by changes in the land use and management regime, called a Land Use Change (LUC).", "According to ISO 14067 [11], GHG emissions through LUC should be integrated, but documented separately in CFP studies.", "The choice of crop can have a high impact on GHG emissions from the whole production system as well as on N2O and NO emissions from fertilized fields [31].", "Therefore, a parameter addressing the type of energy crop should be included in GHG emission calculators.", "A wide range of species can be used as energy crops, but the intensity of crop management depends on the species selected.", "Energy crop production management is in many ways similar to conventional food crop management.", "Crops with rapid growth, a high yield of usable biomass, an ability to grow under adverse weather and poor soil conditions, and with a high resistance against pests and diseases are favored as energy crops [36].", "Sometimes, energy crops can have different crop management requirements than food crops [36], especially if the selected species has not traditionally been grown in the area (e.g. Sorghum in Central Europe) or if perennial crops are used instead of annuals (e.g. Miscanthus sinensis, Silphium perfoliatum).", "If food crops are grown as energy crops, alternative genotypes less suited for food production but with lower input requirements may be used [37].", "However, biomass yield still depends on climate and soil conditions, fertilizer supply, and the timing of sowing and harvesting [38].", "Perennial crops can have several benefits compared to annual crops.", "The inputs of a perennial cropping system are lower because the crop only has to be established once and the long-living roots can interact with the ecosystem, which can be beneficial to the nutrient balance of the soil [36].", "When describing the crop management of perennial crops, the whole life cycle should be taken into account, since the agricultural performance of the crop correlates with the age of the plants.", "During crop establishment and at the end of the crop cycle, productivity is lower than in the years between these two phases.", "Consequently, the CFP of perennial crops may be underestimated when assessing only a single productive cultivation year and ignoring the other cultivation stages.", "Hence, the inclusion of detailed inventories of agricultural management at each stage of perennial crop cultivation would improve CFP calculation and the reliability of the assessment results [17].", "Integrating undersowing crops (sowing a secondary crop underneath the main crop) into crop management may also have a positive influence on the CFP result [39].", "Energy crops can be included in traditional food crop rotations or can be grown in self-contained rotations.", "In general, crop rotation improves soil fertility (by enhancing soil structure, reducing soil erosion and maintaining sufficient content of soil organic matter), nutrient use efficiency (reduced and demand-oriented fertilizer use), and biodiversity (improved crop diversity).", "Crop rotation also tends to reduce the input of crop protection agents and increase crop yields [37].", "The system boundaries in agricultural CFP are typically set at one vegetation period of one single crop [18].", "However, as agriculture systems are highly complex, often not all underlying material flows can be quantified when the assessment is limited to such a short time period.", "Including all interactions (crop rotation effects) between the previous crop and the assessed crop in the CFP was recommended as a possible solution by Brankatschk and Finkbeiner [18].", "When looking at only one vegetation period, it can be difficult to consider the exact nutrient supply, since each crop uses different amounts of nutrients and leaves different residue nutrients in the field.", "Another effect of crop rotations can be the improvement of phytosanitary conditions by reducing the pressure of disease and infestation by parasites.", "Therefore, the previous crop can affect nutrient and pesticide management for subsequent crops.", "By switching crops in a crop rotation, the intensity and timing of farming activities can be influenced, since the soil structure and texture are influenced.", "Crop residues remaining on the field or the introduction of green manure crops or catch crops in the crop rotation can have a major impact on the subsequent crop and on the crop rotation as a whole by affecting the soil properties and fertility, and correspondingly the achievable yield [18,19].", "Today's CFP studies typically assess each crop independently if crop rotations are assessed, but by doing so they lose the ability to reflect the effects of crop rotation itself [18].", "For this reason, the whole crop rotation should be assessed in CFP of energy crop cultivation in order to assess all effects related to crop rotation.", "This includes the consideration of all shifts of inputs in the crop rotation from one crop to the subsequent crop; otherwise the previous crops within the crop rotation carry the GHG emission burden from the following crops.", "Not all 18 identified calculators have assessment of GHG emissions from energy crop cultivation as their only goal, with a target audience of farmers or private companies.", "However, all of them included a possibility for GHG assessment from energy crop cultivation in their goal and scope definition.", "Nevertheless, the objective of each calculator and user group varies (Table 3).", "C-Plan and Farm GAS (both web-based) were mainly designed to raise awareness among farmers, consultants, students and land managers.", "They are both focused on giving an initial overview of farm-related GHG emissions and the impact of farm management decisions on GHG emissions.", "BioGrace, CFF, COMET-FARM, FSGGEC, HGCA 1 and IFSC have the purpose of reporting accurate GHG emissions for subsequent comparisons between farms or countries.", "BioGrace and HGCA 1 are both based on Microsoft Excel, and were developed for the purpose of calculating the entire CFP (“from cradle to grave”) of biofuel production from biomass.", "Both calculators were designed for politicians or consultants to support decision-making and to design national GHG reduction programs, and for farmers to see how changes in management practices could affect the overall GHG emissions of the resulting biofuel production.", "CFF, COMET-FARM and FSGGEC are web-based calculators developed to support farmers to estimate the CFP of their farm or single products with a focus on carbon sequestration and crop management.", "IFSC addresses the same topic, but is Excel-based.", "All four calculators have the goal of identifying mitigation options of GHG emissions on the farm and to report the CFP results voluntarily to national GHG emission reports or for CFP labeling and for comparing CFPs from similar products.", "Only one of the 18 calculators under review, CAPRI, was developed for the purpose of project evaluation.", "CAPRI is a multi-purpose modeling system software for EU agriculture developed for policy-makers and scientists to analyze research questions in relation to specific agricultural policies [40].", "Nine out of 18 calculators were developed for product assessment.", "Four of them (GaBi, openLCA, SimaPro and Umberto) are software solutions and were originally developed to assess the life cycle from industrial products.", "These tools were designed to be used by scientists, companies or policy-makers to assess the potential environmental burdens of a product in its production, use and disposal, and to detect mitigation options in the production chain.", "Agri-LCI models, CFT and HGCA 2 (all Excel-based) and SALCA (software solution) were developed for farmers, companies and policy-makers to assess the LCA from agricultural products and different management systems and to derive recommendations from these results for GHG reduction.", "GEMIS is a life cycle calculating software program developed for companies and policy-makers to model energy, material and transportation flows.", "All 18 calculators under study include the assessment of GHG emission from crop cultivation “from cradle to farm gate” in their system boundaries, but only eight of them (BioGrace, GaBi, GEMIS, HGCA 1, openLCA, SALCA, SimaPro and Umberto) are able to extend the system boundary to the end of the life cycle of the assessed production chain (Table 3).", "Despite the similar system boundary of “cradle to farm gate”, each calculator includes different direct and indirect GHG emissions sources related to crop cultivation (Tables 5, 6).", "OpenLCA, SimaPro, SALCA and Umberto offer assessment of GHG emissions at the global, national and farm scale, GaBi on the global and the national scale, GEMIS at the national level, and CAPRI at the national and regional scale.", "The other 11 calculators were only developed to assess the GHG emissions at the farm scale.", "In 11 of 18 calculators, the time horizon for GHG emission assessment was limited to one crop vegetation period (one year) only.", "However, seven calculators can extend the assessment to multiple crop cultivation periods.", "Different allocation methods are used by the 18 calculators to allocate the GHG emissions to co-products.", "HGCA 1 and 2 expand the system boundaries and substitute the byproducts with other products already included in the CFP study.", "Six calculators share the system's emissions among byproducts by using specific indicators.", "Three of them use only one indicator for allocation: Agri-LCI models and CFT use economic indicators; energy content is used by BioGrace.", "Sometimes in a multifunctional production process it is impossible to find one appropriate indicator which works for all byproducts.", "As a result, two indicators (physical and economic, as used by CAPRI and SimaPro) or three indicators (economic information, area and arable area as SALCA offers) can be used for allocation.", "CFF, COMET FARM, C-PLAN, FSGGEC and IFSC avoid using an allocation method and either allocate all emissions to the main product or choose a suitable functional unit by which the byproduct is outside the system boundary.", "The other calculators provide all allocation methods and the user can choose a suitable method according to the goals defined for the particular CFP study.", "Table 3 provides an overview of the functional units used by the calculators to report the CFP results.", "COMET FARM, CFT, GaBi, openLCA, SimaPro and Umberto provide results per unit product and per unit area.", "All calculators except the Agri-LCI models, CAPRI and COMET FARM provide the GHG emissions separately for each emission category in addition to the total result.", "IFSC and COMET FARM provide the results in imperial units, whereas all the other calculators are metric.", "The amount of data required by each calculator depends on the processes, activities and sources included in the calculator and on the GHG emission calculation pathway (Tier) used.", "The calculators are classified into different Tiers in Table 5 in order to distinguish their degree of complexity of integrated methodology to account for land-based GHG emissions.", "In seven out of 18 calculators, all three Tiers were combined into one approach, e.g. COMET FARM and FSGGEC.", "In GaBi, openLCA, SALCA, SimaPro and Umberto, own more detailed data can be integrated as well.", "Seventeen out of 18 calculators not only use the IPCC guidelines [26], but mix different assessment methodologies.", "However, the assessment methodologies chosen are always on the same complexity level as the Tier methodology applied in the calculator.", "BioGrace, CFF and C-Plan use the Tier 1 approach with global or national default values.", "Seven of the 18 calculators use Tier 1 and Tier 2 approaches for GHG emission calculation and can be modified with country-specific emission values.", "Calculators including the Tier 2 approach focus on regional application, and pedoclimatic and management data are required.", "COMET FARM and FSGGEC provide a country map, with climate and soil data, where the user can locate their farm and run the dynamic process-based crop–soil–atmosphere models.", "The Tier 3 approach requires measurements or high-resolution input data for model simulation, and is locally restricted.", "Seven calculators use Tier 3 approach methods, but only to calculate some processes included in the CFP that are crucial for the result of the CFP; they calculate other processes using approaches of lower complexity.", "When choosing a GHG emission calculator, it is important to know for which region or country it was developed, and consequently which GHG emission default values were implemented.", "Only CFT, GaBi, openLCA, SimaPro and Umberto can be used worldwide.", "All the other reviewed calculators were calibrated for specific countries.", "Six out of the 18 calculators under review use the Ecoinvent Database [41] and other agricultural and product databases (Table 4).", "The Ecoinvent Database provides around 10,000 datasets from agriculture, energy supply, transportation, biofuels, biomaterials and other industrial processes [41].", "The datasets integrated in the models enable users to calculate their production chain by simply combining the individual production steps, which are provided in a kit of modules from the Ecoinvent Database.", "If a specific crop or production step is not available, it is possible to modify an existing dataset or create a dataset from scratch (e.g. new energy crops).", "For most modules, global, European or national mean values are available.", "SALCA uses its own GHG assessment concept for agriculture.", "This covers LCA methods adapted to the agri-food chain, such as GHG emission calculators, and the SALCA life cycle inventory database, based on the Ecoinvent Database [42].", "OpenLCA is an open-source software program into which freely and commercially available databases can be integrated.", "The methodological approach of this calculator is equivalent to GaBi, SimaPro and Umberto, and the same databases can be integrated.", "In a similar way to other software, it works like a kit, in which the individual production steps are provided as freely combinable modules.", "The general methodology of the other calculators is based on global, European or national guidelines (e.g. IPCC and RED) which provide GHG emission default factors for different cultivation-related GHG sources.", "Furthermore, datasets from literature are used to assess specific indirect and direct agricultural GHG emissions.", "COMET FARM integrated the dynamic agro-ecosystem model DayCent (the official U.S. National Greenhouse Gas Inventory model) to estimate emissions on the field and through LUC.", "IFSC uses the COMET-VR soil carbon model from COMET FARM.", "Regarding GWP, the calculators GaBi, openLCA, SALCA, SimaPro and Umberto can determine GWP for 20, 100 and 500 years, and the user can choose the preferred indicator.", "All other calculators only provide GWP for 100 years (Table 4).", "As mentioned above, the selected system boundaries can significantly affect the processes, activities and sources included in each calculator as well as the amount and quality of the input data required.", "Table 5 gives an overview of the included indirect emissions arising from farming processes for each calculator.", "In two calculators (COMET FARM, FarmGas), indirect emissions from the production of operating resources (e.g. machinery, pesticides and fertilizer) are omitted.", "In contrast, in eight out of 18 calculators, GHG emissions for the production of all agriculturally related inputs are embedded in the assessment (CAPRI, CFF, GaBI, GEMIS, openLCA, SALCA, SimaPro and Umberto).", "This group consists of calculators in which a large amount of datasets is provided, allowing the user to decide which indirect emissions should be included in the CFP.", "In GEMIS and CFF, indirect emissions from the production of agricultural inputs are included in the calculators; in CFF, the user can influence the values by modifying the amount of inputs.", "The remaining calculators provide indirect emissions from production for only a limited number of farming inputs – sometimes only one.", "Twelve out of 18 calculators include indirect emissions from manufactured pesticides.", "However, only seven can distinguish among types of pesticides by dividing pesticides into categories: herbicides, insecticides, fungicides and lubricants.", "Furthermore, with GaBi, openLCA, SALCA, SimaPro and Umberto, the user can calculate the CFP of the individual pesticides by aggregating the GHG emissions of the pesticide ingredients provided by the database included in these calculators.", "The other five calculators include pesticide production by aggregating emissions from pesticide use in one category; they do not distinguish between types of pesticides.", "Fourteen out of 18 calculators include indirect emissions from manufactured mineral fertilizer, but only 12 out of these 14 can distinguish among types of mineral fertilizers.", "They all provide a different number of fertilizer types.", "BioGrace, for example, only distinguishes among mineral fertilizer ingredients (N, CaO, K2O and P2O5) while CFT provides 35 different types of mineral fertilizer, and the user can add new types or edit existing ones.", "Table 6 gives an overview of the included direct GHG emissions arising from farming processes for each calculator.", "C-Plan and FSGGEC do not distinguish among mineral fertilizer types; they only take the amount of N fertilizer (sum of N in kg) applied on the field into account.", "The other 16 calculators can distinguish among mineral fertilizers to a different degree of accuracy.", "With the exception of FSGGEC, all calculators included organic fertilizer in their assessment; 13 calculators can even distinguish among different types of organic fertilizers.", "Regarding digestate, only nine calculators take this particular organic fertilizer into account.", "However, they cannot distinguish among application methods (manure chisel plow, drag shoe, drag hose or incorporated in one hour after application) to account for the GHG emission arising from different digestate applications.", "Other than Agri-LCI models, COMET FARM and C-Plan, all calculators of the 18 include direct GHG emissions from crop residues applied on the field.", "Through the use of machinery during energy crop cultivation, GHG emissions arise from fuel combustion.", "All calculators take this into account except COMET FARM and Farm GAS.", "Fourteen calculators can even distinguish among different types of tillage.", "Agri-LCI models and COMET FARM use categories (e.g. reduced tillage, plow-based, direct drilling) to account for different crop management systems and the amount of diesel used, respectively.", "In the other 12 calculators, it is possible to calculate the amount of diesel used by selecting each crop management step within the calculator (e.g. CFT provides a list of management steps) or by adding the actual amount of diesel used (e.g. BioGrace).", "Thirteen out of the 18 calculators account for GHG emissions from LUC, but not all calculators document these results separately and some just account for emissions through land use change and not through management change.", "However, CAPRI, COMET FARM, FSGGEC GaBi, IFSC, open LCA, SALCA, SimaPro and Umberto all feature integrated process-dynamic models to determine emissions from soil carbon change through management changes.", "Only BioGrace, HGCA 1 and GEMIS were originally designed to calculate GHG emissions from energy crop cultivation, but the calibration is limited to traditional energy crops for bioenergy production.", "However, the other 15 calculators provide datasets and calibrations for energy crops in addition to food crops, and also provide the possibility to modify or add crops.", "An overview of the calibrated energy crops in the calculators under study is given in Table 7.", "If the category “other” is selected on the calculator, other energy crops can be calculated without a specific calibration for this crop.", "Datasets from energy crop cultivation are included in CAPRI, GaBi, openLCA, SALCA, SimaPro and Umberto, but they are limited to the traditional energy crops as shown in Table 7.", "Previously unconsidered energy crop species, such as Silphium perfoliatum, could be added by users by modifying existing datasets or creating their own.", "Perennial crops are omitted in most calculators or integrated as an annual average whenever it is impossible to distinguish among the different stages of cultivation.", "CFT, GaBi, openLCA, SALCA, SimaPro and Umberto can calculate the GHG emissions from perennial crop cultivation.", "However, the user has to check if the full life cycle (from the establishment to the end of the crop productivity) of the perennial crop is considered.", "Undersowing crops were not addressed in any of the 18 calculators under review.", "Seven (COMET FARM, FSGGEC, GaBi, openLCA, SALCA, SimaPro and Umberto) of the 18 identified GHG emissions calculators for energy crop cultivation based on the CFP approach can calculate energy crop rotations.", "For crop rotation modeling with GaBi, openLCA, SALCA, SimaPro and Umberto, the existing modules (datasets) from crop cultivation can be combined, e.g. three years of maize cultivation can be calculated by using the same maize cultivation module three times.", "Within the single modules, the management system can be changed by the user.", "With COMET FARM, it is possible to calculate GHG emissions on a farm for a longer period.", "The user can enter management data on an annual basis, which can cause problems if the cultivation period spans over two calendar years (e.g. winter crops).", "FSGGEC offers a simple type of crop rotation calculation to the user: for each year, a single crop can be cultivated and calculated at Tier 1 or 2 level.", "The result is a very simple CFP where only a few GHG emission sources are taken into account.", "COMET FARM is the only calculator which has catch crops integrated.", "It is difficult in all seven calculators to assess crop rotation effects, such as shift of nutrients or reduced farming activities and inputs.", "Most of these calculators generate their crop modules as single annual crops, which makes it difficult to display and to determine the effects of the crops on each other.", "The most important stakeholders for biomass cultivation in bioenergy production are farmers, energy industries, politicians and NGOs.", "All of them require information about GHG emissions and calculators to assess this information for their own purposes.", "None of the calculators discussed here can meet the needs of all target groups, but many calculators are available with varying levels of complexity and target different goals and user groups.", "Raising awareness is the goal of C-Plan and Farm GAS.", "These calculators require little time and knowledge of GHG emissions and climate change.", "They can be used without training and need only small amounts of input data to estimate GHG emissions.", "The results are displayed as simple graphics and guide the user toward identifying mitigation opportunities.", "However, they are not usually designed to assess changes in management and to take into account alternative and more sustainable management practices.", "Results from calculators designed for reporting can be used as the reporting basis for the certification of sustainable biofuel production and for the verification of compliance with sustainability criteria for biofuels of the Renewable Energy Directive and the Fuel Directive [2].", "BioGrace, for example, was developed to harmonize the different European calculators and calculation methods for GHG emissions from biofuel production, which is necessary to comply with the Renewable Energy Directive and Fuel Quality Directive [43].", "The calculation scheme (calculation rules, default values) of BioGrace is often used in combination with other national calculators (national default values and legal frameworks) for reporting the national specific GHG emissions from biofuel production, e.g. ENZO2 in Germany.", "The calculators in this group are either available as an Excel document, in which case calculation functions, emission values and intermediate results can easily be reproduced, or have a web-based user interface where modification can only be rendered manually via input data.", "Calculators designed for product assessment are well suited for revealing the relationship between different production levels.", "The software-based calculators are in general more time consuming and require a basic knowledge of agronomy and basic computer skills.", "Standard values for energy crops are available and different scenarios can be calculated by the user with only a small amount of input data.", "However, these standard modules only contain global or national mean values, and have to be modified by the user for regional calculations.", "In order to model new energy crops, datasets from farm operations, machinery, and mineral and organic fertilizers (including digestate) are available in the integrated databases.", "However, the user must pay particular attention to the inclusion of field emissions and also to which Tier is used to calculate these emissions.", "The goals of a CFP study should always correspond to the goals of the chosen calculator and to the defined target user groups, otherwise the results of the study could be misinterpreted.", "At the very least, the calculator chosen should be in the same purpose category as defined by Colomb et al. [14].", "The user should bear in mind that it is difficult to draw a meaningful comparison of results across similar production chain studies using different calculators with different goals, as these goals affect the system boundaries and the calculation approaches used.", "All 18 investigated calculators can calculate the GHG emissions from “cradle to farm gate”, and these results can be integrated in further CFP of bioenergy production chains.", "The defined system boundary affects the processes, activities and sources included in each calculator.", "System boundaries in CFP studies from agricultural production systems vary greatly within and among the same production chains [17].", "Significant differences in GHG emission results can occur from the same dataset of one bioenergy production chain, depending on the calculator used [55].", "The results show that it is crucial which farming processes are integrated in the calculator, which calculation pathway and allocation method is used, and if the whole cropping cycle (e.g. perennial crops) or crop rotation is included [44].", "Various crop cultivation CFP studies have been based on secondary data from statistics or literature.", "Input data based on global or national statistics can be used to assess the GHG emissions from typical cropping systems at the global or national level, but not to assess the influence of regional pedoclimatic conditions and specific management practices on GHG emissions [17].", "Therefore, the user should identify the type of available input data and the assessment goal and scale for the CFP study before choosing the calculator and the calculation scale, respectively.", "The allocation of GHG emissions among the individual byproducts of energy crop cultivation, as well as the subsequent use of the byproduct's burdens in other production cycles, are major methodological challenges.", "The inaccuracy of the CFP results can increase with each allocation step performed in one LCA, and the results are fundamentally affected by the choice of allocation method [45].", "Six of the calculators in the analysis share the emissions of the system among the different co-products by using a specific indicator as recommended by ISO Standard 14067 [11].", "Physical indicators (e.g. weight or energy content) appear to be most scientifically accurate, as they use physical principles instead of societal values, but economic indicators reflect the driver of the process through product demand.", "However, market prices can differ among countries and can lead to different CFP results [20].", "Expansion of the system boundary can help to foresee the effects on GHG emissions through changes induced by substituting products.", "The integrated allocation method should always be transparent for the user of the calculator and in compliance with the intended purpose of the CFP study.", "According to the scope of the study, the user should select the functional unit carefully, because different functional units can lead to contradictory interpretations of the results.", "Calculators can provide the results in two ways: for the assessed process, or as result of a comparison of two scenarios (baseline vs. end of project).", "Calculators providing the GHG emissions separately for each emission category have a greater potential for identifying mitigation options.", "Users should pay attention if the GHG emissions are reported in CO2 equivalents or as individual GHGs, e.g. N2O, CH4, and CO2, or as N2O-N, CH4-C and CO2-C.", "The simplest reporting unit for energy crop GHG emissions assessment is by area.", "However, this unit is not suitable for reporting GHG emissions in the context of renewable energy sustainability, and cannot be included in the calculation pathways for biofuels or bioenergy.", "In CFP from bioenergy, connecting the GHG emissions to the product is more appropriate.", "However, this includes several units which are associated with production (kg product, MJ product) and several outputs (main product e.g. kg grain and byproduct e.g. kg straw).", "For bioenergy-oriented crop cultivation, results should always be related in some way to the next production phase in the production chain.", "GHG emissions from crop cultivation depend on local conditions [23].", "Therefore, the results from the CFP can be improved by using one of the 14 calculators which take into account national or regional climate and soil conditions.", "Datasets from different databases representing the same process can result in different emission factors (emission assessments) affecting the comparison of CFP studies with datasets from different databases.", "Consequently, for similar inputs (e.g. fertilizer production), emission factors from the same database should be used to quantify different types of inputs (e.g. fertilizer types).", "In addition to the GWP based on the IPCC guidelines [26], GaBi, SALCA, SimaPro and Umberto are able to calculate the GHG emissions for other impact assessment methodologies as well (e.g. ReCiPe, Impact 2002+, Eco-Indicator 99, CML, TRACI and IPCC).", "Furthermore, these calculators offer the possibility of extending the LCA with other impact categories provided, such as acidification, eutrophication, aquatic and terrestrial ecotoxicity, human toxicity, land use and/or ozone depletion.", "Regardless of the calculator chosen, the user should bear in mind that it is difficult to draw a meaningful comparison of results across similar production chain studies using different time horizons in terms of the GWP for 20, 100 or 500 years or using different impact assessment methodologies to translate life cycle flows to the same impact category impact.", "Cultivation of energy crops differs from that of conventional food crops in some aspects which may significantly influence the GHG emissions and their estimation.", "LCA methodologies have been recently adopted for agricultural products to account better for location characteristics and differences in farming practices, focusing on annual crops [17].", "The amount of GHG emissions from energy crop cultivation can be controlled by the choice of crop type, fertilizer, pesticides and machine management and by the design of crop rotations [46].", "Indirect emissions from on-farm operations (e.g. machinery use) have a significant impact on the CFP results [23].", "However, emissions from production of the agricultural operating needs (e.g. seeds, pesticides, fertilizer, machinery, fuel) are sometimes ignored or only partly addressed by the calculators.", "Since each calculator accounts for different GHG emission sources, potential users of these calculators need to check which key sources (e.g. production of fertilizer, pesticides, machinery or seeding materials) are covered by the calculator in order to derive mitigation options for their investigated production chain from the results.", "Furthermore, differences in crop cultivation management can be better detected if the calculator distinguishes among type of fertilizer and pesticides used for crop cultivation, since it has a significant impact on the whole CFP – especially the amount and form of nitrogen (e.g. NH4-N, NO3-N, urea-N) of the fertilizer used [30].", "Optimizing crop management and nutrient use efficiency by adjusting the use of nitrogen fertilizer according to the crop's needs can directly reduce GHG emissions on the field and also indirectly through reduced fertilizer manufacture [46].", "Direct GHG emissions, especially N2O emissions from managed soils, have a significant impact on the CFP result [47].", "However, the calculation of land-based GHG emissions from energy crop cultivation is the stage where the calculators differ most, since different calculation pathways are applied.", "The methodology used to calculate N2O emissions from N-fertilization is the main contributor to the derivation among calculator results [44].", "In most CFPs, the Tier 1 approach [26] is used to calculate N2O emissions from managed crops.", "This approach uses a default emission factor of 1% of nitrogen added to the soil assuming that 1% from mineral and organic fertilizer and crop residues is lost as N2O to the atmosphere.", "Using this method, no distinction is made between fertilizer types, crop types or soil characteristics.", "Calculators following the Tier 2 approach [26] often use the approach from Bouwman et al. [31].", "This approach takes into account different regional conditions as well as different crops and fertilizer types (mineral and organic).", "COMET FARM is the only calculator that adopts the Tier 3 approach and uses the dynamic agro-ecosystem model DayCent to calculate field emissions.", "Fifteen out of the 18 investigated calculators take into account GHG emissions from crop residues, calculating these GHG emissions according to the Tier 1 approach (as described above).", "The accuracy of this calculation method can be improved by including crop residues management in the calculation such as the amount of straw left on the field (e.g. HGCA 1 and HGCA2), the quantity of crop residues or the amount of stubble burnt (e.g. Farm GAS) and by using the real nitrogen content of the above-ground biomass (grain and straw) to calculate the nitrogen content of the above-ground and below-ground biomass (this can be integrated by the user in GaBi, open LCA, SALCA, SimaPro and Umberto).", "Consequently, before choosing one of these calculators, the user should check which nitrogen sources are considered in the calculator and which Tier approach is used, and decide if these are sufficient for their specific goal.", "The amount of NH3 emissions induced by organic fertilizers (i.e. slurry and manure, digestate, poultry manure) depends on the fertilizer type, the fertilizer application rate and method, the daily temperature and a binary variable indicating whether the fertilizer was incorporated within one hour [48].", "Seventeen of the investigated calculators can distinguish among types of organic fertilizer.", "Calculators using the Tier 2 approach of Bouwman et al. [31] for calculating GHG emissions from organic fertilizer application take into account different application methods (e.g. CFT).", "However, none of these calculators take into account the daily temperature or incorporation time.", "The properties of digestate are different from conventional organic fertilizer (slurry ore manure) and are affected by the anaerobic, microbial fermentation process and by the substances used in the process [49].", "During the production, storage and application of digestate, CH4, NH3 and CO2 emissions can occur [50].", "Through organic fertilizer production and storage management as well as the application method, the amount of GHG emissions can be influenced and should be included in the consideration of CFP calculations.", "LUC should be included in the CFP assessment, but should be reported separately in the results (ISO 14067).", "However, some calculators exclude LUC for practical reasons since the methodology used to detect LUC is very complex.", "Models like RothC [51] (Tier 3 level) can calculate the SOC change on a monthly and regional basis, but also require a lot of input data.", "Using the Tier 1 approach [26] is less complex, because global emission factors (CO2 emissions occurring over a period of 20 years) and reference native soil carbon content, depending on soil type and climate region, are provided.", "It is very important to consider the period of time over which emissions occur, since calculators that do not account for time are unable to calculate LUC-induced emissions [14].", "Generally, with a longer time horizon, the yearly rate of SOC change decreases, since SOC change is always faster during the first years after disturbance.", "This aspect has already been highlighted in Petersen et al. [52], where the authors suggested using a 100-year time horizon when simulating SOC change for CFP studies, based on a 100-year GWP calculation.", "However, a 100-year time horizon conflicts with the confidence time of many other factors characterizing the agricultural sector (e.g. land use, cropping systems, management regimes) as their defining framework conditions (e.g. consumer demand, economic trends, societal transformation, public policy) are highly volatile and it is difficult to elaborate predictions in the longer term.", "For agricultural land use decision-making, 20 years should be considered as a more reasonable time horizon, which is why it has been used to include SOC change into CFP according to the Tier 1 approach.", "However, when changing the cultivation system each year, the effect of management change on the SOC content is not stable and may be disregarded when calculating the CFP from annual crops [26].", "In CFP calculation, different tillage systems are accounted for through the different amount of resources used [35].", "New technologies and crop cultivation methods have been shown to reduce the direct fossil fuel (diesel) consumption.", "Diesel consumption is either modeled by the calculator or the user can include the real amount of diesel used.", "Using mean values for diesel consumption estimates can overestimate the amount of diesel consumption by 47% [35].", "Taking real diesel consumption data from the farm is always the most precise way for GHG emission calculations.", "However, if this data is not available, using diesel consumption models which distinguish among farming operations (tillage, seeding, fertilizing and harvesting) and considering the soil characteristic (e.g. CFT) may be a good alternative to simply dividing the results among tillage systems in general (categories as, e.g. reduced tillage, no tillage) or making no distinction whatsoever.", "Not all calculators in this study were designed for specific energy crop calculations.", "Most calculators are calibrated for a small number of crops and it is not possible to integrate new ones.", "Furthermore, characteristics related to energy crop cultivation, e.g. digestate application on the field and whole plant harvest, are often ignored or insufficiently considered.", "New cropping management systems, such as undersowing, were not considered in any of the calculators under review.", "Not only does undersowing offer benefits for reducing GHG emissions by minimizing the farming operations required, thus saving fuel, weeds may be replaced by the undersowing crop and the second crop will be further ahead than if it were sown after the primary crop was harvested [39].", "The GHG assessment of perennial cropping systems is complex, since it is sometimes impossible to gather data for the whole cropping cycle [53].", "Perennial cropping systems are insufficiently considered in the available GHG assessment methods and calculators.", "As previously mentioned, the crop type is a driving factor for N2O emissions, but in most approaches perennial crops are not represented and can only be classified as “other crops” or “grass” [54] or representative data for proper calibration of the models is lacking [17].", "Hence, more research on perennial cropping systems and their field emissions is needed.", "The whole cropping cycle and detailed inventories of agricultural management at each stage of perennial crop cultivation should be included in order to improve the CFP calculation and the reliability of the assessment results [17].", "Including the specific characteristics of energy crop type, cultivation management and new cropping management, e.g. undersowing, in GHG emission accounting calculators can reduce the uncertainty in GHG emission assessment and can help users to detect GHG mitigation options in the cultivation process.", "But to carry out this concept, a high amount of input data with high quality requirements and specific high Tier level GHG emission calculation pathways are necessary.", "Seven of the 18 investigated calculators are able to calculate energy crop rotations, but none of these cover the consequences of optimizing the management, sequence and composition of crop rotations.", "Most of these calculators generate their crop modules as single annual crops, which makes it difficult to display and to determine the effects of the crops on each other.", "For this reason, it seems challenging for the user to have to evaluate new energy crops and their effect at a specific position in the crop rotation and to model crop rotation effects, such as savings in operating resources (e.g. fertilizer, machinery use) and effects on yield.", "Neglecting nutrient shifts from one crop to the subsequent crop leads to free-rider situations for crops that consume nutrients left by preceding crops [18].", "Consequently, the amount of GHG emissions of the subsequent crop decreases, since the crop does not get charged for its true nutrient and fertilizer consumption.", "This points out the need to include the effects of crop rotation in CFP.", "Diverse crop rotations (including the use of catch crops or green manure) can help to reduce the CFP [19].", "Expanding the systems’ boundaries to consider the whole crop rotation could improve the CFP calculations, because in this way all crops (and thus the effects between them) are included in the CFP.", "However, most energy crop cultivation CFP studies are performed for one single crop and therefore for a specific product.", "Hence, the effort for including the whole crop rotation is often too high for users.", "For this reason, new LCA approaches to account for crop rotation effects in single crop cultivation assessment should be developed, such as the agricultural allocation approach developed by Brankatschk and Finkbeiner [18], and integrated in the existing calculator.", "In this paper, we identified 18 calculators for GHG emissions for energy crop cultivation that followed the CFP guidelines [11] and adopted the IPCC approaches [26] for calculating emissions from managed soils.", "However, using the same calculation guidelines does not guarantee the same accuracy of results across all calculators.", "Each calculator addresses different goals and user groups, and consequently has individual advantages and disadvantages.", "This is why users have to work out for themselves the balance between efficiency (time and input data) and accuracy (desired output) when deciding which calculator to use.", "The integrated methodology and default emission factors given by the calculator as well as the amount of farming processes included in an assessment correspond to the level of input data required.", "The main limitations in the assessment of energy crop cultivation management are the failure to account for LUC and to distinguish among fertilizer types including digestate, the lack of distinction among tillage types, and the lack of parametrization of many energy crops in the calculators.", "Furthermore, the impact on the CFP result by using regional GHG emission assessment methodologies is often overlooked.", "The ability of the calculators to detect GHG mitigation options through improvements in cultivation management is therefore limited.", "The methodologies used and the farming operations included in any study have a significant impact on the CFP results, thus emphasizing why CFP results should be carefully interpreted.", "Differences in integrated methodology and accuracy in energy crop cultivation management accounting make any comparison of results from current calculators virtually impossible.", "Only seven calculators are capable of calculating GHG emissions from perennial crops and from energy crops in rotation.", "This may be due to both a lack of methodological guidance to account for crop rotations (or an entire life cycle of a perennial crop, respectively) and a lack of focus on the agronomical specifics of crop rotations systems.", "Expanding the system boundaries of a CFP by taking into account the whole energy crop rotation increases the likelihood of identifying GHG mitigation options.", "However, currently, no reviewed calculator can process the effects from energy crops in rotation as nutrient shifts, reduction in use of agricultural operating needs, sequence and composition of crop rotations as well as integration of catch crops of green manuring.", "To overcome this shortcoming, existing calculators should be extended by integrating energy crop rotations, or new calculators and methods need to be created." ]	[]
Nigella sativa conserved hippocampal oxidative and neurogenic activities to salvage neuro-cognitive integrities in chlorpyrifos insult	Chlopyrifos (CPF)is an organophosphate, implicated in brain damage and cognitive deficits, whose consistence deposit in the environment has contributed to the toxicity burdens of xenobiotics. This study investigated the efficacy of Nigella sativa oil (NSO)against CPF insults on the hippocampus. Thirty-two rats were randomly divided into four groups of eight rats each, exposed to 1 ml/kg of Normal saline, 14.9 mg/kg of CPF, 14.9 mg/kg of CPF plus 1 ml/kg of NSO and 1 ml/kg of NSO respectively for 14 consecutive days. The rats were exposed to 3 trials each on the 11–13 days in the Morris water maze, and subsequently latency to hidden platform and time in the platform quadrant were recorded as measures of long term memory (LTM), short term memory (STM)and reference memory (RM)on the 14th day. The rats were euthanized on day 15, the brains excised, and the hippocampus of five brains removed, homogenized to analyze for total reactive oxygen species (ROS), nitric oxide (NO)levels and acetylcholinesterase (AChE)activities, while the other three were processed for histology and Ki67 immunohistochemistry. CPF caused a marked increase in hippocampal NO and ROS activities, depleted AChE activities and Ki67 expressions, delayed escape latency and reduced visit to the platform quadrant. Intervention with NSO depleted ROS/NO levels, improved neurogenic proteins, AChE activities and neuro-cognitive markers depletions in CPF exposure. Altogether, our findings showed that NSO is a potential therapeutic drug for the treatment of CPF-induced cognitive deficit through its antioxidant property and adult neurogenesis in rats.	[ "MULT" ]	[ "Acetylcholinesterase", "Cognition", "Nigella sativa oil", "Organophosphates", "Oxidative damage" ]	[ "1000" ]	[ "Organophosphates (OPs) are widely used synthetic chemicals that are found in both the domestic and agricultural sectors to assist with pest control [62].", "However, poisoning by these agents has been reported, particularly in developing nations, posing a serious public health problem with several cases reported each year [49].", "Chlorpyrifos (CPF), [O,O‑diethyl-o-(3,5,6-trichloro-2-pyridyl) phosphorothionate] is among the most effective and widely used organophosphate pesticide (OP) pesticides in the world [35,65].", "Acetylcholinesterase (AChE), an enzyme that hydrolyses acetylcholine (ACh) in the cholinergic synapses and in the neuromuscular junctions, is the primary target of CPF.", "It inhibits the enzyme, resulting in the accumulation of ACh in the synapses which results in hyperactivity in the cholinergic pathways [50,58].", "The build-up of ACh in the synapses has been implicated in many conditions, including convulsions, cognitive deficits, movement disorder, respiratory diseases, metabolic deregulations, and neurologic disorders [38,42].", "Nonetheless, it appears that AChE inhibition is not sufficient to result in severe, long-term cognitive deficits observed in CFP-treated rodents as other non-cholinergic pathways may be involved [14,60].", "Understanding other non-cholinesterase pathways of CPF would provide us with a better understanding of the therapeutic strategies for pesticides-induced cognitive impairment.", "In addition, available evidence suggests that CPF-induced toxicity also involved reactive oxygen species (ROS) generation resulting in oxidative stress in affected tissues.", "After oral administration of CPF accumulates in the brain causing long-term brain damage, neurotoxicity [32,59], and in different organs eliciting reproductive toxicity [19,36], hematotoxicity [18], developmental toxicity [13], cardiotoxicity [10], hepatic dysfunction [21].", "In the mammalian brain, the hippocampus is characterized as the primary structure for learning and memory formation [61].", "The dentate gyrus (DG) is responsible receiving and integrating sensory inputs from the entorhinal cortex to the CA3 then to the CA1 in the classical tri synaptic pathway [7], which is clinical for learning and memory [56,64].", "Each hippocampal subfield has specific cell types and plasticity contributing differentially towards the process of acquisition, storage and recall of learning and memory [46].", "Disruption of synaptic and biochemical architecture of different hippocampal subfields as a result of chemical exposure has been extensively implicated in the pathophysiology of cognitive deficits and emotional behavior-related disorders [24,44,45].", "Even though, there exist useful researches on the pathophysiology underlying the development of cognitive deficits, such as dementia, effective treatments remain elusive.", "Nigella sativa (NS), also called the black seed, belongs to the botanical family of Ranunculaceae.", "It is most found in Eastern Europe, the Middle East and Western Asia [20].", "Several studies revealed that NS seeds and oil can be used to treat a variety of diseases, pointing to some of its pharmacological properties.", "Most of its therapeutic efficacies have been strongly linked to composite bioactive compounds, including thymoquinone, alkaloids, riboflavin, piridoksin, niacin, folic acid, minerals and proteins [33,51,52].", "The reports of these pharmacological properties includes but not limited to the efficacy as antioxidant [2,31,32], anti-inflammatory [4], haematoprotective [3], neuroprotective [[27],[28,30]–[32],[34]], improvement of male infertility [40], efficacy in neurodegenerative diseases [5,16] and in memory enhancement [27,28,30].", "Here, we set out to investigate the efficacy of NSO in ameliorating CPF-induced oxidative and neurogenic damages, with subsequent effects on learning and memory functions in rats.", "Chlorpyrifos (PubChem Substance ID 329756699) PESTANAL®, analytical standard was purchased from Sigma (Sigma-Aldrich)(St. Louis, MO, USA), while the Normal saline solution was prepared in our laboratory.", "The Nigella sativa oil (concentration; 100% black seed; HUSNA black seed oil, Fazhab Agency, Karachi, Pakistan) was purchased from a TIBB-medical store in Ilorin, Kwara state, Nigeria.", "Thirty-two adult male Wistar rats weighing between 150 g and 170 g were obtained from the University of Ilorin Biological garden, Ilorin.", "They were housed in cages and fed with standard laboratory diet and water ad libitum, in the animal holding unit of the Faculty of Basic Medical Sciences, College of Health Sciences, University of Ilorin, Ilorin.", "The rats were exposed to a 12 h light/dark cycle at room temperature for 7 days before the commencement of the experiments.", "All rats were handled in accordance with the standard guide for the care and use of laboratory animals.", "The rats were randomly divided into four groups (n = 8) as follows:", "Group 1 (control)– were given Normal saline (1 ml/kg orally) daily", "Group 2– were given CPF (14.9 mg/kg orally) daily", "Group 3– were given CPF (14.9 mg/kg orally) plus NSO (1 ml/kg orally) daily [32]", "Group 4– were given NSO (1 ml/kg orally) daily [3,27,28,30–32].", "The experiments were all conducted in the morning (between the 07:00 and 09:00 hours), and treatments with substances span a period of fourteen consecutive days.", "This research work was approved by the University of Ilorin ethical review committee (UERC) (UERC/ASN/2017/856), following the recommendation of the College of health sciences ethical review committee, in compliance with the Institutional Animal Care and Use Committee (IACUC).", "The rats were subjected to behavioral evaluations on the 14th day of the treatment to assess short-term, long-term and reference memory in the Morris water maze (MWM) paradigm.", "The MWM apparatus in this study was used to assess the changes in memory indices following exposures to CPF and/or NSO in Wistar rats [43].", "Each of the rats (n = 32) were placed in a black, circular pool, which was filled with 23–24 °C water (pool dimensions: 60 cm deep × 136 cm diameter).", "The pool was divided into four quadrants, labeled north (N), east (E), south (S) and west (W).", "It contained a circular platform (10 cm diameter, 28 cm high) that was submerged (about 2 cm below water surface) in the central area of the SW quadrant of the pool.", "The rats swam until they found the platform (total time allowed on the platform = 15 s).", "If the subjects were unable to find the platform after 60 s of swimming, they were gently guided to the platform.", "The rats were then removed from the pool, dried and placed in their cage for 5 min.", "Each of the trials was recorded with the aid of a video system.", "Animals received a training session (three trials per session), for three consecutive days (days 11, 12 and 13 of the experiment).", "Each of the trials was a maximum duration of 60 s.", "The time interval between trials was approximately 30 s in duration.", "24 h after the acquisition phase, the time it took the subjects to find the hidden platform (referred to as ‘escape latency’) was recorded as long-term memory (LTM).", "An average of the escape latency of the two subsequent trials was recorded as short-term memory’ (STM).", "A probe test was also conducted by removing the platform and allowing the rats to swim freely in the pool for 60 s; the time spent in the target quadrant, which had previously contained the hidden platform, was recorded as the reference memory on day 14 of the experiment.", "The time spent in the target quadrant indicated the degree of relative memory consolidation, which had taken place after learning [27–30].", "Once the treatment period was completed, the rats were euthanized with an overdose of Ketamine (10 mg/kg ip) and their brains dissected and weighed.", "Blocks of hippocampal tissue (from Bregma −2.5 mm to −4.5 mm) were removed from the brains of five rats (from each of the four groups), dipped in 30% sucrose solution, homogenized and portions centrifuged at 2500 rpm for 10 min.", "The supernatant was then collected in tubes containing the compounds for NO metabolites and ROS analysis.", "ROS was measured by monitoring the increasing fluorescence of DCFH-DA following a previously described procedure using flow cytometry (Partec, Deutschland) equipped with a 488 nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530 nm band pass filter (FL-1 channel).", "Each determination was based on the mean fluorescence intensity of 10,000 counts [1].", "The remaining tissue homogenate was added to the Griess reagents, sulfanilamide and naphthyl ethylene diamine solutions to measure nitrate/nitrite production (NO metabolites).", "Absorbance was measured with the aid of a microplate reader and the levels of NO metabolites were calculated from a standard curve [9].", "The remaining portions of the homogenized hippocampal tissues were placed in phosphate buffer with 1% Triton-X 100 and centrifuged at 5000 rpm for 10 min.", "The following reagents were used; 35 μL of 5 mM dithio-bisnitrobenzoic acid (also known as Ellman's reagent [DTNB]), 10 μL of 75 mM acetylthiocholine (ATCh) and 50 mM phosphate buffer (pH 8.0).", "Protein concentration in brain homogenates was quantified using a Bradford assay.", "AChE activity was calculated in micromoles of ATCh, hydrolyzed per hour per milligram of protein and was expressed as percentage of control activity and measured values in micromole per hour per milligram of protein.", "After euthanasia and extraction of the three rat brains from each of the groups, the brains were fixed in 10% formalin for 24 h.", "The hippocampal blocks (from Bregma −2.5 mm to −4.5 mm) were removed, dehydrated through ascending grades of alcohol, cleared in xylene and embedded in paraffin blocks.", "Every second hippocampal tissue section (5 μm in thickness) was stained with Cresyl fast violet (CFV) for Nissl substances or immuno-stained to reveal Ki67 protein containing nuclei in the tissues.", "The sections were finally examined under an AmScope 40X-2500X LED Lab Compound Microscope, and photographed using the AmScope 5.0 MP USB Still Photo & Live Video Microscope Imager Digital Camera 5MP, manufactured by iSCOPE corp., USA.", "The Ki-67 is a chromosome-associated protein present during division (G1, S, G2, and M phases but absent from cells at rest, G0).", "Paraffin embedded sections were incubated for epitope retrieval in citrate buffer, pH 6.0, at 90 °C for 40 min, followed by incubation in endogenous peroxidase blocking reagent, 0.6% H2O2 in Tris-buffered saline (TBS)-Triton (0.05% Triton X-100 in TBS, pH 7.4) for 30 min at room temperature.", "Thereafter, sections were pre-incubated in 2% serum (normal goat serum) + 0.1% bovine serum albumin (BSA) + 0.25% Triton in TBS for 60 min at room temperature.", "The sections were then incubated with polyclonal rabbit-anti-lyophilized-Ki-67p antibody (Novocastra, Newcastle, UK; 1:5000 in pre-incubation solution) overnight at 4 °C.", "Incubation with biotinylated goat anti-rabbit IgG (1:1000 + 2% normal goat serum + 0.1% BSA in TBS; Vector lab, CA, USA;1:250) was performed for 2 h at room temperature followed by incubation with streptavidin-biotin complex (Vecta stain Elite ABC kit) and stained with 3,3′-diaminobenzidine (DAB) as chromogen.", "Until incubation with primary antibody, all rinses in between incubations were made with TBS-Triton, afterwards with TBS alone.", "Data from the morphometry, behavior and biochemical were analyzed using one-way analysis of variance (ANOVA) and subjected to post hoc Bonferroni's multiple comparison tests.", "The results are expressed as mean ±SEM.", "Statistical analyses were performed using Graphpad Prism software (version 5.0, La Jolla, CA).", "Values of p ≤ 0.05 were considered statistically significant.", "Exposures of rat to CPF resulted in a significant increase in the level of hippocampal NO when compared to control rats that received Normal saline.", "However, simultaneous administration with NSO significantly reverses the NO outburst to a level comparable to control group.", "Administration of NSO alone significantly (P ≤ 0.05) decreased NO levels when compared with the control and the CPF alone treated rats (Fig. 1).", "CPF exposure caused a marked increase (p ≤ 0.05) in hippocampal ROS activities when compared to controls, however, a simultaneous treatment with NSO showed the depletion of ROS significantly, which was comparable with control group that received Normal saline.", "Rats given NSO only had total ROS level comparable to control group (Fig. 2).", "CPF treated rats showed a significant (p ≤ 0.05) depletion in the hippocampal AChE activity compared to normal control rats and the NSO treated rats.", "Co-administration of NSO with CPF resulted in a considerable but non-significant reactivation of the depleted AChE activities in the hippocampus of the CPF exposed, while a significant (p ≤ 0.05) improvement was observed in the NSO only treated rats compared to the control and other groups (Fig. 3).", "CPF exposure significantly (p ≤ 0.05) caused a delay in the escape latency (STM and LTM), the time required to reach the submerged hidden platform in the Morris water maze (MWM) task, when compared with the saline treated control and the NSO only treated rats.", "Interventional treatment with NSO, alone or in concurrent exposure with CPF, significantly (p ≤ 0.05) shortened the escape latency in the treated rats.", "During the probe trial, CPF rats failed to retrieve the precise location of the hidden platform and spent significantly (p ≤ 0.05) less time in the target quadrant compared to the control group (p ≤ 0.05).", "However, those groups treated with NSO however, spent more time on average in the target quadrant searching for the hidden platform.", "(Figs. 4, 5 and 6)", "Exposure to CPF caused no marked deterioration in the general integrities of the hippocampal subfields (CA 1, 2, 3 and DG), but few necrotic like vacuolations in the cornuammonis pyramidal cells were noted, these effects were however in minimal in the CPF + NSO group.", "Similarly, a marked loss in density and denaturation were observed in the neurogenic proteins (Ki67 + ) cells in the hippocampal subfields, the dentate gyrus and the subventricular zone of the CPF exposed rats, but this was however prevented by NSO intervention (Fig. 13).", "Excessive exposure to CPF has been reported to result in cognitive deficit, depression and anxiety in rodents via oxidation and massive AChE inhibition [11,15,32,57,58].", "To date, effective treatment of cognitive deficit induced by environmental toxic agents still remain a challenge to neuroscience community.", "Neuroprotective measures that can prevent and stop progression of neuronal damage cause by toxic agents would go a long way in improving cognitive performance and other neurological disorders observed in affected patients.", "In this study, we assessed the potential efficacy of NSO, a known antioxidant, in the prevention of CPF-induced cognitive impairment and its possible mechanism.", "By combining biochemical, immune-histochemical and behavioral assay, we showed that NSO efficiently improved cognitive performance in CPF-induced cognitive deficit by increasing antioxidant capacity, anti-inflammatory activity and increased proliferation of adult-born granule cells via adult neurogenesis, evident by Ki-67 expression in the dentate gyrus of NSO treated rats.", "We recently demonstrated that CPF administration resulted in body and brain weight loss, which are considered as indirect markers of metabolic functions, and the recorded oxidative damage was implicated therein [32].", "A similar phenomenon was also observed in the work of [54] and [67] where CPF altered body weight negatively, a change that we herein attribute to the oxidative damage, and confirmed as a possible trend from other OPs [23,55].", "Based on previous reports that have implicated oxidative stress as the major underlying mechanism of CPF-induced toxicity [8,53], we assessed the effect of CPF on total hippocampal ROS.", "Administration of CPF significantly increased the ROS level in the CPF treated groups compared to the control.", "In our study, a marked increase in total ROS in the hippocampal tissues of the CPF exposed rats was recorded, suggesting damage to the cellular membrane of the major cells thus leading to the leak and subsequent oxidative damages in the hippocampus.", "This finding can be strengthened with previous reports that submitted that poisoning (inhalation or oral) from CPF exposure causes oxidative damages and more implicated is the high levels of ROS in the exposed animals [6,32,37,63].", "But, in an interesting outcome, co-administration of NSO with CPF significantly prevented a rise in the level of ROS while the administration of NSO alone maintained an hippocampal ROS level that is comparable to the saline treated control.", "This result further confirmed oxidative stress as one of the mechanism of CPF cytotoxicity.", "In addition, it also showed the efficacy of NSO antioxidant property in combating environmental toxic agent-induced oxidative stress [31,32].", "Furthermore, we evaluated the protective effect of NSO against CPF-induced enhancement of inflammatory marker, have been implicated in the pathophysiology of many neurodegenerative diseases and in neuro-inflammation during systemic inflammation, nitric oxide, NO [66,68], in the hippocampus.", "Animals treated with CPF alone showed a significant increase in the elevation of hippocampal NO, an indication of neuro-inflammatory activities which has been implicated in various neurological disorder [47].", "It was recently reported that, increased neuro-inflammation in the hippocampus resulted in pronounced poor cognitive performance [25], and the induced increase in hippocampal NO by CPF in this study, further suggested it promotes neuro-inflammation.", "Concomitant administration of NSO however, prevented elevation of hippocampal NO levels in the exposed rats, and consequently inferred to reduce neuro-inflammation.", "This data is suggestive of NSO a potent anti-inflammatory, and may be further explored against neurological insults.", "AChE being an enzyme involved in the hydrolysis of ACh at cholinergic synapses, is said to be essential for modulation of neurobehaviors such as learning, memory and movement [17].", "AChE inhibition has been largely reported after CPF administration in rats and suggested as one of the main mechanisms of CPF-induced impaired cognitive performance [48].", "Consistently, our data showed that CPF administration caused a significant reduction in the hippocampal AChE activity.", "Co-administration of NSO with CPF slightly increased hippocampal AChE activity but could not return to the level found in control rats.", "This observation could be due to fact that NSO has less effect on AChE activity or CPF-induced AChE inhibition might be largely irreversible [15].", "However, cholinergic pathway inhibition is said not to be sufficient to produce marked memory deficit observed in CPF treated rats, as other non-cholinergic pathways have been reported to be involved in the modulation of learning and memory.", "We hypothesized that CPF also altered the generation and proliferation of new born neurons in the sub-granular layer of the DG, and subsequently prevented integration of new neurons into learning and memory circuitry resulting in cognitive deficits.", "CFP administration substantially reduced Ki67, a proliferating cell marker and adult neurogenesis, expression in the SGZ which indicated a reduction in the proliferation of adult born neurons.", "Interestingly, rats treated with NSO and CPF showed high expression of Ki67 in the SGZ.", "It is worthy to note that rats given NSO only show significantly higher expression of Ki-67 compare to the controls, indicating NSO is capable of improving cell proliferation in the DG and consequently integration of the adult born new cells into memory and learning circuitry.", "The higher number of Ki-67-immunopositive cells suggests a larger population of dividing progenitor cells [41].", "Learning and memory are the main function of the hippocampus, and these cognitive functions have been reported to be modulated by adult hippocampal neurogenesis in rats [22,39].", "Good learner mice are reported to have high rate of neurogenesis in the adult DG [12].", "Hippocampal neurogenesis has been observed throughout the adult lives of many mammals studied to date [12].", "New neurons in the adult hippocampus originate in the progenitor cells located at the border between the hilus and GCL, which is a region called the sub-granular zone (SGZ) of the DG.", "In conformity with the oxidative damages and ChE dysfunctions enumerated above, coupled with the loss in Ki67 immunoreactive nuclei in the hippocampus, the spatial memory assessed using MWM showed that CPF exposed rats had poor performance in spatial learning as they spent more time in locating hidden platform, and spent less time exploring the platform quadrant during the probe test.", "These activities were significantly mitigated by intervention with NSO, in the CPF and NSO co-administrated rats.", "Consistent with the Ki67 expression in the SGZ in the NSO treated rats, NSO only treated rats showed improve cognitive performance in LTM, STM and reference memory.", "Altogether, our neurobehavioral assessment showed that NSO administration significantly improved the spatial learning and reference memory possibly via improved integration of adult born cells into functional hippocampal circuitry.", "These effects can be supported by its reported potency in enhancing indices of neurocognitive and psycho-cognitive functions in models of neurodegenerative disorders and neurotoxicity [26,30].", "The data presented in this study showed that exposure to CPF caused impaired cognitive performance via multifaceted pathways including oxidative damages, AChE inhibition and reduced potential adult neurogenesis in the hippocampus of rats.", "Interestingly, interventional treatment with NSO shortly after CPF insult mitigated and improved all the neurocognitive indices which may possibly be via the preservation of adult neurogenesis and depletion of stressor markers in the hippocampus of the treated rats.", "Altogether, our findings revealed NSO supplementation as a potentially effective strategy in combating pesticide-induced cognitive deficits." ]	[]
Competition for Space Is Controlled by Apoptosis-Induced Change of Local Epithelial Topology	During the initial stage of tumor progression, oncogenic cells spread despite spatial confinement imposed by surrounding normal tissue. This spread of oncogenic cells (winners) is thought to be governed by selective killing of surrounding normal cells (losers) through a phenomenon called “cell competition” (i.e., supercompetition). Although the mechanisms underlying loser elimination are increasingly apparent, it is not clear how winner cells selectively occupy the space made available following loser apoptosis. Here, we combined live imaging analyses of two different oncogenic clones (Yki/YAP activation and Ras activation) in the Drosophila epithelium with computer simulation of tissue mechanics to elucidate such a mechanism. Contrary to the previous expectation that cell volume loss after apoptosis of loser cells was simply compensated for by the faster proliferation of winner cells, we found that the lost volume was compensated for by rapid cell expansion of winners. Mechanistically, the rapid winner-dominated cell expansion was driven by apoptosis-induced epithelial junction remodeling, which causes re-connection of local cellular connectivity (cell topology) in a manner that selectively increases winner apical surface area. In silico experiments further confirmed that repetition of loser elimination accelerates tissue-scale winner expansion through topological changes over time. Our proposed mechanism for linking loser death and winner expansion provides a new perspective on how tissue homeostasis disruption can initiate from an oncogenic mutation.	[ "AGRI", "BIOC" ]	[ "Hippo", "Ras", "cell competition", "cell intercalation", "cell packing", "differential growth", "epithelial tissue", "tissue mechanics", "tumor progression", "vertex model" ]	[ "1100", "1300" ]	[ "Primary tumors originate from a single transformed cell containing oncogenic mutations despite being spatially confined by surrounding normal tissue.", "It is increasingly apparent that oncogenic cell spread within tissue is driven not only simply by a higher rate of proliferation, but also by a phenomenon called “cell competition” [1, 2].", "Cell competition is a process in which “winner” cells take over the tissue at the expense of neighboring “loser” cells [1–7].", "When oncogenic mutant cells act as winners, the mutant cells eliminate surrounding normal wild-type cells by inducing apoptosis, and thus spread their territory.", "Because inhibiting loser apoptosis is sufficient to halt winner spread [1, 2, 5, 8, 9], loser apoptosis seems to play an active role in promoting winner spread within the tissue.", "However, it is unclear how loser apoptosis impacts surrounding cells to produce selective occupation by winner cells.", "Cell competition can be considered as processes of destruction (loser apoptosis) and reconstruction (winner takeover) of the epithelial tissue structure [1–20].", "Epithelial tissue consists of a sheet of tightly packed cells connected with adhesive junctions.", "This local cell-cell connectivity (“cell topology” [21, 22]) is represented by a distribution of the number of sides (adherent neighbors) of each cell.", "Because the side number positively correlates with the apical surface area (known as Lewis’s law conserved in epithelial tissue [22, 23]), topological change is associated with cellular expansion or contraction.", "In the case of cell competition, the topology of neighboring cells could be changed through loser cell delamination or extrusion, which associates consecutive junctional remodeling (cell intercalation) between a dying cell and its surviving neighbors [7, 24].", "Topological change driven by loser elimination might therefore allow winner cells to spread with increasing their side number.", "This idea prompted us to investigate whether cell topology changes in the vicinity of apoptotic cells contribute to winner takeover during competition.", "Using generalized mechanical simulations and in vivo imaging of Drosophila oncogenic clones, we show when and how winner cells selectively expand to compensate for local tissue volume loss after apoptosis of a loser cell.", "Following induction of locally overgrowing winners, slowly dividing normal cells adjacent to rapidly dividing winners show anisotropic distortion, as reported previously [25–28].", "Immediately following apoptosis of the anisotropic normal cell, the space previously occupied by the apoptotic loser cell was filled by rapid cell expansion of adjacent winner cells.", "The winner cell expansion was unexpected, because previous studies mostly focused on cell division as the compensatory mechanism to fill space (e.g., “compensatory proliferation”) [17, 29–32].", "As a mechanism underlying this compensatory cell expansion of winners, we found that apoptosis of anisotropic losers adjacent to winners causes directionally biased cell intercalations, which re-establishes local cellular connectivity (cell topology) in a manner that selectively increases the apical surface area of winners.", "Importantly, such topological dynamics were verified with in silico and in vivo experiments in two different oncogenic genotypes (activated-Yki and Ras), where the overgrowing oncogenic population triggers apoptosis in surrounding wild-type cells.", "Furthermore, repetition of topological changes through the apoptosis of losers in immediate contact with winners, as observed in many cell competition systems [33], accelerates the tissue-scale expansion of winners in silico.", "Such rapid cell turnover via topological changes may disrupt tissue homeostasis to facilitate tumor initiation.", "To examine the cellular interaction during cell competition from a geometric perspective, we induced clones of GFP-expressing Hippo pathway mutant cells (winners; wtsX1) that rapidly proliferate and outcompete surrounding normal cells (losers; wild-type) in Drosophila wing discs [6].", "Quantitative image analysis of apical cell shapes revealed two geometric alterations.", "First, wild-type cells directly adjacent to wtsX1 clones showed significantly larger anisotropic distortions (Figure 1A, orange) than those directly adjacent to wild-type control clones (Figure 1A, blue and 1B; see STAR Methods for the fly strains and cell anisotropy definition), which was consistent with earlier reports [26, 27].", "Second, the apical cell area of wtsX1 clones was smaller than that of wild-type clones (Figure 1C), indicating that Hippo pathway mutant cells surrounded by wild-type cells are packed at a higher density.", "We next examined whether the ectopic induction of over-proliferating cells was sufficient to cause these two geometric alterations (Figures 1B and 1C) in the vertex model.", "The vertex model is widely used to study mechanical deformation of multicellular tissue in response to cell bond tension and apical area contraction [22, 34–37] (Equation 1 in STAR Methods, Figures S1A and S1B).", "We introduced two clonal cell populations with different division rates into the model (Figures 1D and S1C).", "We confirmed anisotropic deformation of slower dividing cells localized at the clone boundary (Figures 1D, middle, 1E, and S1D), which is consistent with the wild-type cells directly adjacent to the wtsX1 clone in the Drosophila experiment (Figure 1B) and earlier simulation studies [26, 28].", "In addition, faster dividing cells were smaller than surrounding slower dividing cells (Figures 1D bottom, 1F, and S1E), similar to the wtsX1 cells in the Drosophila experiment (Figure 1C).", "These results were consistent irrespective of different mechanical parameter values (Figures S1F and S1G) or formulations (another often used type Equation 2; Figures S2A–S2C).", "These deformations were never observed when two cell populations had identical division rates (Figure 1D, leftmost), and they were more apparent with greater differences in cell division rates (Figures 1E and 1F).", "These simulations suggest that differential proliferation rates are sufficient to reproduce two geometrical alterations in the Drosophila epithelium (Figures 1B and 1C).", "We also examined the anisotropic cell deformation in the setting of differential tension between clonal and non-clonal cells since juxtaposition of wild-type and aberrant cells often affect junction mechanics [38, 39].", "In silico experiments with all tension conditions showed that slower dividing cells at the clone boundary always exhibited higher anisotropy than those with identical proliferation rates (Figures S3A–S3C), demonstrating that differential tension does not have considerable influence on the cell anisotropy.", "Consistently, in vivo observation showed that the wild-type cell anisotropy beside clone was maintained even under modulation of junction contractility or adhesion in Hippo pathway mutant clones (Figures S1H and S1I).", "Collectively, these results suggest that localized over-proliferation induces anisotropic deformation in slower dividing cells at the clone boundary under differential tension.", "Live imaging of cell competition was performed [7, 13, 16, 18, 20] in the Drosophila pupal notum to examine how space previously occupied by apoptotic loser cells is replaced.", "We induced faster dividing winner clones overexpressing an activated Yki (UAS-yki S111A,S168A,S250A:V5; hereafter called Yki clones) [40], a transcriptional co-activator that acts downstream of the Hippo pathway (Figure 2A, left; Video S1).", "We observed that wild-type loser cells near Yki winner clones progressively took on anisotropic shapes (Figures 2A, right, and S1J), especially those between the Yki clones.", "Both anisotropic and isotropic loser cells were subsequently eliminated from the tissue (Figure S1J) by sequential cell intercalations from the shorter edges of a dying cell (Figures 2C–2E; Video S2).", "Although this elimination was observed at several cell diameters away from the clones, the majority of cell elimination occurred in cells immediately contact with Yki clones (Video S1).", "Upon repetitive loser elimination (apoptosis; see STAR Methods for the definition of “apoptosis” in this paper), Yki winner cells beside the clone boundary appeared progressively larger (Video S1).", "At the level of individual apoptosis, winner cells adjacent to apoptotic cells selectively replaced the space originally occupied by apoptotic loser cells through cell expansion; the area of faster dividing winner cells surrounding a dying cell increased, whereas the change in the area of slower dividing loser cells was negligible (Figures 2G and 2H).", "Another oncogenic winner-clone induced by an activated form of Ras (UAS-RasV12), which promotes cell growth and outcompetes surrounding wild-type cells in Drosophila [7, 42], also induced anisotropic distortion in wild-type cells at the clone boundary (Figures 2B and S1J).", "The RasV12 winner cells expanded to compensate for wild-type loser cell apoptosis (Figures 2F and 2I).", "These results indicated that winner compensatory cell expansion was commonly observed upon induction of two oncogenic mutants, Yki and RasV12, which drive localized overgrowth through promoting proliferation or cell growth.", "The compensatory cell expansion of winners following apoptosis was unexpected considering the accepted hypothesis that lost territory is replaced as a result of winner-cell proliferation [29–32].", "Moreover, we found that the winners’ expansion was mediated by the anisotropic deformation of dying loser cells (Figure 1); the acquired area of Yki or RasV12 winner cells positively correlated with anisotropy of dying loser cells (Figures S4A and S4J) that were oriented in parallel to the clone boundary (hereafter we call “clone-boundary-oriented anisotropy”; Figures 2J–2L and S4J).", "Z projection with adherens junctions [E-cad::GFP; white (left), magenta (right)] and clones overexpressing Yki:V5S111A,S168A,S250A [green (right)] shown in Figures 2A and 4I.", "Almost all of the apoptotic cells [blue (left)] were wild-type cells near the clone boundary.", "Upon repetitive apoptosis of the wild-type cells, compacted Yki-overexpressing cells, which might be due to their rapid proliferation, spread as increasing the apical surface area of cells located beside the clone boundary.", "Note that both anisotropic and isotropic loser cells underwent apoptosis.", "Time (hr) after puparium formation is shown in upper left.", "An example of delamination shown in Figure 2C.", "Time (mins) is shown in upper left.", "We then applied a vertex model to determine whether winners used cell expansion to compensate for the volume lost after apoptosis of anisotropic loser cells.", "We incorporated apoptosis into the simulation based on the following experimental observations (Figure 2): (1) the target of apoptosis was slower dividing cells directly adjacent to faster dividing clones, (2) dying cell removal was induced by junctional loss due to sequentially introduced cell intercalations [7, 24] from the shorter edges of dying cells (Figure 3A; Video S3, see STAR Methods for apoptosis in simulations).", "Consistent with our previous experiments, the surrounding winner cells dominated expansion following apoptosis (Figure 3B).", "This winner-cell expansion was repeatedly observed in independent simulations that separately induced apoptosis in each individual loser cell at the clone boundary under differential proliferation rates, but it did not occur in the setting of identical proliferation rates (Figures 3C and S5A).", "This winner-cell expansion was consistently observed in several different model setups (formulation [Equation 2; Figure S2D], delamination process [Figures S3E–S3G], and differential tension conditions in clonal and non-clonal cells [Figure S3D]).", "Moreover, consistent with in vivo experiments, the rapidly dividing winner cells increasingly expanded as clone-boundary-oriented anisotropy of dying loser cells increased (Figures 3D, S2E, and S3H).", "We note that the rapid proliferation of winners in itself was not directly involved in the compensation process following apoptosis, because winner cells took over the territory of apoptosis despite ceasing all cell proliferation during the apoptotic simulation (Figure S3J).", "Therefore, we concluded that the emergent property under differential proliferation (i.e., local anisotropic distortion of apoptotic loser cells [Figure 1]), rather than differential proliferation in itself, has a key role in the compensatory expansion of winner cells.", "An example of delamination in simulation shown in Figure 3A.", "Green or light green cells are faster dividing; gray or white cells are slower dividing cells.", "Green and gray cells locate directly in contact with a dying cell at the onset of apoptosis (magenta).", "We then focused on cells immediately adjacent to an apoptotic cell to examine their contribution to winner-dominated cell expansion, because the territory lost to apoptosis was primarily occupied by the immediate neighbors, and cells located more than two cell distances away from the apoptotic cell acquired much smaller area (Figure S3I).", "We initially hypothesized that rapid winner-cell expansion was the result of a local compression difference between winner and loser cells surrounding the apoptosis (compression hypothesis; Figure 4A), because more compressed cells (Figures 1C and 1F) may generate stronger outward pushing forces that are enough to increase their cell area.", "To address this hypothesis, we tested whether compressed smaller cells adjacent to apoptosis were more likely to expand than larger cells.", "This hypothesis, however, seems not to be the case, because we observed that the cell area before apoptosis and their acquired area after apoptosis only correlated weakly, both in silico (Figure 4B) and in in vivo experiments with Yki and RasV12 (Figures 4C and 4D).", "Additionally, in simulation, winner cells still took over the territory of apoptotic losers even if the tissue-scale pushing forces globally generated by release of winner-clone compression was suppressed during apoptosis by reducing the pressure parameter of all cells in winner clone (Figure S3J).", "These results suggested that local and global compression was insufficient to drive the process of rapid compensatory expansion of winner cells, while winner compression was required for anisotropic deformation of loser cells (Figure 1).", "As another hypothesis that locally mediates expansion, we focused on changes in local cellular connectivity (cell topology) driven by junctional remodeling (cell intercalation) upon apoptosis (Figures 2C and 3A).", "In epithelial cells in non-competitive tissue, there is a positive correlation between cell area and the side number representing local cellular connectivity (Lewis’s law; Figure S1B) [22, 23, 43].", "Based on this Lewis’s law, an increase or decrease of side/edge number in each cell adjacent to apoptosis would be associated with cell area expansion or contraction (topology hypothesis; Figure 4E).", "We first confirmed that both winner (Yki or RasV12) and loser (wild-type) cells in competitive tissues obey this law (Figures 4I, 4J, S4D, and S4E), similar to non-competitive normal tissue.", "We then examined the topological changes following apoptosis and found that cells adjacent to apoptosis showed a strong positive correlation between the captured area and change in side number after apoptosis both in silico (Figure 4F) and in in vivo experiments with Yki and RasV12 (Figures 4G and 4H).", "This suggests that cell intercalations during delamination lead to simultaneous increases in the side number and area of winner cells.", "These results prompted us to further test the topology hypothesis as a possible mechanism for winner-cell compensatory expansion.", "To understand how intercalations associated with apoptosis selectively increase winner side number and area, we first analyzed where cell intercalations took place relative to the clone boundary.", "Pupal notum junctions are reportedly subject to stochastic fluctuations of edge length that trigger cell intercalations by chance as edge lengths decrease to zero [44].", "This framework suggests that shorter junctions are more likely to undergo intercalation, and our in vivo observations consistently showed a higher probability of intercalation at shorter edges (Figures 2E and 2F).", "In anisotropic dying cells caused by differential proliferation rates (Figure 1), edges perpendicular to the clone boundary were shorter than other edge types (Figures 5A–5C, S4B, and S5B; see Figure 5D for edge classification), suggesting that these perpendicular edges were more likely to be eliminated by cell intercalation.", "Indeed, both in silico and in vivo experiments with Yki and RasV12 indicated that cell intercalation likely occurred in a manner to eliminate the perpendicular edges (perpendicular intercalation) during anisotropic loser cell delamination (Figures 5F–5H, S2F, S4C, and S5C; see Figures 5D, 5E, and S4F–S4I for the definition of normalized frequency of cell intercalation).", "Moreover, the increased frequency of perpendicular intercalation during an apoptotic event increased the side number and area of winner cells after apoptosis, but not that of loser cells (Figures 5O–5Q, S2I, S4M, and S5F).", "We did not observe this effect for the two other types of intercalation that occurred at boundary and non-boundary edges (Figures 5I–5N, S2G, S2H, S4K, S4L, S5D, and S5E).", "Thus, perpendicular intercalation frequency during each apoptosis event uniquely correlates with selective increases in winner side number and occupied area.", "We next asked how the perpendicular intercalation selectively drives winner-cell expansion.", "Of the four cells involved in intercalation upon apoptosis (one dying cell and three surviving cells; Figure 6A, upper), two surviving cells gain a single side, whereas the other surviving cell and the dying cell each lose a side after an intercalation event.", "Based on this rule, we comprehensively calculated topology changes (side number) in winner and loser cells for all geometrically possible intercalations (Figure 6A, table).", "For example, in a subtype of perpendicular intercalation (second row from bottom of table in Figure 6A), a winner cell increases side number by one, whereas each of two loser cells increases or decreases by one, respectively, and therefore these two cancel out the change in side number of losers in total.", "Notably, all the subtypes of perpendicular intercalation uniquely increase winner-cell side number and area in total, whereas those of boundary and non-boundary intercalations mainly increase for loser cells in total (Figure 6A, table).", "In silico experiments were performed to determine whether perpendicular intercalation is sufficient to drive winner-dominated cell expansion.", "In the simulation, we introduced a directionally biased event of boundary, non-boundary, or perpendicular intercalation upon apoptosis under identical proliferation conditions (Figures 6B and S5I) where intercalation is typically unbiased (Figures S5G and S5H).", "This allowed us to examine the contribution of directionally biased intercalation to winner-cell expansion (topology hypothesis; Figure 4E) in the absence of winner compression (compression hypothesis; Figure 4A).", "By introduction of biased perpendicular intercalation during apoptosis, clonal cells directly in contact with apoptosis consistently increased both their side number and area averaged for each apoptotic event, whereas non-clonal cells in direct contact with apoptosis did not (Figure 6B, right).", "In contrast, neither side number nor area of clonal cells increased upon the prioritized induction of boundary and non-boundary intercalations (Figure 6B, left and middle).", "This indicates that frequent perpendicular intercalation upon apoptosis directly increases side number and area in winner cells.", "Finally, we examined the long-term topological change upon sequential induction of apoptosis in multiple loser cells at clone boundaries (Figure 7A).", "After simulating repetitive apoptosis where intercalations occur from shorter edges according to in vivo regular rule as shown in Figures 2E and 2F, winner clones took over almost 100% of the total cell area that was lost to apoptosis (Figure 7D, purple), and consequently, winner cells spread more rapidly than in the absence of apoptosis (middle and left panels of Figure 7B and Video S4; Figure 7C, purple and black).", "This long-term expansion of winner cells was ensured by stably maintaining anisotropy of loser cells during multiple apoptosis events (Figure 7E) in which loser cells that were newly faced with clone boundaries after apoptosis subsequently became anisotropic (Figure S6).", "Due to the maintenance of a high-degree of cell anisotropy, the frequency of perpendicular intercalation remained high during apoptosis (Figure 7F).", "This allowed winner cells at the outermost layer of the clone persistently increased their side number and area for a long-term during clone development (Figures S7A–S7C).", "Additionally, winner cells inside clones (i.e., more than two cell distances away from the clone boundary) also slightly expanded on a longer timescale (Figure S7E, inside) without changing side number (Figure S7D, inside), possibly due to an accumulation of minor relaxations of mechanical compression (Figure S3I, acquired area of winner cells localized to two- or three cells away from apoptotic cells).", "We note that this increase in cell area by mechanical relaxation was relatively modest compared to the dramatic area expansion as a result of cell topological change at the clone border (Figures S7D and S7E).", "These results suggest that topological change driven by frequent perpendicular intercalation is still critical for long-term winner-clone spreading.", "Accordingly, we incorporated random directionality into intercalations associated with apoptosis under differential proliferation rates to assess whether winner-clone spreading is disrupted by the unbiased occurrence of three intercalation types (randomized intercalation; right panels of Figure 7B; Video S4).", "We found that clone spreading speed was reduced (Figure 7C, light-blue) due to equal and simultaneous increases in the side number and cell area of both winner and loser cells (Figures S7B and S7C, bottom panel).", "As such, the net increase in winner-clone area after loser apoptosis was reduced to approximately 50% of the total area lost to apoptosis (Figure 7D, light blue), confirming that topological change at the clone boundary significantly contributed to winner-clone spreading.", "Without apoptosis (left), with apoptosis where intercalations occur from shorter edges according to in vivo regular rule (middle; regular intercalation), and with apoptosis where three intercalation types randomly occur with equal probability (lower; randomized intercalation).", "Change in number of sides (upper).", "Log of ratio of the final cell area over initial cell area (lower).", "See also Figure 7B.", "Because topological change due to apoptosis is limited to cells in direct contact with apoptotic cells, loser death restricted nearby winner clones, as seen in many cell competition systems [33], would promote winner-clone spreading.", "We examined in silico whether the restricted death actually improves winner expansion by comparing it to a scenario in which apoptotic cells were spatially scattered in slower dividing cell population.", "We found winner clones most effectively occupied the apoptotic territory when loser apoptosis was restricted to the clone boundary (Figures S7F–S7H, up to 1-cell distance).", "On the other hand, occupation efficiency was reduced when apoptosis had a more scattered spatial distribution (Figures S7F–S7H, up to 3- or 5-cell distance) and further dropped down to baseline (no apoptosis) when death had a completely random distribution (Figures S7F–S7H, all slower dividing cells).", "Collectively, we can conclude that repetitive cell topology changes due to frequent perpendicular intercalation at the clone periphery accelerates tissue-scale occupation by winner cells.", "It has long been acknowledged that loser apoptosis is limited to within several cell diameters away from winner clones in many cell competition systems [33].", "However, it was unclear how this localized apoptosis influences subsequent winner tissue occupation.", "Here, we showed that localized apoptosis adjacent to the clone boundary efficiently accelerates winner-clone expansion through cell topological changes, indicating that regulating topology might have a major contribution to tissue takeover by winners (Figures 7 and S7).", "We also identified the mechanisms underlying these topological alterations (Figure 7G): anisotropic loser cells driven by localized overgrowth (Figures 1, 2A, and 2B) undergo apoptosis, producing a non-cell-autonomous increase in the side numbers and areas of surrounding winner cells (Figures 2 and 3).", "This topological alteration was verified with in silico and in vivo experiments in two different oncogenic genotypes (activated-Yki and Ras), which targets different signaling pathways but commonly show overgrowth phenotype.", "These results indicate their common feature, differential growth, may be the origin of the topological dynamics leading to winner-cell expansion, while Yki can be activated by Ras signaling [45, 46] and winner-cell expansion may be driven at least in part through Yki activation.", "We further showed in silico and in vivo that such topological dynamics leading to selective winner expansion is regulated by directionally biased cell intercalation (Figures 5 and 6), which occurs as a consequence of perpendicular edge shortening in anisotropic apoptotic cells (Figures 2E, 2F, and 5A–5C).", "Although polarized actomyosin-dependent mechanical tension is well known to bias intercalation direction in many morphogenetic processes [47, 48], the Hippo pathway mutant clone in the Drosophila epithelium did not accumulate myosin II or increase tension at the radial junction (i.e., perpendicular edge) but did at the circumferential junction (i.e., non-boundary edge) of wild-type cells surrounding clones [26, 27].", "The compensatory expansion of winner cells upon apoptosis should therefore be achieved by stochastic junction loss at the shorter perpendicular edges rather than greater tension due to active myosin enrichment.", "In addition to perpendicular intercalation, our theoretical analysis indicates that a specific boundary intercalation subtype increases winner-cell side number and area following apoptosis (top row of table in Figure 6A).", "Because the subtype only occurs when all surviving cells involved in the intercalation were winners, it should be frequent when an apoptotic cell shares more sides with winner cells.", "This situation in which the boundary intercalation subtype is more frequent than perpendicular intercalation might be rare in Yki- or Ras-activating clones with fewer shared sides due to cell sorting [38], but frequent in Myc- or minute-dependent competition, where winner and loser cells actively mix to increase their shared sides [17, 20, 49].", "Moreover, regarding the expansion process, intercalation-mediated space filling does not require cell division (Figure S3J).", "This fact is supported by live notum imaging that shows winner expansion even after growth arrest (approximately after time = 28.5 in Video S1).", "Therefore, intercalation-mediated winner expansion might contribute to the competition scenario without proliferation (e.g., post-mitotic tissue [50]).", "Future examination of the relative contributions of intercalation subtypes in various cell competition scenarios would clarify the topological dynamics that lead to selective winner expansion.", "Given the knowledge that a striking increase in cell apical area stimulates proliferation through feedback regulation by mechanical signaling pathways in non-competitive tissue [51, 52], we expected that the dramatic increase in winner-cell area that follows loser apoptosis (Figure 7) would facilitate winner proliferation.", "As such, the increase in cell area by topological change is transformed into the increase in cell number, which could further accelerate long-term clone expansion.", "It was recently suggested that mechanical or morphological consequences of cell loss affect the division orientation of winner cells, which colonize lost territory [17, 49].", "Therefore, examination of the contribution of positive feedback on winner division rate and orientation in silico [36] and in vivo would provide a more comprehensive understanding of winner-clone spreading.", "Although it was difficult to detect this positive feedback effect on cell division with our live imaging in notum, due to growth arrest during terminal differentiation, it will be interesting to monitor the contribution of cell expansion to division in actively growing tissue.", "Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Koichi Fujimoto ([email protected]).", "Strains used for imaging fluorescently-labeled mitotic clones [56, 57] in fixed larval imaginal discs: UbxFLP; Act > y+ > Gal4, UAS-GFP; FRT82B, Tub-Gal80 (82B tester), UbxFLP; FRT42D, Tub-Gal80; Act > y+ > Gal4, UAS-GFP (42D tester), UbxFLP; Tub-Gal80, FRT40A; Act > y+ > Gal4, UAS-GFP (40A tester) [53].", "Additional strains used were as follows: wtsX1 (from T. Xu), hpo42-47 (from D.J. Pan), UAS-sqh-RNAi (P{TRiP.HMS00437}attP2) (Bloomington Stock Center 32439), UAS-shg-RNAi (P{GD14421}v27081) (Vienna Drosophila Resource Center #27081).", "Strains used for notum live imaging: y, w, hs-flp; DE-Cad::GFP; Act > CD2 > GAL4, P{UAS-CD8.", "mCherry}, p{tubP-GAL80ts}20 (Bloomington Stock Center 7019), UAS-ykiS111A,S168A,S250A:V5 (Bloomington Stock Center 28817), UAS-RasV12 (from T. Xu).", "Crossed offspring for experiments carried a single copy of the transgenes as shown in Experimental genotypes.", "Both male and female larvae and pupae were used for experiments.", "Fly stocks were kept in vials or bottles at 18 or 25°C and transferred every 4 or 3 weeks, respectively.", "Fly food is a standard corn-sugar-yeast recipe.", "For cross experiments, healthy adult virgin females and males were placed in a vial and flipped every 1–3 days, and vials containing offspring were raised at 18, 25, or 29°C (see Sections in “METHOD DETAILS” for incubation temperature) until dissection or imaging, unless otherwise stated.", "For heat shock experiments, vials containing offspring were transiently transferred to 33°C or 34°C for 30 min to induce somatic clones (see Section “Live imaging of pupal notum”).", "Cross and offspring were kept at 25°C until dissection.", "We used both male and female larvae for experiments.", "Larval tissues were stained with standard immunohistochemical procedures using rat anti-Drosophila E-cadherin antibody DCAD2 (Developmental Studies Hybridoma Bank (dshb.biology.uiowa.edu), 1:20) and Alexa 647-conjugated phalloidin (Invitrogen, 1:50).", "Specimens were mounted with 4’,6-diamidino-2-phenylindole (DAPI)-containing SlowFade Gold Antifade Reagent (Thermo Fisher Scientific).", "Images were taken on Leica TCS-SP8 or Zeiss LSM700 confocal microscopes.", "Images shown in Figures 1A and S1H are sum projections.", "Live imaging was performed in the pupal notum rather than the wing disc because the notum does not require ex vivo cultivation and is easy to handle.", "We used both male and female pupae for experiments.", "Cross and offspring were kept at 18°C for 7–9 days and then subjected to heat shock at 33°C or 34°C for 30 min to induce somatic clones.", "Larvae were then kept at 18°C.", "78.5–81.5 hr or 94–98 hr after heat shock, white prepupae (WPP) were collected.", "Collected WPPs were transferred to 29°C to express the UAS-ykiS111A,S168A,S250A:V5 transgene, while WPPs were raised for 8 hr at 18°C and transferred to 29°C to express the UAS-RasV12 following earlier studies [7].", "The pupae were then imaged at 29°C between 15.5–33.5 hr (Yki) or 16–27 hr (RasV12) after temperature shift.", "Pupae for image acquisitions were prepared using a modified previously described protocol [58] or [59].", "Pupal dissection procedure is as follows:", "Rinse staged pupa with water and dry it on a tissue (e.g., KimWipe).", "The following procedure is different between protocol [58] or [59].", "(Protocol described in [58])", "Mount the washed pupa on a glass slide using double-sided tape.", "Remove the pupal case carefully with forceps at the top of the notum.", "Place filter paper wetted with water around the pupa to avoid desiccation.", "Enclose the pupa with silicon (Shin-Etsu) and place a coverslip on it to seal the pupa.", "(Protocol described in [59])", "Paste double-sided tape (upper) to single-sided tape (lower) to enhance the strength by increasing the thickness.", "Mount the washed pupa on the tape.", "Remove the pupal case carefully with forceps at the top of the notum.", "Place the exposed notum directly on a coverslip.", "Videos of the nota close to the scutum region were taken on Leica TCS SP5 or SP8 confocal laser scanning microscopes.", "Z stacks (0.8 μm/slice) were taken every 4 min.", "Images shown in Figures 2A, 2B, 2C, 4I, 5E, S4D, and S4F and Videos S1 and S2 were maximum projections.", "For Figures 2B and S4D, the maximum projection was performed by subdividing the picture in 256 × 256-pixel windows to avoid reflecting cuticle wrinkles in the projected image.", "Tissue-scale images shown in Figures 2A, 2B, 4I, and S4D and Video S1 were processed through the subtract background function in Fiji [55].", "E(i) is the set of edges incident to vertex i, and F(i) is the set of faces to which vertex i belongs.", "The tensional force Ftension is exerted to a vertex i by the connecting edges between vertices i and j, where the edge length dij converges to the preferred length l0.", "The internal pressure Fpressure is exerted on a vertex i by the cell face α to which a vertex i belongs, where Fpressure increases as the area of cell α (Aα) decreases and the edge length (dij) increases.", "The magnitude of Ftension and Fpressure are controlled by the parameters Gij and nH, respectively.", "For all figures in this paper, except for Figures S1F, S1G, S3A–S3G, and S3J (examining the parameter dependency) and Figure S2 (using the other model formulation), the parameter values were set at Gij = 5, l0 = 0.0001, and nH = 1.", "The parameter accounted for cell packing geometry of non-competitive tissue in Drosophila (Figures S1A and S1B), and did not dramatically affect the emergence of cell deformation due to differential proliferation rates (Figures S1F, S1G, and S3A–S3C) or asymmetric area expansion induced by apoptosis (Figures S3D, S3F, and S3J).", "Both models (Equation 1 and Equation 2) were integrated numerically using the Euler method with free boundary conditions.", "To achieve a mechanical equilibrium of tissue state, the position vector of each vertex was calculated after each step until the total velocity of all vertices dropped below a threshold of 1.0.", "Cell intercalation was incorporated when edges with a length below the threshold 0.001 were maintained for 8.3 × 10−4 normalized time [/loser cell cycle], noting that the intercalation during apoptotic cell delamination was introduced independent of this rule (see Section “Apoptosis simulations”).", "All simulation results were visualized by Processing language (https://processing.org/).", "Positions of vertices (junctions) of wild-type cells located at the clone boundary were manually extracted with ImageJ [54] or Fiji [55].", "Because epithelial cells in the wing disc are columnar in shape, each vertex was measured at the focal plane of the apical surface, identified by anti-E-cadherin or phalloidin staining.", "The cell area was calculated from the positions of the vertices by considering a cell a polygon.", "Strength of cell shape anisotropy was calculated using Equation 5 (Figures 1A, 1B, and S1I).", "Epithelial cells were segmented using Fiji plugin Tissue Analyzer [41, 61].", "Segmentation procedure is as follows:", "Save projected video with RGB Color as image sequence (TIF format) with Fiji.", "Drag and drop image sequence files into the List.", "Select the channel of adherens junctions (E-cad::GFP).", "Select “Segmentation” tab and click the “Detect Bonds (preview only)” or “Detect bonds V3 (preview only)” and optimize blur values to obtain images with roughly proper segmentation.", "Click “Detect bonds (save watershed)” or “Detect bonds V3 (save watershed)” to segment all images in the List", "Select “Correction” tab and manually correct the mistakes of the automated segmentation for all images.", "Select “PostProcess” tab and click “Finish all” and “Check finish all” to finalize segmentation.", "Select “Recenter” tab and click “Autocenter based on 2D correction.”", "Select “Cell tracking” tab and click “Track cells (dynamics tissue)” to identify the same cells in time.", "If tracking errors (e.g., cell swapping errors, cell pairing errors) occur, click “Correct/edit cell tracks” and fix them.", "Click “Check track” and “Update track mask” to finalize cell tracking.", "Select “Virtual cloning” tab to distinguish different types of cells (e.g., wild-type, Yki, RasV12).", "Select “Original” and mark cells of interest by left click and click “Drawing -> clone.”", "Click “Check virtual clone” and “Update virtual clone mask” to finalize clone registration.", "Select “Cell divisions” tab and click “Check divisions” and “Update cell division mask” to detect cell division.", "Select “Cell death” tab and click “Track dying cells,” “Check death” and “Update cell death mask” to detect cell death.", "Select “Track bonds” tab and click “Track bonds,” “Check track bonds” and “Update track bonds” to identify the same bonds (edges) in time.", "Select “SQLite DB” tab and click “Generate/Update database.”", "Select “Plots” tab and click “Export cell data” for cell data and “Export bond data” for junction data to save them as csv format.", "In this paper, we call “apoptosis” when cells are delaminated from the tissue during live imaging.", "We therefore do not strictly distinguish delamination “with apoptosis” and “without apoptosis” (i.e., live cell delamination [24]).", "We expect that the delamination in Yki-induced competition is due, at least in part, to apoptosis, because caspase3-positive wild-type cells are observed during Hippo pathway-induced cell competition [6, 62].", "Temporal change in cell area for apoptotic cells and surrounding cells (Figures 2, 4, 5, and S4) was quantified by segmentation and manually identifying cells of interest as “virtual clone” in Fiji plugin Tissue Analyzer [41, 61] (see Section “Image segmentation”).", "When cells surrounding a dying cell were delaminated or underwent mitosis during quantification (40 min before until 24 min after delamination complete), the contribution of a single apoptotic event on the captured area of surviving neighbors cannot be precisely determined; therefore, we excluded the apoptosis event from the quantification analysis.", "We also exclude apoptosis inside of the midline [inside of the two most central lines of sensory organ precursors (SOP)], where spontaneous cell delamination occurs independently of cell competition.", "Cell anisotropy (Figures S4A, S4B, S4C, and S4J) was calculated using Equation 5.", "Clone-boundary-oriented cell anisotropy (Figures 2J–2L, 5B, 5C, 5G, 5H, and S4J) was calculated using Equation 6.", "The normalized frequency of each type of intercalation (Figures 5G, 5H, 5J, 5K, 5M, 5N, 5P, and 5Q) was obtained by dividing the number of occurrences of each intercalation type by the total number of intercalations during a single apoptosis event (Figure 5D).", "When multiple intercalations (T1 transition) and/or T2 transition take place at a time (during a single time interval: 4 min), the type of cell intercalations were distinguished by estimating intercalated edges from the arrangement of cell neighbor relations.", "When more than two types of edges are estimated, the probability for occurrences for each intercalation type was calculated as n/nALL (n: number of estimated edges for a given intercalation type, nALL: total number of estimated edges).", "See Figures S4F–S4I for details.", "Note that, when additional intercalations were observed soon after apoptosis (from 16 to 24 min after delamination complete), the cell topologies were not stably determined; therefore, we excluded the apoptosis events from the current intercalation analysis.", "Clonal cells were identified in the Tissue Analyzer and projected onto the segmented image (Figures 2A, 2B, 4I, and S4D).", "Cell anisotropy (Figures 2A and 2B right, “cell-stretch” in “Viewer” tab), side number (Figures 4I and S4D, “packing-no cutoff” in “Viewer” tab), cell area (Figures 4I and S4D, “image color coder” in “Misc” tab) and apoptosis (Video S1, “apoptotic cells” in “Viewer” tab) were visualized by Tissue Analyzer.", "For the plots of normalized area as a function of side number (Figures 4J and S4E), we excluded SOPs and their primary neighbors and pupal midline region from the data.", "In addition, we did not plot data points when the sample number was less than 10.", "Note that the definition of cell anisotropy in Figures 2A and 2B (see [41]) is different from the one shown in Figures S4A, S4B, S4C, and S5J (see Equation 5) and Figures 2K, 2L, 5B, 5C, 5G, 5H, and S4J (see Equation 6).", "Scatter plots: Error bars represent standard deviation (s.d.) or standard error (s.e.) of the mean as specified in figure legends.", "Boxplots: The upper/lower hinge and middle line represent the 25th/75th and 50th percentiles (“boxplot” function in R environment).", "Statistical test: Fisher’s exact test (two-sided), Wilcoxon rank sum test (two-sided), Wilcoxon signed rank test (two-sided), Welch’s t test (two-sided, normality and homoscedasticity were tested by Kolmogorov-Smirnov test and F-tests, respectively).", "Smirnov-Grubbs test was performed to detect outliers (P-values < 10−4).", "Correlation coefficients (described as R in Figures) and significance test were based on Pearson’s product-moment correlation coefficient.", "All calculations and plots were performed with Microsoft Excel or R environment." ]	[ "Rapid cell expansion of oncogenic mutants fills space of normal cell apoptosis", "Topological change by directionally biased cell intercalations drives cell expansion", "The topological dynamics is verified with in silico and in vivo experiments", "Repetitive rapid cell turnover via topological changes accelerates mutant expansion" ]
Investigating the state of physiologically based kinetic modelling practices and challenges associated with gaining regulatory acceptance of model applications	Physiologically based kinetic (PBK) models are used widely throughout a number of working sectors, including academia and industry, to provide insight into the dosimetry related to observed adverse health effects in humans and other species. Use of these models has increased over the last several decades, especially in conjunction with emerging alternative methods to animal testing, such as in vitro studies and data-driven in silico quantitative-structure-activity-relationship (QSAR) predictions. Experimental information derived from these new approach methods can be used as input for model parameters and allows for increased confidence in models for chemicals that did not have in vivo data for model calibration. Despite significant advancements in good modelling practice (GMP) for model development and evaluation, there remains some reluctance among regulatory agencies to use such models during the risk assessment process. Here, the results of a survey disseminated to the modelling community are presented in order to inform the frequency of use and applications of PBK models in science and regulatory submission. Additionally, the survey was designed to identify a network of investigators involved in PBK modelling and knowledgeable of GMP so that they might be contacted in the future for peer review of PBK models, especially in regards to vetting the models to such a degree as to gain a greater acceptance for regulatory purposes.	[ "PHAR" ]	[ "Alternatives methods", "PBK models", "Physiologically based kinetic models", "Regulatory", "Risk assessment", "Toxicology" ]	[ "3005" ]	[ "Physiologically based kinetic (PBK1) models describe the body as a set of interconnected compartments that represent plasma and various organs, and characterize a chemical's fate within the body in regards to pharmacokinetic properties including absorption, distribution, metabolism and elimination (ADME).", "The development and use of PBK models have significantly increased over the last two decades, as is reflected in the rise of published literature referencing PBK models (Fig. 1).", "A wide variety of institutions have expressed a high degree of interest in PBK model applications, including academia, regulatory agencies, pharmaceutical and chemical industries (Schuck et al., 2015).", "PBK models have been deemed an effective means to aid in in vitro to in vivo extrapolation (IVIVE) (Blaauboer, 2010; Kramer et al., 2015), route to route extrapolation (Bessems et al., 2017), high to low dose, and inter- and intra-species extrapolations (Punt et al., 2016).", "The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), which is part of the European Commission, published its Toxicokinetic Strategy (2015)2 outlining a plan for identifying opportunities to apply new approach methods (NAM) to generate and more effectively use toxicokinetic data.", "The central feature of this strategy is using PBK models to integrate ADME data and to predict whole-body toxicokinetic behaviours.", "To facilitate the acceptance of PBK models for which development is based solely on non-animal data, good modelling practice (GMP) focusing on this new generation of PBK models is required for further development and recognition at an international level.", "As a starting point, GMP should include relevant information from existing guidance documents (WHO/IPCS, 2010; US EPA, 2006; EMA, 2016; US FDA, 2016).", "For example, development of PBK models can be accomplished through a six-step approach (Rietjens et al., 2011), regardless of the use of conventional methods or NAM to parameterize models.", "Briefly, these steps include (i) defining a simplified representation of the biological system for which model compartments can be included (i.e., a conceptual model); (ii) translating the conceptual model into a mathematical model by formulating a differential equation for each compartment; (iii) defining equation parameters as values derived either from literature or from experiments; (iv) solving the equations by calculating the concentrations of relevant compounds and their metabolites in the specific compartments, or the extent of their adverse reactions with the toxicological receptor; (v) evaluating model performance, with adjustments and improvements to the model when necessary; and (vi) using simulations to make predictions (Rietjens et al., 2011).", "The process involving each of these six steps should be transparent and documented.", "In addition, GMP may involve the development of standard reporting formats, which is equivalent in purpose to the QSAR Model Reporting Format (QMRF) and QSAR Prediction Reporting Format (QPRF), for presenting sufficient details of model construction and application.", "Such detailed reporting will aid kinetic modellers in accounting for all the necessary components that constitute a proper model and is expected to facilitate acceptance of kinetic modelling approaches by decision makers.", "Finally, the formation of a panel comprised of international PBK modelling experts and a knowledgeable group of peer-reviewers would not only expedite the drafting of GMP, but also promote future applications of PBK models in regulatory risk assessment.", "As toxicity testing and risk assessment shift more towards approaches involving NAMs such as in vitro techniques, PBK models are being used more frequently to convert in vitro points of departure (PoDs) to in vivo exposure (i.e., reverse dosimetry) for risk screening.", "Several examples are outlined in the review by Punt et al. (2011), including those for neurotoxicity (DeJongh et al., 1999; Forsby and Blaauboer, 2007), acute and repeated dose toxicity (Gubbels-van Hal et al., 2005; Rotroff et al., 2010; Pery et al., 2013; Gajewska et al., 2015), developmental toxicity (Verwei et al., 2006; Louisse et al., 2010), and genotoxicity (Paini et al., 2010).", "Among these examples, Paini et al. (2010) converted a threshold for a molecular initiating event (e.g., DNA binding) to an external exposure; Pery et al. (2013) and Gajewska et al. (2015) linked in vitro cell viability to an external dose; Wetmore et al. (2012a,b) incorporated in vitro metabolism measurements in IVIVE and reverse dosimetry to estimate external doses that are relevant to in vitro PoDs.", "One of the major limitations for a broader application of PBK models highlighted by Punt et al. (2011) is that development of new PBK models can be labour intensive processes that require generation of a large range of data through in vitro, in silico, or in vivo analyses to construct and parameterize a model.", "A more efficient approach is starting with the structure of a previously developed, well-parameterized, and thoroughly-vetted model for a close analogue; and adjusting chemical-specific parameters based on in vitro or in silico data (e.g., metabolism rates, absorption rates, partition coefficients between blood and tissues) (Lu et al., 2016).", "Such approach also directs the modellers to obtain necessary in vitro or in silico data that are relevant to a specific chemical.", "In order to facilitate acceptance and use of the new generation of PBK models, which rely on non-animal data, in the regulatory domain, experts were invited by EURL ECVAM to participate in a workshop on “Physiologically-based kinetic modelling in risk assessment – reaching a whole new level in regulatory decision-making” (Ispra, Italy, November 16–17, 2016) to identify challenges in (i) applying PBK modelling to support regulatory decision making; (ii) constructing PBK models without the use of in vivo kinetic and dynamic data, instead, relying solely on in vitro or in silico methods; and (iii) assessing model credibility and validity.", "The workshop participants concluded that an updated GMP requires inclusion of strengths and limitations encountered when parameterizing PBK models using in vitro measurements and in silico predictions, as well as processes for evaluating these types of PBK models to support regulatory decision-making.", "Other outcomes of the workshop and recommendations of the workshop participants are summarized by Paini et al. (2017).", "Prior to the workshop, in October 2016, the organizers sent invited experts a questionnaire containing 11 questions, to understand the use of PBK models from these experts (Paini et al., 2017).", "Due to the agreement among the experts that this exercise should be expanded to a broader level, the survey entitled “Applications of Physiologically Based Kinetic (PBK) models in science and regulatory submission” was created and extended to a larger, international PBK modelling and user community.", "The objective of this article is to outline and report the results from this survey to provide insight into the current applications of PBK models in research and regulatory communities, as well as challenges in regards to applying and evaluating PBK models that are constructed using data generated by NAMs.", "The survey findings and our analyses of the results provide valuable information on current practices in model development, application, and evaluation using a variety of modelling platforms.", "This information will be used to identify the key elements that should be included in the updated guidance on GMP for PBK models designed to support regulatory risk assessment applications.", "A survey was created by the European Commission - Joint Research Centre (JRC), EURL-ECVAM (https://eurl-ecvam.jrc.ec.europa.eu/) to understand the frequency of use and applications of PBK models in research and regulatory communities.", "The survey was published on the 11th of January 2017 and remained opened for 7 weeks until the 28th of February.", "The survey was made available to the participants at the workshop entitled “Applying exposure science to increase the utility of non-animal data in efficacy and safety testing” hosted by the National Centre for the Replacement Refinement & Reduction of Animals in Research (NC3R; https://www.nc3rs.org.uk/) in February 2017.", "To increase the size of the participant pool, the survey was then reopened for the members of the Biological Modelling Speciality Session of the Society of Toxicology (SOT-BMSS; https://www.toxicology.org/groups/ss/BMSS/index.asp) from the 14th to the 30th of April, 2017.", "The survey was conducted using an online questionnaire via the European tool EUSurvey (https://ec.europa.eu/eusurvey/home/welcome) and was comprised of 18 primary questions; 14 with YES/NO or multiple choice answers and 4 in free text format.", "Some of the YES/NO questions were expandable to a secondary related question, depending on a participant's reply.", "In some cases, additional comments could be entered as free text.", "The survey was grouped into four main areas: PBK model construction, evaluation, applications, and acceptance by regulatory agencies.", "In addition, the country of residence, professional affiliation, and expertise of the respondents were collected by the survey.", "The full questionnaire is available in the supplementary material.", "For the purposes of this analysis, responses to the questionnaire were aggregated and individual responses were kept anonymous.", "The target group for this survey was those involved in PBK model development and/or application in both research and regulatory communities, starting with approximately 200 individuals who had publications in PBK modelling.", "No individuals could be identified for Brazil, Australia, New Zealand; and thus, the survey was sent to the respective Toxicology and Pharmacology national associations for these countries.", "In addition, recipients of the survey were encouraged to forward it to interested parties.", "The aim was to reach as many scientists within the field as possible, as well as to understand the various geographic locales in which experts reside, so that a network of scientists involved in PBK modelling might eventually be created.", "However, the authors did not receive feedback on the degree to which the survey was forwarded to other scientists or regulators, and thus, it was not possible to determine the exact number of recipients of this survey.", "The answers collected by the EUSurvey tool were exported to a Microsoft Excel file for downstream analysis and interpretation.", "Data analysis and graphical representation was conducted using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA).", "Furthermore, dissemination of the survey replies was elaborated upon by means of an interactive infographic using graphical mapping built by Klimeto (http://klimeto.com/).", "This visualization approach was chosen to allow for transparency and for sharing of answers in an appealing manner for any parties interested in retrieving such information, while allowing the identities and contact information of the participants to remain anonymous.", "The interactive infographic was built on Google Chart API as a HTML5 and JavaScript web app, and is currently hosted by the Bolegweb project, (Kliment et al., 2016; http://bolegweb.geof.unizg.hr).", "The survey results can be accessed at http://bolegweb.geof.unizg.hr/questionaire/pbk/.", "This paper presents results across the international modelling community, and the web-based results are divided by country.", "The results are captured in a tabular format and can be visualized by means of pie-charts.", "For those wishing to access the individual results by country for the PBK model survey using the infographic, use of the Google Chrome browser is advised, and double click on the country of interest, the results will then be displayed.", "For those using Microsoft Internet Explorer or Mozilla Firefox, if the map does not load, it is recommended to use ctrl+r to refresh.", "This infographic is also mobile friendly.", "A total of 93 survey recipients filled out the online survey from the EUSurvey tool.", "One recipient preferred not to reply to the survey, due to possible conflicts of interest, but gladly shared experience using PBK models which will be taken into account where appropriate.", "The 93 replies were received from 19 countries (Fig. 2): Australia (N = 2, 2%), Belgium (N = 2, 2%), Canada (N = 7, 8%), Finland (N = 1, 1%), France (N = 6, 6%), Germany (N = 4, 4%), India (N = 2, 2%), Indonesia (N = 1, 1%), Italy (N = 2, 2%), Japan (N = 1, 1%), Jordan (N = 1, 1%); The Netherlands (N = 8, 9%), Norway (N = 1, 1%), Spain (N = 1, 1%), Sweden (N = 2, 2%), Switzerland (N = 3, 3%), Taiwan (N = 1, 1%), United Kingdom (N = 13, 14%), and the United States of America (N = 35, 38%).", "Survey participants represented experts from governmental organizations (N = 22, 24%), industries (N = 26, 28%), academia (N = 19, 20%), regulatory agencies (N = 11, 12%), small medium enterprises (N = 6, 6.5%), and other institutions (N = 6, 6.5%); three participants (3%) declared work in multiple sectors (Fig. 3A).", "Other sectors identified were: consulting firms, non-government organizations, retired, and a software company.", "From the industrial sector, nearly half (N = 12, 46%) of participants were in pharmaceutical sector, followed by those in chemical industries (N = 9, 30%), agrochemical (N = 2, 8%), consumer products (N = 2, 8%), cosmetics (N = 1, 4%), and food (N = 1, 4%) sector.", "From the regulatory agencies, representatives involved in human medicine (N = 7, 44%), chemicals (N = 3,18%), food (N = 3,18%), and environment (N = 3,18%) replied to the survey, in addition to one representative whose work involves regulation of chemicals, food, and human medicine.", "The professional background and expertise of the survey participants is summarized in Fig. 3B. Nearly half of the respondents have experience in multiple fields (N = 45), and the most common field of expertise is mathematical model development (N = 50, 27%), followed by risk assessment (N = 44, 24%), laboratory science (N = 44, 24%), statistical analysis (N = 17, 9%), and risk management (N = 3, 2%).", "Other specific expertise (N = 25, 14%) included molecular modelling, clinical pharmacology, pharmacokinetics, pharmacometrics, physiologically based pharmacokinetic modelling, in vivo toxicology, software development, education & mentoring, biopharmaceutics, absorption and formulation effect prediction, epidemiology, regulatory risk assessment, genetic toxicology, food safety, and drug development.", "Of the 93 respondents, 60 (65%) built their own PBK models in-house, and of those, 48 (80%) had experience in coding the models.", "Of these 48 respondents with coding experience, 21 (44%) followed some guidance documents when building and coding their PBK models.", "The primary guidance documents followed were the US Environmental Protection Agency's Approaches for the Application of Physiologically Based Pharmacokinetic (PBPK) Models and Supporting Data in Risk Assessment (US EPA, 2006) and the World Health Organization International Programme on Chemical Safety's Characterization and Application of Physiologically Based Pharmacokinetic Models in Risk Assessment (WHO/IPCS, 2010).", "Two participants reported that they used the US Food and Drug Administration's (FDA) draft guidance on Physiologically Based Pharmacokinetic Analyses – Format and Content, Guidance for Industry (US FDA, 2016) and European Medicines Agency's draft Guideline on the qualification and reporting of physiologically based pharmacokinetic (PBPK) modelling and simulation (EMA, 2016).", "One participant used examples of “best practices” in literature (Loizou et al., 2008; Rietjens et al., 2011), and another used the US FDA's Guidance for Industry and Other Stakeholders Toxicological Principles for the Safety Assessment of Food Ingredients (US FDA, 2000).", "There are 19 programming languages/software platforms reported being used by the respondents (Fig. 4), and the most commonly used programs include Berkley Madonna (Berkeley, CA) (N = 24), acslX (The AEgis Technologies Group, Huntsville, AL) (N = 21), MATLAB (MathWorks, Inc., Natick, MA) (N = 19), and R (N = 18), followed by GastroPlus (Simulations Plus, Lancaster, CA) (N = 11) and Simcyp (Certera, Princeton, NJ) (N = 10).", "Most participants used multiple platforms.", "PBK models include both physiological and chemical-specific parameters, and respondents often employ more than one source to obtain model parameter values (Fig. 5).", "Literature data were found to be the primary sources for obtaining physiological parameters (N = 84, 43%), followed by in vivo measurements (N = 32, 16%), databases (N = 25, 13%), in vitro measurements (N = 25, 13%), in silico predictions (N = 21, 11%), and others (N = 7, 4%).", "Literature data were also found to be the primary sources for obtaining chemical-specific parameters (N = 78, 33%), followed by in vitro measurements (N = 20, 8%), in silico predictions (N = 43, 18%), in vivo measurements (45%), and databases (22%).", "Some respondents also provided additional information on databases they used to obtain values for model parameters, and their inputs are summarized in table SM1, in the supplementary material.", "Respondents were asked to provide their input as to which ADME properties should be measured using in vitro approaches, rather than predicted, when constructing a PBK model.", "The top responses to this free-text question included metabolism/clearance, active transport, absorption, and chemical properties such as pKa (Fig. 6).", "In the early stage of model development, in silico approaches (e.g., QSAR models) may be used to estimate parameter values that can be replaced later with experimental measurements.", "Sensitivity analysis (SA) is an essential step in model validation, as it evaluates the impact of input variations on variability of model outputs.", "SA can be local or global.", "A local SA addresses sensitivity related to point estimates of parameter values, while a global SA addresses sensitivity related to distributions of parameter values.", "For the question of whether SA was conducted as part of the PBK model evaluation routine, 18 (19%) of the respondents answered no. For those who answered yes, most respondents commonly conducted only local SA (N = 32, 34%) or both types of SA (N = 32, 34%), compared to 8 (9%) who conducted only global SA.", "About 3% of the respondents (N = 3) used other means, such as their own sensitivity metrics.", "Of interest, one participant noted that most of the models he reviewed did not include SA, even though he believes that SA should be conducted routinely for PBK models that are developed to inform decision-making.", "For one of the open questions concerning methods and criteria used to evaluate model performance, 71 (76%) participants responded, and most responses centered around comparing model simulations with observed in vivo data such as peak concentration, area under the curve, time to reach peak concentration, and half-life.", "Various criteria are used to determine goodness of fit.", "Most participants stated that they visually inspect how well the model fits the data and calculate the fold difference, while others use statistical methods to calculate coefficient of determination, mean absolute percentage error, or root mean-squared error.", "Respondents also noted that it is important to confirm mass balance and to conduct sensitivity/uncertainty analysis.", "These individual's answers can be found on the web-based infographic described in methodology section.", "For another open question on defining GMP, 66 (71%) respondents answered, and two examples are listed below.", "The other responses can be found on the web-based infographic.", "1.", "“GMP is less to do with how well a model performs and more to do with how a model is created, and documented and its ability to be reproducible.", "To me, good modelling practice for PBK modelling means that a model is created in a physiologically meaningful manner using quality and reliable data, with transparency in model structure and parameters and the ability to reproduce simulation results.”", "2.", "“GMP to me includes: transparent declaration of model assumptions; listing available data for model development; choice of appropriate model structure by hypothesis testing; decisions made during model development stated clearly along with their justifications; approaches and results of model performance evaluation reported; both the domain of applicability and inapplicability for the model be identified; model limitations in its application front (for example, data-gap reducing the strength) and the development front (for example, a chance of over parameterization) identified; clear documentation of the code; sensitivity analysis (local and global), uncertainty analysis, and variability analysis conducted; finally, an estimate of overall model confidence metric for its performance.”", "Most participants were found to be involved in PBK modelling from several times a month (N = 28, 30%) to less than once a month but several times a year (N = 32, 34%).", "About 22 (24%) of the participants incorporated PBK modelling in their daily work, while the remaining participants rarely used PBK modelling all of whom are non-modellers (N = 11, 12%).", "The primary fields in which the participants apply PBK modelling approaches include human health (N = 49, 25%), human medicine (N = 29, 15%), industrial chemicals (N = 29, 15%), occupational health (N = 22, 11%), environmental health (N = 21, 11%), and cosmetics (N = 21, 11%), (Fig. 7A).", "Other fields included food & feed safety (N = 16, 8%), veterinary medicine (N = 5, 3%), and others (N = 2, 1%) such as mathematical biology, predictive toxicology, and quantitative systems toxicology (Fig. 7A).", "For specific applications, PBK modelling approaches were found mainly to support human health risk assessment (N = 70, 29%), exposure assessment (N = 58, 24%), chemical safety assessment (N = 39, 16%), and experimental design (N = 31, 13%) (Fig. 7B).", "Applications in drug design, ecological health risk assessment, and veterinary health accounted for 6% (N = 15), 5% (N = 11), and 1% (N = 3), respectively (Fig. 7B).", "Other (N = 11, 5%) applications of PBK modelling approaches included informing pharmaceutical dose selection, evaluation of pharmaceutical dose adjustment for specific populations, clinical trial design and optimization, drug labelling, and IVIVE to investigate concepts for animal-free risk assessments.", "Twenty-seven (N = 27, 25%) of the 93 participants declared use of PBK models for submission of dossier/opinions/risk assessment to regulatory agencies, and most of them submitted to agencies in several countries.", "Submission was done in the following countries: USA (N = 19,40%); Europe (N = 8, 17%); Japan (N = 4, 8%); Australia (N = 3, 6%); Canada (N = 2, 7%); China (N = 2, 7%); The Netherlands, Norway, Spain and UK (N = 1, 2% each); Rest of the World (ROW, N = 1, 2%).", "Many of those submitting a dossier did so for multiple countries.", "Nearly half (N = 13, 48%) of the participants submitting a dossier for regulatory purposes using PBK models followed specific guidelines for reporting PBK model development and application.", "There were two primary sources of guidelines:", "Report template or internal best practice documents prepared within organizations;", "Published guidance documents (US EPA, 2006; WHO/IPSC, 2010; CEN, 2015);", "Of those participants that have experiences submitting PBK models to support regulatory risk assessment, only 39% (N = 36) reported that they have faced challenges in gaining regulatory acceptance of PBK models.", "Replies can be summarized into the following four main challenges:", "Lack of expertise in PBK modelling and its applications within the regulatory agencies;", "Validation of the model requires testing various scenarios to reassure the agency that due diligence has been undertaken in assessing the model assumptions and predictions, but data for testing are often not available;", "Lack of user friendly software for testing the model by risk assessors who are not programmers;", "Difference in acceptance criteria between agencies and countries.", "Through the International Programme on Chemical Safety (IPCS), the World Health Organization (WHO, http://www.who.int/ipcs/en/) establishes the scientific basis for management of chemicals, and strengthens national capabilities and capacities for chemical safety.", "A WHO chemical risk assessment network is in place to enhance global efforts to assess the risk to human health from exposure to chemicals by developing tools and guidance documents.", "A participant reported in our survey that the Chemical-specific Adjustment Factor (CSAF) working group, as part of the WHO Chemical Risk Assessment Network, released a manuscript that discusses recent challenges and experiences in gaining regulatory acceptance of chemical-specific adjustment factors and PBK models (Bhat et al., 2017).", "The majority of the participants (N = 83, 89%) agreed that PBK models should be subject to an independent peer review system in order to gain more acceptance in regulatory settings.", "More than half of the participants (N = 52, 57%) stated that they are willing to act as independent peer reviewers for the regulatory agencies.", "The survey concerning applications of PBK models in research and regulatory submission of chemicals was completed by 93 international individuals occupying a wide range of working sectors.", "The equally high distribution among participants primarily involved human health and exposure assessments using mathematical modelling (N = 50) and risk assessment (N = 42), with nearly half involved in both (N = 22), indicate a balanced set of responses from both model builders and end users.", "The results provided in this article present a snapshot of the current state of PBK model construction, evaluation, and application.", "Although a majority of individuals stated that they use multiple modelling platforms or coding languages, preference of use among sector types was apparent.", "For example, those involved in academia primarily use Berkeley Madonna and MATLAB, while those associated with government and regulatory agencies primarily use R and acslX, and those in industry primarily use programs such as Simcyp or GastroPlus.", "Among these listed programs, R is the only open source coding language, and it has the capability to incorporate user-developed packages for a variety of modelling applications.", "The program acslX provides an intuitive platform that has been used for over 30 years for conducting a wide array of PBK and pharmacodynamic (PD) analyses, but further development and support for this program ceased in November 2016.", "MATLAB is a computing software that is widely used by scientists and engineers for algorithm development, data analysis, visualization, and numeric computation.", "In addition to MATLAB, MathWorks Inc. also produces nearly 100 additional products, some of which are ancillary to MATLAB and others that act as stand-alone toolboxes for specialized tasks.", "Berkley Madonna is known for its ability to rapidly solve differential equations and is widely used amongst the PBK modelling community.", "These four platforms/languages (R, acslX, MATLAB, and Berkeley Madonna) may be preferred by users in academia or government due to familiarity or costs.", "In addition, acslX and Berkeley Madonna are used in many PBK modelling training courses, and thus, the comfort level with these four platform types remains high within the PBK modelling community.", "Familiarity with these platforms also allows peer reviewers, especially those who have attended these training courses but are not programmers, to have the necessary background to review or evaluate models developed using these software programs.", "On the other hand, models developed using software programs that are unfamiliar to reviewers may not be properly vetted.", "Both Simcyp and GastroPlus are proprietary programs that offer numerous options for model parameterization (e.g., built-in in silico models or user-specified input) in a user-friendly graphical interface.", "These programs are primarily used within the pharmaceutical industry because screening-level models can be developed much more quickly through the menu choices, default settings, and internal algorithms for model parameterization.", "Interestingly, Simcyp was the top platform used for dossier submission to regulatory agencies as reported by the participants.", "This result may be due to several regulatory agencies, including the US FDA, the European Medicines Agency (EMA), and Japanese Pharmaceuticals and Medical Devices Agency, being affiliates of the Simcyp Consortium, which is a collaborative research centre for PBK and mechanistic modelling.", "Another unique aspect of these two generic PBK modelling platforms is that the proprietary nature of drug development processes necessitates a formal agreement between the pharmaceutical companies and software developers to allow those software developers to use confidential preclinical and clinical data provided by the companies for supporting software development.", "At the same time, the software developers ensure the software products are relevant to the needs of companies, such as providing recommendations on dosing regimens or formulations, examining drug-drug interactions, or designing clinical trials.", "In addition to modelling platform uses, the survey also gathered information regarding the sources for obtaining values of model parameters.", "Physiological parameters such as body weight, organ weight, cardiac output, respiratory parameters, and milk secretion were found to be primarily retrieved from the literature and from in vivo studies.", "Chemical-specific parameters (e.g., metabolism rate, absorption rate, partition coefficient, fraction unbound in plasma) or properties that are used to estimate these parameters (e.g., polarity, pH, log Kow) were also found mostly to be taken from the literature.", "Secondary sources of chemical-specific data included in silico predictions, which were primarily used by those in academic and governmental sectors; or in vitro measurements, which were primarily used by those in the industry sector.", "Such differences may be due to industries having more resources to conduct in vitro experiments, compared with academia or government agencies that have limited resources.", "An excellent summary of these various approaches for model parametrization can be found in Bessems et al. (2014).", "A list of available databases reporting parameters for building PBK models provided by the respondents of the survey is available in the supplementary material.", "Although several of these databases are open source, most require a license for access.", "Some participants indicated that data required to fully understand ADME properties is sorely lacking, as is data required to understand the physiology of cells (e.g., volumes, diameters, etc.).", "Other participants suggested that clinical data may act as appropriate sources for examining the limitations of IVIVE.", "Often, it is the modeller's purpose to build the most parsimonious or simplest model for the data at hand.", "Traditionally, this approach required optimizing certain parameters so that model outputs agree with available in vivo data.", "Usually, these parameters are related to characteristics that impact entry or disappearance of the chemical in plasma (i.e., absorption or metabolism/excretion), and simply optimizing these parameters in accordance with allometric scaling would often be sufficient for model evaluation.", "However, as the number of chemicals in commerce has continued its precipitous rise (Binetti et al., 2008; Egeghy et al., 2012), the limitations of this approach have become obvious.", "Specifically, it is often the case that most chemicals do not have in vivo PK data available for comparing against model predictions.", "Moreover, policies in the US and EU are shifting towards reductions in animal studies; a good example is the full ban of testing cosmetic ingredients and final cosmetic products in Europe, in force since 2013.", "In these cases, model development and evaluation must rely on better mechanistic understanding of ADME properties, as well as NAMs to derive values for model parameters required to describe these ADME properties.", "It was found from the survey that, among different NAMs, in vitro methods are often preferred over in silico methods.", "This result could be explained by a higher degree of confidence that in vitro measurements may confer over in silico predictions, in addition to the need of having measurement data for training in silico models.", "There is universal consensus among individuals and working sectors that metabolism and clearance properties should be derived experimentally.", "Those participants in both government agencies and academia expressed a second priority to experimentally measure partition coefficients, followed by transporter processes.", "Alternatively, those in industry consider transporter processes to be a secondary focus, followed by partition coefficients.", "Regardless, it is apparent that for greater acceptance of models by regulatory agencies, the properties of metabolism, partitioning, and transport are of high priority.", "PBK models developed for pharmaceutical products focus primarily on the fate of the parent compound, along with its distribution in the body and arrival at the site of action; thus determining an appropriate clearance value is essential to achieve these goals.", "On the other hand, metabolism is key in understanding mode of action for environmental chemicals, and availability of data on kinetic constants (Vmax and Km) is essential.", "Several efforts are underway to map exposure knowledge (Teeguarden et al., 2016) and adverse outcomes (Ankley et al., 2010; https://aopwiki.org/wiki/) with high throughput in vitro dose-response data to significantly expand our understanding of chemical fate and effects (Wittwehr et al., 2017).", "However, little attention has been placed on experimental identification of metabolites to enable testing of their ability to trigger adverse outcome pathways.", "Quantification of the generation and clearance rates of these metabolites can be accomplished in vitro through whole-cell experiments, or through studies on individual phase I and phase II microsomal or cytosolic enzymes.", "Measuring chemical responses upon interaction with the individual enzymes can allow for determining kinetic constants like Vmax and Km for different transformation pathways (e.g., oxidation, hydroxylation, epoxidation).", "Enzyme fractions can be purchased for different organs (e.g., liver, lungs, kidney, skin), and the complexity of PBK models will vary as a function of the number of metabolites accounted for, the number of organs compartmentalized, and differences between and within species (Punt et al., 2008, 2009, 2016).", "Overall, the advancement in these in vitro technologies are contributing to generation of biotransformation data (e.g., Vmax and Km) necessary for the proper function of PBK models.", "Most respondents agreed that GMP should be followed during model evaluation (EFSA, 2014; Loizou et al., 2008).", "GMP involves a combination of several considerations: (1) a transparent description of how the model is created; (2) appropriate documentation, including model assumptions, sensitivity analysis (local and global), uncertainty analysis, and variability analysis; and (3) an estimate of overall model performance and reproducibility.", "These considerations were reiterated at an EMA workshop (London, UK, November 2016) in which participants discussed the key elements that should be included in a draft document on qualification and reporting of PBK analyses.", "During the EMA workshop, the need for SA was central to the discussion, as some attendees debated the need for a global SA when using a commercial PBK platform to develop a PBK model, and that focus should be placed on parameters with less uncertainty (Zhao, 2017).", "Currently, most evaluation of PBK models is conducted by visually comparing model predictions with experimental data, such as area under the curve (AUC), peak concentration (Cmax), time to peak concentration (tmax), and plasma half-life.", "Besides visual examination, other criteria are used to examine goodness of fit, such as setting error thresholds of 0.5–2-fold, or even 0.8–1.25-fold.", "Pharmaceutical companies often use this latter threshold in their determination of the bioequivalence between two drugs.", "In other words, bioequivalence in this case is defined to be no more than a 20% difference between the AUC and Cmax of two medicines, usually a generic medicine compared to the innovator medicine (MedSafe, 2015; Bpac, 2009).", "Only half of the participants who have experience in submitting PBK models for regulatory agencies review stated that they used any sort of guidelines when developing or reporting their models.", "Since most guidelines generally contain common modelling practices, even though survey participants and many modellers may not explicitly follow published guidelines, it is likely the case that their familiarity with modelling etiquette has resulted in their adhering to a majority of the guidelines.", "Despite a lack of clearly established GMP guidelines, certain pharmaceutical and industrial sectors are collaborating with regulatory agencies on a more frequent basis in an effort to apply NAMs and PBPK modelling approaches in evaluation of their products’ safety and to receive permission to be released onto the market.", "For example, in 2015, the results of a survey focusing on preclinical pharmacokinetic/pharmacodynamics (PK/PD) revealed that pharmaceutical companies have increased the use of preclinical PK/PD analysis in all therapeutic areas (Schuck et al., 2015).", "This trend is reflected through an increasing number of submissions containing PBK model analysis to regulatory agencies, in the US to FDA and in Europe to EMA (Rowland et al., 2015).", "More specifically, PBK models are applied to support benefit–risk evaluations by providing a mechanistic basis for extrapolation beyond the clinical trial population, by reducing uncertainty, and by predicting drug–drug interactions (DDIs) in specific groups of populations (e.g., elderly, pediatric) (Shepard et al., 2015).", "Furthermore, PBK models are developed within a systems pharmacology context to efficiently extrapolate PK/PD properties in various healthy and patient populations (Jamei, 2016), for both adults and children, for better precision medicine (Wagner et al., 2015).", "Studies have shown that application of PBK models has the potential for improving pediatric drug trials (Leong et al., 2012) and oncology treatments (Yoshida et al., 2017).", "EMA and the US FDA both released draft guidance intended to help pharmaceutical companies to report PBK model analyses to the respective regulatory agency in a harmonized format (EMA, 2016; US-FDA, 2016).", "These more recent movements show that there is an increasing interest in applications of PBK models in several branches of science (medicine, pharma, environmental, exposure) and that these models may gain more regulatory acceptance.", "Perhaps it is the combination of a lack of established standardized GMP guidelines and the wide range of computing platforms used for model development that serve as the two greatest obstacles for PBK model use in regulatory risk assessment.", "The unique needs of each regulatory agency add further difficulty in establishing GMP guidelines that can be used universally in risk assessment.", "In most cases, a standard checklist to evaluate the quality of a PBK model and the appropriateness of its applications is unavailable for decision makers who possess little knowledge of the PBK modelling process.", "Establishment of GMP across the modelling community can act as an excellent start for developing such a standardized checklist among scientific peers, which can then be communicated to and adapted by regulatory agencies.", "Over time, these agencies can revise the checklist to meet their specific needs, while still maintaining the standardized approach for reviewing model development and evaluation that are generalizable among different regulatory agencies.", "The lack of a common set of computing platforms for PBK modelling, especially in the field of environmental health, has been a challenge for many years.", "Harmonizing the general structure of model code among different platforms can serve as an initial step to address this issue.", "The programming command and code sorting will certainly remain unique within each platform, but the general structure for coding differential equations and organizing various sections of the model (e.g., defining physiological and chemical-specific parameters separately) tend to be conducive to forming a template that can be easier to understand by reviewers who are not familiar with a specific software.", "The PBK models published by academia and industry often are developed in an attempt to seek an answer to a specific scientific question related to a certain chemical or chemicals (e.g., interpreting biomarker data, organizing animal time-concentration data), and the purpose of such a study can differ significantly from that of a regulatory agency drafting safety guidelines for those chemicals.", "In many cases, it may be perfectly suitable to use a PBK model to help answer these scientific questions through various academic exercises, with some supporting in vivo data to verify such predictions.", "However, when the purpose is to use a PBK model to support regulatory decision-making, the model requires careful vetting of selected parameter values, model code and structure, and methods of model evaluation, before confidence can be gained in applying that model for regulatory risk assessment purposes.", "In addition to proper documentation and quality control, the validity of a model and its application heavily relies on attaining the proper data to derive the most appropriate values for model parameters.", "Over time, the advancements in molecular biology, in vitro testing systems, and in silico techniques have led to generation of new knowledge regarding biological systems, in addition to data the necessary for facilitating more mechanistic-based model development and improving a model's capability to predict and extrapolate.", "Inclusion of transporter processes and -omics data will have the potential to further improve confidence in model parameters (Poulin et al., 2016).", "The modelling community should be aware that although these newer generation PBK models may possess increased predictive capability due to inclusion of mechanistic processes and emerging data, additional challenges are introduced to risk assessors attempting to review these more detailed and complex models in support of regulatory decision making.", "Especially in the case which experimental in vivo data are not available for comparison to model predictions, regulatory agencies are likely to remain reluctant to involve such models in their chemical safety assessments, highlighting a need to develop new guidance documents to help regulatory agencies in reviewing and accepting these type of models.", "This paper describes how PBK models are currently constructed, evaluated, and applied in various working sectors and countries.", "This analysis provides insight into the current state of the PBK modelling and user community, in addition to a cursory contact list of modellers available for peer review.", "Several limitations of acceptance of PBK models into the regulatory risk assessment process still remain due to lack of guidance, lack of data, or lack of expertise.", "However, the modelling community is continuously expanding, and with it, PBK models are gaining ground for use in various scientific and regulatory risk assessment applications.", "A similar PBK modelling survey should be updated periodically to keep pace with evolving databases, available software, and new challenges.", "The U.S. Environmental Protection Agency has provided administrative review and has approved this paper for publication.", "The views expressed in this paper are those of the authors and do not necessarily reflect the views of the U.S. Environmental Protection Agency or the European Commission." ]	[ "Physiologically Based kinetic (PBK) models are used widely in academia, industry, and government.", "Good modelling practice (GMP) for model development and evaluation continues to expand.", "Further guidance for establishing GMP is called for.", "There remains some reluctance among regulatory agencies to use PBK models.", "The next generation of PBK models could be developed using only data from in vitro and in silico methods." ]
Data in support on the shape of Schwann cells and sympathetic neurons onto microconically structured silicon surfaces	This article contains data related to the research article entitled "Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth" in the Biomaterials journal [1]. Scanning electron microscopy (SEM) analysis is performed to investigate whether Schwann cells and sympathetic neurons alter their morphology according to the underlying topography, comprising arrays of silicon microcones with anisotropic geometrical characteristics [1]. It is observed that although soma of sympathetic neurons always preserves its round shape, this is not the case for Schwann cells that become highly polarized in high roughness microconical substrates.	[ "MULT" ]	[]	[ "1000" ]	[ "Many studies report the response of different cell types which do not form extensive cell projections (i.e., fibroblasts, osteoblasts, Schwann cells, etc.) on artificial micro-and/or nanostructured surfaces [2–4].", "In such cell types, the underlying topographical cue rearranges and reorganizes the cytoskeleton of the whole cell.", "In contrast to the above mentioned cell types, nerve cells respond to topographical cues in a quite distinct manner, mainly due to their unique morphology.", "The neuron consists of a network of long neurite projections (i.e., axons and dendrites), which extend from the cell body.", "When cultured on different substrates, the neurite projections have been shown to rearrange their network shape according to the imposed topography, while the cell body does not necessarily follow a similar adjustment [5].", "We have shown that discontinuous anisotropic topographies such as this of microconically structured silicon surfaces influence cell growth orientation of Schwann cells and sympathetic neurons [1].", "More specifically, neuronal axons and Schwann cells follow the directionality of the major axis of microcones with elliptical shape while there is a random orientation for substrates with microcones of arbitrary shape.", "In this article, we demonstrate via SEM microscopy on the different cell types the effects of the above mentioned topography on cell shape.", "Schwann cell primary cultures were prepared from sciatic nerves of P2-P4 rat pups.", "Upon dissection, nerves were enzymatically triturated in a Trypsin 0.25% and 1% collagenase Type 1 (Sigma C0130) in HBSS solution for 30 min at 37 °C.", "The enzymes were deactivated by adding Dulbecco׳s modified Eagle׳s medium (DMEM; Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Biosera, Sussex, UK) to the cells and the sciatic nerves were mechanically dissociated into single cell suspensions and plated to dishes.", "Before the first passage, Schwann cells were purified from contaminating fibroblasts using anti- thy1.1 antibody (AbD Serotec) and rabbit complement (Calbiochem) according to Brockes et al. [6].", "Then 105 cells were seeded onto micropatterned silicon scaffolds and cultured for 4–6 days until fixation for further analysis, with medium replacement every 2 days.", "In some experiments, PDL coating has been used.", "Superior cervical ganglia (SCG) were dissected from newborn (P0–P1) rat pups and dissociated in 0.25% trypsin for 30 min at 37 °C.", "After dissociation, SCG neurons were re-suspended in Roswell Park Memorial Institute (RPMI 1640, GIBCO/Life Technologies 32404-014) culture medium 1640 containing 10% FBS, 100 units/ml penicillin and 0.1 mg/ml streptomycin (p/s) (GIBCO/Life Technologies 15140-122), 10 mM antimitotic agent FdU with uridine, and 50 or 100 ng/ml Nerve growth factor (NGF; 2.5 S, Millipore, Billerica, MA, USA).", "104 cells were plated on collagen coated or micropatterned silicon scaffolds in 48-well plates and cultured for 7 days until fixation for further analysis.", "Medium was changed every 2 days.", "Mouse embryos (embryonic day (E) 13.5) were dissected from pregnant mice.", "DRGs from the lumbar region (L1–L4) were collected in cold PBS, then seeded onto the micro-structured Si substrates (2–3 DRG per substrate) and incubated in growth medium (DMEM)-F12, (GIBCO/Life Technologies 22320-022) supplemented with 10% FBS and 100 ng/ml NGF.", "Explants were incubated 5 days in vitro (DIV) until fixation for SEM analysis.", "The morphology of the cells growing on the patterned surfaces was analyzed by SEM.", "After culture termination, the cells were washed with 0.1 M sodium cacodylate buffer (SCB) and then incubated in the same solution for 15 min.", "After repeating this step twice, the cells were fixed using 2% glutaraldehyde, 2% formaldehyde in 1% SCB fixative buffer for 1 h at 4 °C.", "All surfaces were then washed twice (for 15 min each time) with 1% SCB at 4 °C, dehydrated by immersion in serially graded ethanol solutions (50–100%) and incubated for 15 min in dry 100% ethanol.", "Prior to electron microscopy examination, the samples were sputter-coated with a 10 nm gold layer (Humme Technics Inc, Alexandria, Virginia USA).", "SEM analysis used a JEOL 7000 field emission scanning electron microscope at an acceleration voltage of 15 kV.", "Although arbitrary Schwann cell growth takes place on the microconically structured silicon surfaces of arbitrary shape (low laser energy, low roughness), Schwann cells tend to follow a preferential orientation when grown on substrates of microconically structured silicon surfaces of elliptical shape (medium and high laser energy, medium and high roughness) [1].", "SEM study revealed a differential cell shape on the different substrates.", "Schwann cells grown on the surfaces comprising microcones (MCs) of arbitrary shape were mostly polygonal-shaped (Fig. 1a).", "On the contrary, the majority of the cells on the surfaces comprising MCs of elliptical shape were mostly spindle-shaped (Fig. 1b).", "In the case of the whole DRG explants, when these were grown on the microconically structured silicon surfaces of elliptical shape (medium and high laser energy, medium and high roughness), SEM analysis revealed that the migrating Schwann cells were oriented parallel to the elliptic MCs (Fig. 2).", "Furthermore, most of the axons were grown on top of the aligned Schwann cells [1].", "It is therefore postulated that the elliptical MCs of medium and high roughness substrates dictate a virtual path to Schwann cells, which in turn guide axonal outgrowth.", "In the case of sympathetic neurons, although the axons on the microconically structured silicon surfaces of arbitrary shape (low laser energy, low roughness) grew randomly, neurons on microconically structured silicon surfaces of elliptical shape (medium and high laser energy, medium and high roughness) exhibited a directional growth providing a network of parallelly aligned axons [1].", "SEM microscopy revealed that the somas were always of round shape, regardless the type of the culture substrate used, i.e., flat or different microstructured surfaces (Fig. 3)." ]	[]
Sigma-2 receptor ligands QSAR model dataset	The data have been obtained from the Sigma-2 Receptor Selective Ligands Database (S2RSLDB) and refined according to the QSAR requirements. These data provide information about a set of 548 Sigma-2 (σ2) receptor ligands selective over Sigma-1 (σ1) receptor. The development of the QSAR model has been undertaken with the use of CORAL software using SMILES, molecular graphs and hybrid descriptors (SMILES and graph together). Data here reported include the regression for σ2 receptor pKi QSAR models. The QSAR model was also employed to predict the σ2 receptor pKi values of the FDA approved drugs that are herewith included.	[ "MULT" ]	[]	[ "1000" ]	[ "The sigma-2 (σ2) receptor is a peculiar target overexpressed in several tumor cell lines and its ligands are actually under clinical evaluation as positron emission tomography radiotracer and as fluorescence imaging agents [1–3].", "Few selective ligands have been found for the σ2 receptor and in some cases, their finding occurred through an accidental discovery [1,4].", "Data here reported provide information about a set of σ2 receptor ligands, taken from the Sigma-2 Receptor Selective Ligands Database (S2RSLDB), and selective over σ1 receptor, together with their pKi (–logKi) [5].", "These latter have been used in building up the first hybrid QSAR model embracing the all set of known σ2 receptor selective ligands [6].", "The model has also been used to predict the σ2 receptor pKi for the Food and Drug Administration approved drugs.", "These latter predicted σ2 receptor pKi data are also here reported.", "The dataset consists of 548 σ2 receptor selective ligands which were randomly split three times and then divided into training (209 compounds), invisible training (209 compounds), calibration (65 compounds) sets for model development and a validation set (65 compounds) for invisible model validation.", "The three splits and four sets have been randomly generated, and their pKi minimum, maximum and middle are reported in Table 1.", "QSAR models have been developed with the use of the software CORAL [7].", "Once the splits and sets were determined, nine models were developed and statistical quality recorded.", "Differences of these models consist in the way molecular structures have been depicted the software.", "Thus, in Table 2 regressions for the σ2 receptor pKi models using SMILES, molecular graphs and hybrid descriptors (SMILES and graph together) are reported.", "While in Table 3 is reported the statistical quality of models of the σ2 receptor pKi.", "Fig. 1 shows a CORAL screenshot with settings for hybrid model split 1.", "While in Table 4, the complete list of SMILES and their distribution into the sub-training (+), calibration (–), test (#) and validation (*) sets for σ2 receptor pKi hybrid model split 1 is reported.", "These data may be prospectively used in finding novel models for σ2 receptor affinity.", "The endpoints of the FDA-approved drugs were determined in order to additionally validate the model.", "The whole set composed of 1428 drugs was refined in order to remove quaternary ammonium salts, and compounds with too long SMILES (not elaborated by CORAL), and compounds containing atoms not enumerated in the model (Al, Fe, Gd, etc.).", "Overall, the whole set was reduced to 1376 compounds and these were evaluated with hybrid model resulting from split 1.", "Over 1376 compounds, 925 have been defined as outliers by the model since they fall outside the domain of applicability.", "Table 5 reports the SMILES and predicted σ2 pKi for these FDA approved drugs evaluated with the hybrid model split 1." ]	[]
Vitamin K2-Dependent GGCX and MGP Are Required for Homeostatic Calcium Regulation of Sperm Maturation	A low-calcium microenvironment is essential for spermatozoa to mature in the epididymis; however, it remains unclear how dysregulation of epididymal luminal calcium is associated with male infertility. Using a warfarin-induced vitamin K2 deficiency rat model, we found that vitamin-K-dependent γ-glutamyl carboxylase (GGCX)and matrix Gla protein (MGP)were essential in extracellular calcium signaling of the intercellular communication required for epididymal sperm maturation. We found that GGCX and MGP co-localized in the vesicular structures of epididymal cells and spermatozoa. Calcium-regulated MGP binds to proteins in a biphasic manner; sub-millimolar calcium enhances, whereas excessive calcium inhibits, the binding. Bioinformatic analysis of the calcium-dependent MGP-bound proteome revealed that vesicle-mediated transport and membrane trafficking underlie the intercellular communication networks. We also identified an SNP mutation, rs699664, in the GGCX gene of infertile men with asthenozoospermia. Overall, we revealed that the GGCX-MGP system is integrated with the intercellular calcium signaling to promote sperm maturation.	[ "MULT" ]	[ "Cell Biology", "Developmental Biology", "Specialized Functions of Cells" ]	[ "1000" ]	[ "Proper intercellular communication is essential for multicellular organisms to function and survive normally, and aberrant exchange of information may result in pathological conditions and diseases.", "Extracellular vesicles (EVs; also known as exosomes and microvesicles) are the information carriers for cell-to-cell communication in most cell types (Schneider and Simons, 2013; Tkach and Thery, 2016).", "One cell type that heavily relies on intercellular communication is the spermatozoon, which is generated in the testis and whose transcription is halted, for the cell cycle is arrested until fertilization (Braun, 1998).", "Spermatozoa have to exchange numerous cellular components by interacting with the epithelial cells that nurture and protect them while they transit through and mature in the epididymis (Dacheux and Dacheux, 2014; Hermo and R.B, 2002; Robaire and Hinton, 2015).", "The cell-cell cross talk among spermatozoa and the various types of epididymal cell takes place in the luminal microenvironment, in which a unique composition of epididymal fluid is formed and regulated for special needs of these cells, such as metabolic demands (Cornwall, 2009; Robaire and Hinton, 2015; Turner, 2002; Zhou et al., 2018).", "The epididymal epithelium, at the front line in contact with spermatozoa, is responsible for the formation of the epididymal luminal microenvironment and conveying the epigenetic and environmental factors to the spermatozoa and thus plays a critical role in the regulation of male fertility as well as the health of offspring (Chen et al., 2016a, 2016b; Conine et al., 2018; Hansen et al., 2010; Latchoumycandane et al., 2002; Sharma et al., 2016, 2018; Smythies et al., 2014; Sun et al., 2018).", "However, the physiological roles of the constituents of the epididymal luminal microenvironment, the medium for the communication between spermatozoa and epididymal cells, remain largely unknown.", "One feature of the epididymal luminal microenvironment is a low level of calcium (Cornwall, 2009; Levine and Marsh, 1971; Turner, 2002).", "An aberrantly high Ca2+ level in the lumen can result in defective spermatozoa and impaired fertility, without affecting spermatogenesis (Luconi et al., 1996; Schuh et al., 2004).", "The epithelial calcium channel TRPV6 has been reported to be important for epididymal luminal calcium homeostasis (Weissgerber et al., 2011).", "However, we have found that TRPV6 co-localizes with the calcium-activated anion channel TMEM16A in some epididymosomes of the very proximal and the very distal epididymis.", "This co-localization is absent from the caput and proximal corpus epididymidis where supposedly most of the calcium is reabsorbed (Gao da et al., 2016).", "This suggests the presence of another calcium reabsorption mechanism in addition to the TRPV6-mediated pathway.", "It is known that vitamin K (VK) is a key modulator of extracellular calcium homeostasis in maintaining bone health and preventing ectopic calcification in blood vessels and other systems (Schurgers et al., 2013; Wallin et al., 2008; Weber, 2001).", "We therefore examined whether the vitamin-K-dependent (VKD) calcium regulation mechanism also functions in the regions of epididymis where the TRPV6-TMEM16A coupled complex is absent, and, if so, how VKD calcium regulation is involved in the cell-cell communications that take place in the epididymal luminal microenvironment.", "The epididymal epithelium actively secretes and absorbs epididymosomes, the EVs in the epididymal luminal microenvironment (Chen et al., 2016a; Conine et al., 2018; Sharma et al., 2016, 2018; Sullivan et al., 2005).", "Interestingly, EVs are also known as matrix-calcifying vesicles (Lotvall et al., 2014), an annotation suggesting that EVs and extracellular calcium homeostasis are functionally correlated.", "EVs are released in response to environmental stress by factors promoting calcification (Kapustin et al., 2011, 2015; Reynolds et al., 2004).", "In the vasculature, released EVs are loaded with MGP, a VK2-dependent calcification-inhibiting protein that requires the carboxylation by GGCX to prevent extracellular calcification (Reynolds et al., 2004).", "We speculate that GGCX and MGP are important for homeostatic calcium regulation in the epididymis, a mechanism that has not previously been studied.", "To sustain the carboxylation activity, GGCX relies on the VK cycle, in which VK is reduced by vitamin K epoxide reductase (VKOR) and continues the carboxylation reaction (Rishavy and Berkner, 2012; Schurgers et al., 2013; Stafford, 2005; Tie and Stafford, 2016).", "Warfarin is the specific inhibitor of the VKOR complex subunit 1 (VKORC1) able to interfere with the VK cycle (Hirsh et al., 2001; Piatkov et al., 2010).", "We therefore used the warfarin-induced VK2 deficiency model of adult rats to examine if GGCX and MGP participate in homeostatic calcium regulation in the epididymis.", "Overall, our data reveal a role of VK2-dependent GGCX-MGP pathway in extracellular calcium-dependent vesicle-mediated cell-to-cell communication during sperm maturation.", "In extending the biological relevance of VKD-MGP signaling from rats to humans, we also identified an SNP mutation of GGCX rs699664, which has been reported to cause a change from 325Arg to Gln, in association with idiopathic asthenozoospermia in infertile patients.", "Our study suggests that mutations of GGCX or dysfunction of the GGCX-MGP system in calcium-regulated epididymal sperm maturation is the pathological cause in some infertile men.", "As a first step to determine the expression level of the VK-cycle-associated proteins in the epididymis, real-time PCR method was used, and results showed that the mRNAs of GGCX, MGP, and VKORC1 were all expressed in the wild-type (WT) rat epididymis, whereas VKORC1L1 was detected at a negligible level (Figure 1A).", "The mRNA levels of the GGCX substrate MGP were about 10-fold higher in the epididymis than in the testis and other organs.", "High MGP mRNA expression was also detected in the kidney, an organ sharing many similar physiological properties with the epididymis.", "GGCX mRNA expression was stable throughout postnatal development of the epididymis, whereas MGP mRNA increased moderately at early ages then decreased slightly toward adulthood (Figure 1B).", "Western blots of rat epididymis and kidney showed the expected band for GGCX at about ∼88 kDa and for MGP at about ∼12 kDa (Figure 1C, arrows).", "The bands below the expected size of GGCX might represent its degradation products, as demonstrated in other studies (Tie et al., 2016).", "Noteworthy, a strong band was detected above the expected size of MGP at ∼32 kDa, which is a common characteristic of immunoblotting for MGP observed in other studies (Lomashvili et al., 2011), and in this study it was attributable to an MGP-mediated calcium-dependent protein complex, described in the subsequent section.", "To determine the cellular distribution and subcellular localization of the proteins, we performed confocal imaging and immunofluorescence labeling experiments.", "The results showed that both GGCX and MGP proteins were absent when the spermatozoa first entered the initial segment, but they appeared strongly in the epithelial cells of the caput and corpus epididymidis, then slightly decreased in the cauda epididymidis of WT males (Figures 1D and 1E).", "No MGP was detected in basal cells (Figure S1).", "Higher-magnification images showed that GGCX was mainly located in the intracellular granular organelles of epithelial cells, particularly enriched in the apical stereocilia of principal cells as well as in the cytoplasmic droplets of spermatozoa in the lumen (Figure 1F, arrows and asterisks).", "Consistent with the notion of a secretory protein on EVs, MGP immunolabeling was accumulated in the vesicular structures of the apical membrane of principal cells and in the luminal contents, especially in the corpus region.", "The MGP-positive vesicle-like structures were not only enriched in the apical membrane punctae of principal cells but were also aligned in rows on the surface of the sperm head (Figure 1G, double arrows).", "To determine the cellular colocalization of MGP and GGCX, double-immunofluorescence labeling of rat epididymal cryosections were performed (Figure 2).", "Colocalization patterns of MGP and GGCX were enriched in the corpus and cauda epididymidis, but scarce co-localization was observed in the initial segment and caput regions (Figure 2A).", "Three-dimensional projections of higher-magnification micrographs showed that the colocalization was found in vesicular structures of the epithelial cells and the cytoplasmic droplets of spermatozoa in the lumen (Figure 2B).", "These data suggest that in situ MGP carboxylation by GGCX occurs not only in epididymal epithelial cells but also in the epididymal luminal microenvironment.", "To investigate the reproductive function of the VKD proteins GGCX and MGP in vivo, we established a warfarin-induced VK2-deficient rat model by using a modified protocol supplemented with VK1 to avoid VK1-deficiency-induced hemorrhage, as described previously (Price et al., 1998).", "Compared with the WT rats, all rats in the VK1 control (VK1 CTL) and VK1 co-administered with 15 mg (+15 mg) and 30 mg (+30 mg) warfarin showed normal body weight (Figure 3A) and expected organ-to-body ratios for the testes, epididymides, kidneys, and spleens (Figure 3B).", "To evaluate fertility (Figure 3C), adult male rats in each group were mated with WT female rats.", "The mean litter size in the +15 mg and +30 mg groups was significantly smaller in a dosage-dependent manner than in control groups, whereas the rats in the VK1 control group had normal litter sizes, as did those of the WT rats.", "To elucidate the defects underlying the subfertility, the numbers of spermatozoa from the caput and the cauda epididymidis were measured (Figure 3D).", "The results showed that warfarin treatment reduced the cauda sperm count in a dosage-dependent manner, whereas the caput sperm count showed no significant difference.", "The assessment of cauda epididymidal spermatozoa by computer-assisted semen analysis (CASA) revealed that the percentage of total and progressively motile spermatozoa, straight line velocity (VSL), and averaged path velocity (VAP) were significantly decreased in the warfarin-treated groups compared with those of the control groups (Figures 3E–3H).", "Images captured during CASA showed that cauda but not caput spermatozoa were sluggish and frequently aggregated (Figure 3I).", "Numerous granules were accumulated around spermatozoa in the cauda epididymidal lumen of the warfarin-treated groups, also in a dose-dependent manner.", "The cauda spermatozoa of +15 mg and +30 mg treated rats displayed a significantly higher percentage of missing heads or a bent head wrapped around the neck when compared with controls (Figure 3J).", "Gross anatomical analysis of epididymal cryosections showed that there were some detached epithelial cells in the lumen of the caput, corpus, and cauda regions of warfarin-treated rats, whereas they were seldom found in the initial segment (Figure 3K).", "It was noted that the F-actin cytoskeleton immunofluorescent signals were also significantly reduced in the smooth muscle cells of warfarin-treated rats, whereas no obvious difference was found in the control groups (Figure 3L).", "Spermatogenesis in the testis was unaffected in any group (Figure 3M).", "The male subfertility, reduced sperm count, defective sperm motility, and exfoliation of the epididymal epithelium in warfarin-treated rats point to a potential disorder of the epididymal microenvironment in these rats.", "To evaluate this possibility, we performed MGP and GGCX double-immunofluorescence labeling, as well as TUNEL staining of the epididymis.", "The intracellular co-localization of MGP and GGCX in the epididymal epithelium was decreased, and there was an accumulation of detached cells and larger granules, especially in the epididymis of +30 mg warfarin-treated rats (Figure 4A).", "The MGP immunofluorescent signals from clear cells and principal cells in the epithelium were reduced dramatically in the +15 mg and +30 mg groups in a dose-dependent manner, suggesting that warfarin treatment caused serious epididymal epithelial cellular dysfunction.", "TUNEL analysis showed a few apoptotic cells in the epididymal lumen of the +15 mg group and numerous cells in the +30 mg group (Figure 4B).", "Unstained epididymal cryosections were analyzed by X-ray fluorescence (XRF) to reveal the relative Ca2+ intensity in different groups (Figure 5A).", "Results were correlated with optical histological images to assess the distribution of elements in the same area of the tissue.", "The Ca2+ concentration tended to be higher in warfarin-treated +15 mg and +30 mg groups than in control rats.", "Each XRF image was divided into an epithelial portion and a luminal portion for quantification (Figure 5B).", "The relative Ca2+ intensity was about 1.5 and 3times greater, respectively, in the epithelium and luminal area in the +30 mg group than in the control groups.", "To confirm the Ca2+ distribution change in the epididymal lumen after warfarin treatment, epididymal fluid collected from the vas deferens (VD) was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (Figure 5C).", "The Ca2+ concentration within the VD fluid was not significantly different in any group.", "However, after eliminating the interference of Ca2+ in the spermatozoa, the Ca2+ density ratio of VD fluid versus spermatozoa was significantly elevated by about three times in the +15 mg group and four times in the +30 mg group when compared with control groups.", "To examine the MGP carboxylation status in the epididymis after warfarin treatment, double-immunofluorescence labeling was performed on WT rat epididymal cryosections with an anti-full length MGP antibody and the conformation-specific anti-Gla residue antibody that recognizes proteins containing active γ-carboxyglutamyl (Gla) residues (Figure 6A).", "Most of the MGP in the WT rat epididymis was recognized by the anti-Gla residue antibody, both in the epithelium and in the lumen.", "Western blot results confirmed the presence of an anti-Gla residue antibody identified-MGP band at ∼12 kDa (Figure 6B).", "These results suggest that the extent of binding of the anti-Gla residue antibody reflects the MGP carboxylation status.", "The immunolabeling of the Gla-residues in the epididymis from the different groups was undertaken to assess the MGP carboxylation level (Figure 6C).", "The signals from Gla residues were reduced in warfarin-treated epididymides, confirming that warfarin treatment promotes the inactive uncarboxylated form of MGP (ucMGP).", "Western blotting of MGP in the WT rat kidney and epididymis showed two bands at ∼32 and ∼12 kDa.", "To determine if the ∼32 kDa band was caused by nonspecific binding of anti-MGP antibody, we conducted western blots with MGP antigen peptide (Figure 7A) and found both the 12 and 32 kDa bands were almost diminished, which could mean that the 32 kDa bands contain MGP antigen but are tightly aggregated to other proteins with a size roughly around 20 kDa.", "Western blot analysis in the presence of extra EDTA, Ca2+, and Mg2+ (Figures 7B–7D′) showed that the intensity of the ∼32 kDa bands was increased in the presence of 250 mM EDTA (a calcium chelator) in the homogenates, whereas increasing Ca2+ from 2 to 10 mM reduced the intensity.", "The intensity seemed slightly decreased in the presence of Mg2+.", "Increasing the Ca2+ concentration from 25 to 100 mM further diminished the amount of the 32 kDa band, as well as most of other proteins including actin, but not the 12 kDa MGP protein (Figure S2).", "All these results suggest that the MGP aggregate formation with target proteins is dependent on the presence of low calcium concentration and is inhibited by excessive calcium and magnesium.", "The biphasic calcium dependence of MGP-bound aggregation was confirmed with western blot of protein lysates from immortalized mouse epididymal DC2 cells in the presence of various calcium concentrations (Figures 7E and 7E′).", "To identify the target proteins, we analyzed the 32 kDa bands excised from SDS-PAGE gels by in-gel digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS).", "The analysis yielded over 1,000 proteins (detected in at least two of five trials of three rounds of proteomic experiments of three animals) in both the 32 kDa bands of epididymis and kidney with molecular sizes ranging from 10–530 kDa (data not shown).", "Of these proteins, 102 and 112 proteins were identified with molecular size between 20 and 25 kDa in the 32 kDa bands of the epididymis and kidney, respectively (Figure 7F).", "Bioinformatic analyses using STRING free online software on these proteomes from the epididymis and kidney and their common genes, respectively, were performed.", "Results showed that sorting complex required for transport (ESCRT) and Rab proteins responsible for vesicle-mediated transport and membrane trafficking, as well as the response to oxidative stimulus, are the key protein-protein interaction networks (Figures 7F’, S3, and S4).", "As proof of MGP binding with the identified proteins, we picked LCN2 (∼22 kDa) for the verification for its commercially available antibody and high expression level in the epididymis (Figure S5).", "Double immunofluorescence labeling of MGP and LCN2 revealed the co-localization in the luminal content but not in the epididymal epithelium (Figures 7G and 7G′).", "To check whether mutations of human VKD MGP and GGCX affect male fertility, we screened for potential mutations in the exons of MGP and GGCX in a cohort of 199 patients with idiopathic asthenozoospermia and 110 fertile controls.", "The sperm samples collected from fertile healthy donors exhibited normal motility, morphology and sperm counts.", "We observed a significant association of an SNP mutation rs699664 in GGCX with male infertility under the recessive model (Figure 8A).", "The rs699664 is a non-synonymous T/C SNP in the eighth exon of GGCX (Figure 8B), and the allele T is considered regressive (Kinoshita et al., 2007).", "As reported in the National Center for Biotechnology Information (NCBI) database, this mutation leads to a change from 325Arg to Gln in GGCX, which is in the transmembrane region of GGCX.", "Our results suggest that the VKD GGCX-MGP system could be a target for clinical screening and potentially for the treatment of idiopathic male infertile cases.", "In this study, we demonstrate that the VKD enzyme GGCX and its substrate MGP play a crucial role in the extracellular calcium-dependent vesicle-mediated cell-cell communication involved in the homeostatic regulation of luminal microenvironment and sperm maturation in the epididymis.", "Both GGCX and MGP proteins are present in vesicular structures in the cytoplasm of epididymal cells and on the surface of spermatozoa in the lumen.", "This specific cellular localization pattern suggests that GGCX and MGP proteins function in the epididymal lumen microenvironment via the regulation of vesicle-mediated intercellular communication.", "It is likely that both GGCX and MGP proteins on the sperm surface and in the cytoplasmic droplets arise from epithelial cells via epididymosomes.", "Consistently, our proteomic analysis of these biphasic calcium-dependent MGP-binding proteins indicate that vesicle-mediated transport and stress-induced membrane trafficking are the key underlying protein-protein interactions.", "We speculate that the spermatozoa exert metabolic demands on the epididymal cells as well as provide survival signals to them to sustain a microenvironment that meets the needs of the spermatozoa during their transit through epididymis.", "We found that optimal extracellular calcium is the mediating cofactor.", "By using the warfarin-induced VK2 deficiency rat model, we found that VK cycle disruption resulted in epididymal sperm defects, epithelial exfoliation, and macro-granules accumulation in the epididymal lumen and male infertility.", "We attribute these phenotypes to the excessive calcium in the epididymis as a result of inactive ucMGP caused by GGCX inhibition after warfarin treatment.", "The increased ucMGP in the epididymal lumen loses the ability to bind Ca2+, which eventually leads to local calcium deposition and promotes calcium phosphate crystal nucleation, as reported from in vitro studies (Schurgers et al., 2007; Yagami et al., 1999).", "Our results therefore showed that VK2 is essential for epididymal sperm maturation and male fertility.", "Alternatively, they indicated a potential use of warfarin as a male contraceptive by targeting GGCX-MGP.", "One unanticipated finding was that, in the MGP western blot assays of epididymal and kidney lysates, we always observed an unknown band at around 32 kDa, a band approximately 20 kDa larger than that of MGP.", "Our results indicated that this band is a protein complex containing MGP.", "Moreover, we found that the 32 kDa band was intensified by lowering calcium concentration with the chelation of EDTA and diminished by increasing the concentration of calcium or magnesium in the crude lysates.", "Although EDTA binds with higher affinity to magnesium than calcium, the higher sensitivity of the 32 kDa MGP band toward calcium suggests that the complex is mainly regulated by calcium rather than by magnesium.", "The binding of MGP to targeted proteins can also be explained by its special chemical property as well as the unique microenvironment in the epididymis.", "The inactive form (ucMGP) is a protein with an isoelectric point of 8.8, so that at the acidic pH of ∼6.8 in the epididymal lumen (Breton et al., 1996; Levine and Marsh, 1971; Shum et al., 2009), the ucMGP protein carries five positive charges; the active cMGP would indeed be charge neutral after the addition of five negatively charged gamma-carboxyl groups by GGCX.", "Thus, cMPG is likely water insoluble, whereas ucMGP is soluble.", "However, cMGP (carboxylated MGP) has a much stronger binding affinity for divalent calcium ions than ucMGP, and five calcium ions can interact with each MGP molecule (Hackeng et al., 2001; Huang et al., 2003).", "After calcium binding, cMGP carries ten positive charges, which dramatically increases its water solubility.", "We speculate that this highly charged cMGP can bind to the negatively charged moieties in the extracellular matrix, including phosphate-containing macromolecules and phosphatidylserine-containing phospholipid membranes, which are present in EVs (Skotland et al., 2018).", "In another scenario, cMGP could also bind to the inorganic calcium compound hydroxyapatite (Hackeng et al., 2001; Huang et al., 2003), whereas magnesium can interfere with the affinity of cMGP for calcium (Roy and Nishimoto, 2002).", "It is likely that the high-calcium condition triggers the formation of hydroxyapatite-like compounds that competitively bind to MGP with the organic proteins and subsequently lead to pathological conditions (Tan et al., 2012).", "Consistently, our result of titrating calcium concentrations versus the density of the 32 kDa MGP band in cultured DC2 cell lysates (see Figure 7E) revealed that sub-millimolar levels of calcium favor MGP-protein complex formation, which would occur in the sub-millimolar calcium environment of epididymal fluid (Levine and Marsh, 1971; Turner, 2002; Weissgerber et al., 2011).", "Moreover, when the calcium but not magnesium concentration was increased to as high as 25 to 100 mM, proteins in the lysates could not be electrophoresed normally in the PAGE gel, but the intensity of the ∼12 kDa band was indeed increased compared with actin and the 32 kDa bands, which is believed to be due to the increased solubility of cMGP with excessive calcium in the protein extracts.", "This also suggests that most MGP is in the active form under physiological conditions of the epididymis and kidney.", "The diminished 32 kDa bands under high calcium concentration also suggest that the MGP-complex is not susceptible to the reducing conditions of western blot analysis but to the availability of the divalent ions.", "Taken together, our results show that MGP-mediated protein-complex aggregation is dependent on sub-millimolar calcium levels, whereas excessive calcium levels inhibit aggregation.", "We propose that, under the physiological low calcium microenvironment of the epididymal lumen, MGP-meditated calcium-dependent macromolecules transport is essential for sperm maturation and plays a critical role in epididymosome-mediated transgenerational information transfer from father to offspring.", "To extend our rat study on VKD proteins to human reproductive health, we identified a significant difference in the GGCX SNP mutation rs699664 in the asthenozoospermic samples compared with controls, but we found no differences in MGP.", "It is known that the GGCX rs699664 SNP mutation results in the amino acid change from 325Arg to Gln in GGCX (De Vilder et al., 2017) and is associated with bone density in elderly women (Kinoshita et al., 2007; Rieder et al., 2007).", "Asthenozoospermia is considered a common cause of male infertility characterized by a reduction in both sperm motility and normal sperm morphology (Sanchez-Alvarez et al., 2012).", "Although the causes of asthenozoospermia are not yet fully understood, an abnormal redox state could be involved in its pathology (Balercia et al., 2004; Sun et al., 2014).", "Biochemically, the VK cycle is a redox cycle (Ivanova et al., 2018; Stafford, 2005) and it may regulate not only the homeostasis of calcium but also the redox status of cells and tissues.", "Previous investigations indicate that MGP's carboxylation status does not affect its secretion into the extracellular environment (Price et al., 1994; Wajih et al., 2004).", "Consistently, not all MGP in the epididymal lumen is carboxylated.", "The co-localization of GGCX and MGP suggests that both proteins are secreted in vesicular forms and that uncarboxylated MGP can be activated by GGCX carboxylation in situ in the lumen.", "Therefore, both GGCX carboxylation and the prevention of MGP mineralization may be regulated by the epididymal luminal microenvironment.", "We speculate that dysregulation of such luminal GGCX-MGP system is the underlying cause of sperm maturation defects from warfarin administration and from the GGCX rs699664 mutation.", "Since the amino acid in position 325 of GGCX is near the MGP's binding site, which is located outside the ER lumen (De Vilder et al., 2017; Tie and Stafford, 2016), it is believed to stay outside of the vesicles and encounter ucMGP in the epididymal luminal microenvironment after its release.", "It is speculated that the substitution of 325Arg/Gln affects the enzymatic activity of GGCX (De Vilder et al., 2017).", "Since ucMGP is a peptide containing five positive charges and the 325Arg amino acid of GGCX is singly positively charged and, in addition, it has been hypothesized that all the five γ-glutamyl residues of MGP are carboxylated in one round by GGCX in sequential order (Rishavy and Berkner, 2012), the function of 325Arg in GGCX is therefore likely to stabilize the MGP γ-glutamyl residues binding by dynamic charge-charge interaction with GGCX promoting the sequential carboxylation.", "The mutation of positively charged 325Arg to a neutral 325Gln moiety in the GGCX rs699664 SNP mutation might thereby result in an unfavorable condition for MGP binding during carboxylation, leading to unsuccessful MGP carboxylation and eventually male infertility.", "In summary, our study demonstrates that the VK2-dependent protein MGP and its carboxylation enzyme GGCX play an essential role in epididymal sperm maturation and male fertility in rats and that the mutation of GGCX is associated with one form of human male infertility.", "Our study also suggests that the VKD signaling axis is a promising target for the development of diagnostic products and therapies for male infertility.", "We also showed that the GGCX-MGP system participates in vesicle-mediated transport, presumably between epithelial cells and spermatozoa in the epididymis and that it is subject to the regulation by luminal calcium in a biphasic manner.", "In view of the versatile function of EVs in many physiological processes and the ubiquitous expression of MGP, our findings also provide insight into the physiology of VKD GGCX-MGP-dependent calcium signaling in other organs, such as kidney.", "Although this study provided insights into the VKD regulation of epididymal sperm maturation, subsequent studies are necessary to elucidate clearly the molecular mechanism of the GGCX-MGP system for sperm maturation in men.", "Such studies include providing the direct evidence for the exact roles and contributions of the GGCX mutations, defining the calcium-dependent MGP-mediated intercellular interaction networks, determining the physiological roles of biphasic calcium dependence of MGP-protein complexation, as well as identifying the potential transepithelial transportation pathways for these complexes across epididymis.", "All methods can be found in the accompanying Transparent Methods supplemental file." ]	[ "Epididymal sperm maturation requires VK2-dependent GGCX-mediated MGP carboxylation", "A GGCX SNP mutation is found in infertile men suffering from asthenozoospermia", "Carboxylated-MGP regulates intercellular calcium signaling in the epididymal lumen", "Calcium-regulated MGP binds to proteins in a biphasic-manner and favors low levels" ]
Is there a place for human fetal-derived stem cells for cell replacement therapy in Huntington's disease?	Huntington's disease (HD) is a neurodegenerative disease that offers an excellent paradigm for cell replacement therapy because of the associated relatively focal cell loss in the striatum. The predominant cells lost in this condition are striatal medium spiny neurons (MSNs). Transplantation of developing MSNs taken from the fetal brain has provided proof of concept that donor MSNs can survive, integrate and bring about a degree of functional recovery in both pre-clinical studies and in a limited number of clinical trials. The scarcity of human fetal tissue, and the logistics of coordinating collection and dissection of tissue with neurosurgical procedures makes the use of fetal tissue for this purpose both complex and limiting. Alternative donor cell sources which are expandable in culture prior to transplantation are currently being sought. Two potential donor cell sources which have received most attention recently are embryonic stem (ES) cells and adult induced pluripotent stem (iPS) cells, both of which can be directed to MSN-like fates, although achieving a genuine MSN fate has proven to be difficult. All potential donor sources have challenges in terms of their clinical application for regenerative medicine, and thus it is important to continue exploring a wide variety of expandable cells. In this review we discuss two less well-reported potential donor cell sources; embryonic germ (EG) cells and fetal neural precursors (FNPs), both are which are fetal-derived and have some properties that could make them useful for regenerative medicine applications.	[ "BIOC", "NEUR" ]	[ "Cell therapy", "Fetal-derived neural precursors", "Human embryonic germ cells", "Huntington's disease", "Neural transplantation", "Stem cell" ]	[ "1307", "2804" ]	[ "Most neurodegenerative conditions are currently untreatable and, for the majority, treatments able to positively influence the underlying pathogenesis are likely to be a long way off (Bartus and Johnson, 2016; Gribkoff and Kaczmarek, 2016; Wild and Tabrizi, 2014).", "This makes strategies such as cell replacement therapy attractive, because a condition may be a target for cell replacement as long as there is relatively focal (at least in the early stages) loss of defined groups of neurons.", "There has been interest over the last couple of decades in treating Huntington's disease (HD) with cell replacement therapy.", "HD is a slowly progressive condition in which there is relentless deterioration of cognitive, motor and psychiatric faculties over a 20–30year period.", "Currently there is no available disease-modifying treatment, but it represents a good target for cell replacement therapy as it is a well characterised monogenetic condition in which there is relatively focal loss of medium spiny striatal neurons (MSN) (Rosser and Bachoud-Lévi, 2012).", "Furthermore, it is anticipated that progress made towards achieving functionally effective grafts in HD will be applicable to other neurodegenerative conditions (Dunnett and Rosser, 2014).", "One of the key requirements for cell replacement therapy to be functionally effective is that the donor cells have the capacity to be precisely differentiated into the target cell type, i.e., MSNs for HD (Precious and Rosser, 2012).", "The most convincing evidence so far that cell replacement can be effective in HD comes from both animal and human studies using donor cells derived from the whole ganglionic eminence (WGE) in the fetal brain (Pauly et al., 2012; Mazzocchi-Jones et al., 2009; Döbrössy and Dunnett, 2003).", "The WGE is the area that will eventually become the adult striatum and is where MSNs are born and develop (Deacon et al., 1994; Olsson et al., 1995, 1998; Marin et al., 2000; Evans et al., 2012; Straccia et al., 2016).", "Thus, MSNs differentiating from WGE have been committed to an MSN lineage during the process of normal development.", "Such cells are currently regarded as the “gold standard” for cell replacement in HD.", "Optimal grafts result when transplants are derived from fetal WGE collected during the peak period of MSN neurogenesis (i.e., approximately embryonic day 14 in rat and 8–10 weeks gestation in human) (Dunnett and Rosser, 2011).", "Transplantation of developing MSNs into the degenerating striatum has been shown to ameliorate motor and cognitive deficits in animal studies, primarily in rats and primates (Schackel et al., 2013; McLeod et al., 2013; Paganini et al., 2014; Yhnell et al., 2016).", "Such studies have allowed the mechanisms underlying the functional improvement to be explored, and have shown that implanted cells can integrate into the circuitry and make functional synaptic connections, providing that they are of the appropriate phenotype (i.e., destined to become MSNs) and were procured within the appropriate developmental window (Dunnett and Rosser, 2014).", "Preliminary evidence of functional efficacy in human transplants comes from a seminal French study that reported human fetal-derived graft survival and significant improvements in both motor and cognitive function in three patients over an approximately six-year period (Bachoud Lévi et al., 2000; 2006).", "Enhanced FDG-positron emission tomography signal in the frontal cortex of these individuals suggested that the implanted cells had integrated into the striatal neural circuitry and made functional connections with relevant cortical regions (Gallina et al., 2014).", "The proof-of-concept provided by this study is encouraging and demonstrates that transplantation of “native” developing MSNs into the damaged striatum can produce functional improvements in at least some patients with HD.", "Nevertheless, there is still a pressing need to undertake further studies of fetal WGE transplantation both to confirm the ability of transplanted WGE cells to improve function and to identify the parameters necessary to increase the reliability of the process and understand which patients are most likely to benefit.", "For the longer term, however, it will be necessary to identify expandable sources of donor cells for clinical application, as primary fetal cells present several challenges: they are scarce (an issue compounded by the fact that bilateral transplants in HD require cells from approximately four fetuses, i.e., eight WGEs); they cannot be stored long-term (thus causing logistical problems for coordinating cell collection, surgery and pathological screening of cells); and they are difficult to standardise.", "Thus, in addition to continuing primary fetal transplants for the reasons outlined above, it is also important to identify cells that can be expanded in number in vitro and stored to facilitate GMP (Good Manufacturing Practice) production, whilst maintaining the capability to generate striatal MSNs.", "Expandable sources of cells, including human embryonic stem (ES) and human adult-derived induced pluripotent stem (iPS) cells, which can be directed down neural lineages and specified to the required cell type are reviewed extensively elsewhere (Bachoud-Lévi and Perrier, 2014; Choi et al., 2014; Ross and Akimov, 2014; Chen et al., 2014).", "Here we discuss the potential of two types of expandable cells derived from human fetal tissue; embryonic germ (EG) cells and fetal neural precursor (FNP) cells, as potential donor cells for cell replacement therapy in HD.", "The reasons for being interested in these cell sources are first, that human fetal tissue will need to be collected for some time to come in order to supply cells for proof-of-concept and optimisation studies as outlined above, and secondly that both cell types have theoretical advantages over hES and hiPS cells for regenerative medicine applications, as discussed further below.", "EG cells are derived from primordial germ cells (PGCs) that reside in the gonadal ridge of first trimester embryos.", "PGCs are induced from pluripotent epiblast cells very early in embryonic development (Ohinata et al., 2005), and continue to express markers of pluripotency such as Oct4, Nanog and SSEA-1 (Pashai et al., 2012).", "In vivo, PGCs are unipotent and destined to give rise exclusively to gametes, however, in vitro exposure of mouse PGCs to exogenous fibroblast growth factor (FGF) 2, leukaemia inhibitory factor (LIF) and stem cell factor (SCF) can cause conversion to EG cells, which proliferate rapidly and form colonies similar to those observed when culturing ES cells (Durcova-Hills et al., 2008).", "EG cells show all the hallmarks of pluripotency, including differentiation into cell types from all three germ layers, as well as formation of teratomas and chimeras (Labosky et al., 1994; Stewart et al., 1994).", "So far, only a small number of laboratories have reported successful conversion of human PGCs to human EG (hEG) cells using FGF2, LIF and membrane-bound SCF provided through feeder cells (Pan et al., 2005; Turnpenny et al., 2003; Shamblott et al., 1998; Liu et al., 2004).", "However, most groups report that, unlike their mouse counterparts, hEG cells do not form teratomas and cannot be maintained in culture long-term, due to their high tendency to differentiate rather than preserving pluripotent traits (Turnpenny et al., 2006).", "Although resistance to pluripotency and indefinite culture presents some challenges, it also presents a theoretical advantage in that neuronal precursors derived from such sources may be less likely to overgrow or form tumours post-transplantation; something that is currently a problem for many hES cell derived donor cells (Master et al., 2007).", "To explore the possibility that hEG cells could be used for cell replacement applications, we attempted to generate our own hEG cell cultures.", "Human fetal tissue was collected through the South Wales Initiative for Transplantation (SWIFT) program in accordance with the Polkinghorne and Department of Health guidelines, with full ethical committee approval, and under the Cardiff University Human Tissue Act 2004 research licence (Kelly et al., 2011).", "We harvested gonadal ridges from over 100 human embryos, ranging in age from 6 to 12 weeks gestation, and subjected them to established methods in order to first generate cultures of PGCs.", "Harvested tissues gave rise to proliferating cells that were positive for alkaline phosphatase (AP), regarded as a marker of pluripotency as well as of PGCs (Fig. 1A).", "Some also expressed SSEA-1 and SSEA-4 (Fig. 1B and C, respectively), but very few were Oct4 positive (Fig. 1D).", "Over 14 days in culture the proportion of cells expressing AP and SSEA-1 increased, with AP staining consistently more abundant than SSEA-1 (Fig. 1E).", "Conversely, expression of Oct4 fell (Fig. 1E), suggesting that AP staining, whilst convenient, is probably not a reliable PGC marker on its own.", "Having obtained PGCs in culture, the next step was to convert them to stable EG cell lines.", "In line with the literature on hEG cell derivation, this proved more difficult.", "Despite using numerous tissue samples of various ages and exploring numerous different parameters including media components, growth factors, feeder cells, passaging techniques and cell isolation, we failed to convincingly generate hEG cell colonies from any of our PGC cultures.", "The manipulations tested are summarised in Table 1 and included:", "Growth factors.", "All our cultures contained both FGF2 (based on reports of successful conversion of PGCs to EG cells in both mouse and human (Shamblott et al., 1998; Turnpenny and Hanley, 2007)) and either LIF or oncostatin, another member of the interleukin-6 family that has overlapping activity (Mosley et al., 1996).", "We also tested: soluble SCF, required for survival and proliferation of PGCs (Tu et al., 2007; Dolci et al., 1991; Godin et al., 1991; Matsui et al., 1991); GDNF, found in female germ cells and needed for germ cell survival in male mice (Miles et al., 2012; Farhi et al., 2010); EGF, which induces proliferation in chick PGCs (Ge et al., 2009); and the nodal signalling molecule activin A, which is critical for maintenance of pluripotency in human cells and stimulates Oct4 transcription in spermatogonial stem cells (Vallier et al., 2005; He et al., 2009).", "None of these factors, either alone or in combination, led to the appearance of pluripotent hEG cells.", "High dose (100 ng/ml) SCF supplementation, added to either StemPro 34 medium or standard medium containing 15% fetal calf serum (FCS), as this has been reported to promote colony formation of germ cells in mixed fetal gonadal cultures grown without feeder cells (Tu et al., 2007).", "We observed that when small tissue fragments were present in these cultures, Oct4 positive cells appeared on the outside of the fragments and formed visible clusters on the surface, irrespective of the culture medium used (Fig. 1F).", "With continued culture, the somatic cells of the gonadal fragments also attached to the dish and spread out to form a monolayer on which PGCs migrated out.", "Twenty-four hours after plating, PGC clusters could be removed under a dissection microscope and transferred to feeder-coated plates, where they remained visible for several weeks but did not convert into hEG cell colonies.", "Various feeder layers.", "The STO mouse embryonic fibroblasts cell line (ATCC CRL-1503) has been reported as the most successful cell line for this purpose (Turnpenny and Hanley, 2007; Shamblott et al., 1998), but is known to drift with passage, such that sub-clones can vary considerably in their properties, including expression of membrane bound SCF.", "We used several cloned STO lines, the spontaneously immortalised testicular stromal cell line JK-1 (Kim et al., 2008), and several non-transformed lines of human fetal somatic fibroblast-like cells (He et al., 2007).", "PGC purification.", "A practical obstacle to maintaining cultures of PGCs was the high proliferative capacity of somatic gonadal cells.", "We attempted magnetic bead purification (MACS) using the pluripotency marker CD133, which is expressed in the germline (Gashaw et al., 2007).", "However, this did not result in a sufficiently pure population of human PGCs that avoided contamination with somatic cells.", "As germ cells are highly prone to apoptosis in vitro, preventing isolation by FACS, we attempted a gentler approach by adapting methods developed for isolating spermatogonial stem cells from adult testis samples using a three-step matrix selection process on plastic, collagen and laminin, followed by culturing on feeder cells to induce conversion to pluripotent stem cells (Conrad et al., 2008).", "However, PGCs did not form clusters under these conditions.", "Despite the numerous different manipulations carried out in a large number of fetal samples, and the successful (albeit short-term) culture of PGCs, we were unable to generate any hEG cell lines.", "We did, however, observe an effect of high SCF concentrations on human PGC adhesion and migration, which could lead to future tools for isolating this highly vulnerable cell type.", "It seems likely that the factors which can induce de-differentiation of mouse PGCs to a pluripotent state are not sufficient for the human equivalent.", "This is congruent with the fact that no long-term expandable hEG cell lines have become available to researchers, despite their generation first being reported in 1998 (Shamblott et al., 1998).", "Fundamental differences between mouse and human gamete formation may further prevent establishment of stable hEG cell lines, the timing and regulation of epigenetic re-programming being obvious candidates.", "It has long been known that proliferative cells with the capacity to differentiate into cells of a neural lineage (FNPs) can be isolated from the developing fetal WGE, and other brain regions.", "This was initially shown in rodents (Reynolds et al., 1992) and since then numerous papers have demonstrated similar findings in both rodent and human (Murphy et al., 1990, 1994; Bartlett et al., 1995; Fricker et al., 1995).", "There are clear species differences in the responses of FNPs to specific proliferation media, with murine cells tending to proliferate for much longer periods of time than rat and porcine cells (Svendsen et al., 1998; Ciccolini and Svendsen, 1998; Armstrong et al., 2000, 2002).", "In addition, if hFNPs could survive cryopreservation this would ease current practical constraints associated with scheduling the neurosurgery and would also permit at least some standardisation of the cells, which cannot currently be achieved for primary hWGE.", "Specifically, hWGE can only be reliably held in culture (using media to reduce metabolic processes, ie “hibernation”) for a short period of time (up to 8 days; Hurelbrink et al., 2000).", "This is not a long enough period of time to permit full quality control of the tissue.", "Furthermore, FNPs are multipotent, rather than pluripotent, and are lineage restricted.", "Thus they are less likely to give rise to fast-growing tumours following grafting, in contrast to grafts of pluripotent-derived cells.", "A major problem for use of FNPs for regenerative medicine has been the tendency for hFNPs to produce progressively fewer neurons with increasing periods of time in culture.", "Furthermore, hWGE-derived FNPs that have undergone passaging in vitro produce fewer MSN-like neurons and survive poorly following transplantation into rodent HD models (Caldwell et al., 2001; Zietlow et al., 2012).", "We have previously suggested that the loss of neurogenic potential may be due to loss of positional information as the hFNPs continue to proliferate in the absence of developmental signals to which they would normally have been exposed during development in the embryo (Zietlow et al., 2005).", "Indeed, PCR analysis of mouse WGE-derived FNPs, compared to primary WGE, shows a dramatic fall in the levels of striatal-specific gene expression (unpublished observations), although in human cells, at least over the first few passages, the expression of striatal markers appeared relatively stable (Martín-Ibáñez et al., 2016).", "Despite this apparent phenotypic change, short term proliferation of hFNPs still results in a modest increase in cell number (1.6 fold increase in cell number over 10 days in culture), which could have important practical applications (Kelly et al., 2007).", "Previous data of ours suggests that, in contrast to transplanted long-term expanded hFNPs, transplants of short-term (10 day) expanded WGE-derived hFNPs retain the ability to survive and integrate into the host brain (Kelly et al., 2007).", "This study reported that grafts of 10 day expanded hFNPs analysed after 12 weeks survival in vivo sent out fibre projections that were target-specific for cells of that origin.", "Furthermore, the hFNP grafts produced more profuse outgrowths than did primary WGE grafts (Kelly et al., 2007).", "This data was encouraging in that it suggested that short-term expanded FNPs had a potential to reconnect neural circuitry that is as good, if not better, than that of primary fetal cells, but their low proliferative potential made them less attractive than the highly proliferative pluripotent stem cells.", "However, recent reports have highlighted that the challenge of directing hES cells to a genuine MSN phenotype capable of ameliorating functional deficits has so far been greater than initially anticipated.", "There are sporadic reports of limited improvements in small preclinical studies, for example some improvement in rotation following ES cell-derived MSN grafts (Delli Carri et al., 2013), but to date this falls considerably short of the sorts of improvements seen in rat allografts and the more successful human allograft studies.", "Thus, we suggest that it is important to continue exploring alternative donor cell sources and recently we have begun again to consider the potential of short-term expanded hFNPs as donor cells for regenerative medicine.", "These cells may have the capacity to more readily respond to developmental signals that would allow them to maintain their identity over the period of expansion in culture.", "Such ‘epigenetic memory’ has been suggested from work in several adult somatic tissues (Hiler et al., 2015) and may be an important aspect of these cells and allow a more adaptable approach for generating large populations of MSNs for clinical application.", "In a comparison of hFNP and hWGE grafts 20 weeks post-transplantation, graft-derived DARPP32 positive cells (a classic marker of MSNs) were seen both in primary WGE and expanded WGE grafts (Fig. 2), and stereological analysis revealed no significant difference in the number of graft-related DARPP-32- positive neurons (Fig. 2A).", "Both primary and expanded FNP grafts were also positive for calbindin (stains both MSN and striatal interneurons).", "Interestingly, hFNP grafts differed from hWGE grafts in an important aspects of morphological structure; WGE grafts are typically organised into “P zones” (containing mostly striatal-like cells), which stain for a variety of markers, including acetylcholinesterase (AChE), and “non P zones” containing mostly non-striatal (probably cortical-like) cells (Graybiel et al., 1989; Wictorin, 1992).", "The hFNP grafts appeared to show very weak, or no organisation into such zones, as shown by AChE staining (Fig. 2B).", "Our own interpretation of this is that the hFNP grafts may present an advantage in their lack of non-P zones; non-P zones are thought to result from cortical progenitors which migrate through the WGE during development and thus may ‘dilute’ the effect of the graft.", "However, it is important to emphasise that this is speculation and further studies are needed to resolve this issue.", "To date, there is very limited literature reporting on functional assessments of hFNP grafts in HD models.", "McBride et al in 2004 reported improved functional effect on the cylinder task following transplantation of human cortical derived FNPs in the quinolinic acid lesion model of HD (McBride et al., 2004).", "However, this was not supported by histological evidence of graft-derived MSNs, based on DARPP-32 expression 8 weeks post transplantation.", "This relatively short time course for graft maturation may be the reason for the lack of DARPP-32 expression along with the fact that the cells were derived from the developing cortex, rather than WGE, and were expanded in culture for a long period of time, which as we have shown previously is not supportive to good graft survival (Zietlow et al., 2005, 2012).", "Studies using human fetal tissue-derived neural precursor immortalised cell lines have also reported survival, differentiation and integration of transplanted cells with modest behavioural recovery in some cases (for example Roberts et al., 2006; Ryu et al., 2004).", "However, it is clear that systematic studies examining the functional effects of WGE-derived FNPs in HD models are required.", "In summary, there is some evidence that hFNPs retain the ability to default to an MSN-like phenotype following modest expansion in vitro, and also that they are capable of projecting to target specific brain regions.", "It is important to acknowledge that most studies to date, including the ones presented here, have relied on default neuronal differentiation of the transplanted FNPs post-transplantation.", "That is, FNPs are precursor cells, and in these experiments they have not undergone directed differentiation towards specific neuronal phenotypes.", "An important next step will be to subject FNPs to molecules known to be important for MSN differentiation.", "In conclusion, we review the potential of two major sources of fetal-derived stem cells as donor cells for regenerative medicine in HD.", "At this stage in the development of CNS regenerative medicine it is important to properly consider all possible donor cell sources, as there are factors associated with each that make them more or less suitable for individual conditions.", "For example, another strategy is the use of fetal-derived iPSCs, on the basis that they may possess a different range of properties to adult-derived iPSCs, but there is currently very little literature in this area.", "Here we discuss our extensive attempts to produce hEG cells, which we were unable to convincingly generate and maintain, and this is consistent with the small numbers of reports of successful conversion in the literature.", "Further exploration of this cell type will need to await a better understanding of the biology of hPGCs.", "In contrast, we suggest that FNPs hold more promise.", "Although they cannot as yet be regarded as potential donor cells for neural transplantation, we suggest that there is sufficient positive data to support further exploration of their potential." ]	[ "Cell replacement therapy for neurodegenerative disease.", "The shortage of primary human fetal tissue that necessitates the search for alternatives.", "Potential of fetal derived neural precursor cells.", "What about embryonic germ cells?" ]
Survey strategies to quantify and optimize detecting probability of a CO2 seep in a varying marine environment	Designing a marine monitoring program that detects CO2 leaks from subsea geological storage projects is challenging. The high variability of the environment may camouflage the anticipated anisotropic signal from a leak and there are a number of leak scenarios. Marine operations are also costly constraining the availability of measurements. A method based on heterogeneous leak scenarios and anisotropic predictions of chemical footprint under varying current conditions is presented. Through a cost function optimal placement of sensors can be given both for fixed installations and series of measurements during surveys. Ten fixed installations with an optimal layout is better than twenty placed successively at the locations with highest leakage probability. Hence, optimal localizations of installations offers cost reduction without compromising precision of a monitoring program, e.g. quantifying and reduce probabilities of false alarm under control. An optimal cruise plan for surveys, minimizing transit time and operational costs, can be achieved.	[ "COMP", "ENVI" ]	[ "Detection", "Marine", "Monitoring", "Seeps" ]	[ "1712", "2302", "2305" ]	[ "All geological CO2 storage projects need a surface monitoring program with four main objectives; 1) maximize assurance of storage integrity, 2) assure that a leak will likely be detected, 3) continue to build an accurate baseline to capture trends and natural variability, and 4) to prevent unjustified accusations of adverse effects from the storage project (EU Commission, 2011; Blackford et al., 2015a).", "The 2011 incident at the Weyburn project is an example of the latter (Boyd et al., 2013).", "With proper selection and operational procedures geological CO2 storage projects will be designed not to leak.", "Geological monitoring of the reservoir, complex and overburden will be the primary monitoring strategy to assure operations according to plans and to detect any adverse events.", "Still, due to precision and resolution, there will always be an uncertainty that CO2 migrates toward the surface undetected.", "Transport of the CO2 within the formation might find other potential pathways like fractures and/or faults or the CO2 might create new pathways (Oldenburg and Lewicki, 2006).", "Leaks following weakness zones in geological structures or old wells might cause high flux rates of CO2 far away from the injection site.", "More diffusive leaks can be caused by CO2 migrating through the overburden reaching the surface over a relatively large area.", "Leak scenarios will be an intrinsic component in site characterization and lay the ground for risk and impact assessments necessary for obtaining a permit.", "The site specific spatial and temporal evolution of the CO2 plume within the storage complex determines the area to be monitored.", "The anisotropic evolution of the Sleipner plume (see e.g. Boait et al. (2012)) illustrate that subsurface monitoring is a prerequisite for designing a marine monitoring program.", "For offshore storage projects a monitoring program for the seafloor and the water column can be costly, and the marine environment is hostile for instrumentations.", "It is therefore suggested that the seafloor monitoring program has three levels of modus operandi; 1) anomaly detection modus, 2) confirmation and location modus, and 3) seep quantification modus.", "Blackford et al. (2015a) suggest a fourth step; impact assessment.", "All modes will have different needs with regards to instrumentation and data.", "As an example, location mode will require current predictions in real time to be able to move upstream from a signal, as opposed to the statistical current conditions that is sufficient for the detection modus.", "The focus here is the detection phase, in which the monitoring program looks for anomalies in the environment.", "How can a potential seep be detected, and is it possible to quantify the certainty of detecting a leak?", "Answering these questions relies on 1) where will a leak most likely occur, 2) how will a seep trail materialize in the water column, and 3) will it be possible to distinguish the signal from the background variability?", "Leak of CO2 shallower than 500 m will rise in the water column as gas bubbles (Alendal and Drange, 2001).", "Depending on the flux rate, it might create individual bubbles, bubble trains or bubble plumes.", "The dynamics of these regimes are different, with the plume dynamics being the most challenging to model due to the two way coupling with the surrounding seawater.", "In all cases an increase in CO2 concentration is expected in the vicinity of a leak, with subsequent possible environmental impacts (Blackford et al., 2010).", "The spatial extent of area influenced is expected to be limited (Dewar et al., 2013, 2014), but might become severe depending on the size of the leakage flux and the dilution rate in the water column.", "Environmental changes might serve as indicators that a leak is occurring, either through changes in bottom fauna or in the pelagic ecosystem, and detection of bubbles can be made from ship sonars (Brewer et al., 2006; Noble et al., 2012).", "In addition the elevated concentration of dissolved CO2 can be used as indicators of a leak.", "One main challenge will be to define the degree of anomaly needed in order to mobilize the more expensive confirmation and localization step, balancing the need for confirmation with the cost of false alarms.", "Changes in flux or new occurrences of natural gas seeps, changes in biota or CO2 concentration can only be distinguished if a proper baseline statistic is available.", "In the present study elevated CO2 concentrations is used as the methodology to design a monitoring program.", "CO2 is naturally present as dissolved into seawater and an increase of CO2 concentration is expected according to the acidification of marine waters (Caldeira and Wickett, 2003).", "Botnen et al. (2015) demonstrated a stoichiometric approach for detection of small CO2 concentrations that might stem from seeps, lowering the concentration threshold for a signal to become statistically significant.", "This method lies in between the statistical power analysis for obtaining an environmental baseline (Yang et al., 2011a) and the pure process based monitoring approach suggested by Romanak et al. (2012).", "The marine waters are in constant motion and are characterized with high variability; Tides change current directions, wind alters the amount of mixing, and local topography change local current conditions (Alendal et al., 2005).", "Footprints of leaks are thus a varying and highly anisotropic signal depending strongly on the local oceanic and atmospheric conditions (Alendal et al., 2013; Greenwood et al., 2015; Ali et al., 2016).", "This makes designing a monitoring framework considerably harder compared to when assuming isotropic signal (Yang et al., 2011b).", "Hvidevold et al. (2015) presented a procedure for optimizing the sensor placement on the seafloor that partly accounts for footprint anisotropy.", "The highly anisotropic footprint signal prediction from a General Circulation model (Alendal et al., 2013; Ali et al., 2016) was simplified through a least square approximation to an ellipsoid, generating orthogonal major and minor directions.", "It was observed that this procedure reduced the area of detectable concentration compared to the average field itself.", "In this study the same GCM simulations is used and a method to use the predicted footprints directly is introduced.", "This method combined with predicted time-series in an array of 51 × 51 grid points around the seep location open for other approaches in designing a monitoring program.", "Three different methods will be presented; i) an average method to compare with results from the previous study (Hvidevold et al., 2015), ii) an event method taking into account the occasionally elevated concentrations and iii) a single measurement method.", "The two former represent fixed installations, with different approach in data analysis, while the latter can represent measurements taken during cruises and surveys.", "By combining the stoichiometric approach in (Botnen et al., 2015) and the statistical approach suggested has the potential to become a powerful tool to discover small leaks from CCS.", "Such leaks matters on an environmental scale and may become harmful for the environment and contribute to ocean acidification.", "Very small natural CO2 leaks from the Jan Mayen fracture zone is proven to be discovered by using the stoichiometric approach (Botnen et al., 2015).", "Here the focus is to arrive to the optimal placement of sensors on the sea floor to obtain an early detection of a potential leak from subsea geological storage of CO2.", "It is not the scope to perform a detailed site specific design, nor is a proper assessment of possible leakage pathways available for candidate CCS storage sites.", "Hence, in this study a synthetic area of 31 × 31 km containing 16 randomly placed wells is used.", "In reality geological weakness zones needs to be included as well.", "The value p0 is the probability of leakage through the well and σ describes how quickly this probability levels off.", "It is assumed that all wells have the same p0 and σ ≈ 0.6 km.", "This is a simplification, in reality some wells may reach the storage formation and some will not.", "To account for potential unknown pathways a low background probability is added to the map, assuming a ∼10 times higher probability for leakage in a well compared to the background.", "Under the assumption that a leak is ongoing within the area probability field is normalized i.e., the probability that a leak occurs is 1.", "Fig. 1 shows the resulting probabilities.", "Simulating a seep is challenging, especially due to the many different scales that are involved, ranging from 10−2 m (bubble scale) to 102 − 103 m (scale of dynamically active concentration field) (Alendal and Drange, 2001; Sato and Sato, 2002; Dewar et al., 2013, 2014).", "Away from the dynamically active seep zone the CO2 concentration behaves as a passive tracer, being transported and further mixed by local currents and turbulence.", "Locally there might be strong variations in current conditions and mixing, influenced by a number of factors such as surface wind, tide (Davies and Furnes, 1980) and topography (Alendal et al., 2005).", "To obtain a footprint a 800 m resolution regional ocean model (Bergen Ocean Model; BOM) set-up for the North Sea advects and disperse CO2 as a passive tracer (Alendal et al., 2013; Ali et al., 2016).", "The model forcing comprises tide (Davies and Furnes, 1980) (4 tidal constituents; M2, S2,K1, and N2), river runoff data, and initial and lateral boundary conditions for salinity and temperature taken from the UK Metoffice FOAM 7 km model published at (http://www.myocean.eu/).", "The atmospheric forcing is based on spring 2012 data, collected and interpolated from The European Centre for Medium-Range Weather Forecasts (ECMWF, the Centre).", "In the vertical 41 sigma-coordinate layers is used, distributed with higher resolution (1 m) near the free surface and the sea floor.", "In this study a source term representing a leak rate of 1.74 kg/s, with a flux area representing one grid cell of (800 × 800) m2 located closest to the seabed, i.e. the grid-cell closest to the seafloor.", "A two months simulation of CO2 seep has been used to gather time series, with 1 min resolution, in a 51 × 51 grid cell array in the vicinity of the leakage.", "Fig. 2 show time series for nine grid cells closest to the leakage point (blue lines).", "The reported precision for measurements of total inorganic carbon (ct) is ∼0.090·10−3 kg/m3 (Dickson et al., 2007), shown as green line in Fig. 2.", "In the North Sea the natural variability of ct between seasons varies between 2.260·10−3 kg/m3 and 4.520·10−3 kg/m3 mainly caused by e.g. changes in biological processes (photosyntheses and respiration), air-sea exchange, and hydrography (salinity variations and mixing between watermasses) (Omar et al., 2005; Salt et al., 2013).", "Omar et al. (2005) and Botnen et al. (2015) developed a conceptual model correcting for the natural variability and the anthropogenic trend based upon a stoichiometric approach.", "When contrasting measurements with a reference location it is possible to determine changes in ct that must stem from other sources, like seeps of CO2.", "The threshold for detecting anomalies after applying this conceptual model is ct = 0.226·10−3 kg/m3 (red lines in Fig. 2).", "The different concentration lines in Fig. 2 clearly illustrate the threshold reduction offered.", "As a further simplification the constant leakage rate and the resulting seafloor concentration are independent on leak location.", "In reality flux will be dependent on leak location and be time dependent, and local topography and current variability might alter the characteristics.", "For a better description seasonal, spatial variability and long term trends will have to be accounted for.", "This will require availability of the aforementioned current statistics and a much more comprehensive simulation study.", "The main challenge is how and where to do measurements with the purpose of detecting a leak.", "Based on the GCM time series predictions, three different approaches is presented.", "Each building on a detection probability function, G(x − xl;ct), that gives the probability of detecting a leak at xl if a measurement is taken at x and with a given a detection threshold ct.", "In Hvidevold et al. (2015) this footprint was used to approximate an analytical function, introducing additional approaches and simplifications to the problem.", "Here the simulated footprint is used directly.", "In reality this method will not be used, it is shown here for comparison with the previous study (Hvidevold et al., 2015).", "To wait until the average concentration becomes statistically significant will delay the detection, or even prevent, the leak to be detected.", "Rephrased, the function gives the probability of a single measurement at x to be above ct.", "For instance in the grid cell located at the leakage point, the center plot in Fig. 2, the concentration is always above the threshold concentration ct = 0.226 × 10−3 kg/m3, hence G(xl;ct) = 1.", "While in the lower left plot the concentration is often below this ct and the probability for a single measurement to detect the leak is 0.23.", "Calculating GS(x;ct) for all 51 × 51 grid cells gives a probability field around the leakage, Fig. 4.", "This represent a combination of the two former approaches, continuous measurements at fixed locations and utilizing GS(x;ct).", "A fixed installation might experience periods of increased CO2.", "The green contour line in Fig. 4 represent pt = 0.1, bounding the area for which a sensor can detect a leak GE(x;ct,pt) = 1.", "All three approaches use the assumption that a leak is occurring within the area shown in Fig. 1 and the predicted CO2 concentration footprint time series, exemplified in Fig. 2, is independent of leak location.", "One problem is to invert the probability functions defined to the area monitored by a measurement taken at a given location.", "Given a measurement at a fixed xa, the probability of detecting a leak at xl will be G(xa − xl;ct), where G is any of the probability functions defined earlier.", "Treating xl as the independent variable the probability fields of detecting a leak at r = x − xa will be G(− r;ct), representing a 180° rotation of the anisotropic G contours.", "If measurements taken at xa show no indications of a leak the leak probability map can be updated this new information.", "Fig. 5 illustrates this for a single measurement point in a section of the leak probability map, Fig. 1.", "The probability of any coordinates being the site of a leak prior to any measurements (a) is updated with the new belief after measuring no indication of a leak at xa, white cross, for the different methods.", "The average method (b) and the event method (d) rule out a leak in the area monitored, with the area monitored by the average method being considerably smaller.", "If a single measurement stays below the threshold concentration a leak can not be ruled out completely, but the probability map may be updated by subtracting G( − (x − xa);ct) as shown in Fig. 5(c).", "The design problem is to localize where to take n measurements in an optimal way.", "For instance, placing a measurement in a location already covered will give low use of that measurement.", "This is solved using Matlab's built-in genetic algorithm, ga, with a population size of 300 individuals (collections of measurement coordinates).", "The genetic algorithm will evolve the population of measurement collections toward higher probable detection.", "The benefit of this method is the global search space offered, avoiding getting trapped in local optimums.", "Fig. 6 show the probability to detect a leakage as function of number of measurements for the different methods, demonstrating the ability of the procedure to quantify the leak detection capability of a measurement grid.", "The average method is very conservative in the sense that average concentration must become statistically significant before a leakage is detected.", "In reality a fixed installation will detect a leakage immediate once the measurements becomes significantly different from the natural variability.", "This is also illustrated by the small area covered in Fig. 5(b).", "Fig. 6 also show that fixed installations (event) gives much higher detection probability than single measurements, given similar amount of measurements.", "For instance placing 2 fixed installations is better than taking 7 single measurements, and requiring a detection probability of 80% only 12 fixed installations is needed compared to 60 single measurements.", "This is due to the fact that fixed installations represent many measurements taken at the same position, and hence increase the probability of capture periods of elevated CO2 concentrations.", "Due to logistics and cost it might still be beneficial to rely on cruises and taking point measurements and the method has the ability to locate where these should be taken.", "Due to the many simplifications and assumptions made the numerical values should be used with caution.", "Still it is interesting to see how much better an optimal layout of measurements will perform compared to an intuitive approach with successively selecting locations based on highest remaining probability of leaks, Table 1.", "For instance, the event based method using 10 locations with an optimal layout is much better than using 20 measurements with the intuitive layout.", "The optimal layout for the three methods, using four and ten measurements, is shown in Fig. 7.", "For the average method the area covered by the measurement is almost as big as the leakage probability area surrounding a well, and the optimal layout is to cover a new well location for each new measurement, therefore it is almost similar to the intuitive strategy as can also be read from Table 1.", "One measurement can cover several wells if the event based method is used.", "Therefore it is not necessarily optimal to place measurements directly on well locations.", "Single measurement method require clustering of measurements in order to optimize detection.", "In addition the preferred location is downstream due to the anisotropy seen in Fig. 4.", "The results presented here are based on many simplifications and short cuts due to lack of information and data.", "Still it is demonstrated that combining proper site characterization, environmental baseline statistics and leak footprint predictions can be combined to optimize the required marine monitoring program.", "At the moment the necessary information to perform a real design at a real site is lacking and hence the possibility of demonstrating the concept in-situ.", "To perform a more comprehensive design for a future storage project will require a thorough geological survey identifying potential leakage pathways, including mutual propensity to leak, to get a more realistic probability map (Fig. 1).", "In addition a proper environmental baseline is needed for better detection limit, as part of this baseline spatial and temporal current statistics should be gathered for reliable footprint prediction.", "The aim must be to get involved in future storage demonstration projects with the purpose of assuring that necessary information is gathered during site characterization and selection.", "However, these demonstration storage sites will be designed to not leak and cannot be used to test the concept.", "Such test will require in-situ experiments, like the QICS experiment (Blackford et al., 2015b), or use of natural gas seeps.", "In any case it will require resources that are not available for the moment.", "Higher confidence in the monitoring program is achieved through quantification of the degree of certainty when a leak is indicated.", "Such indication will mobilize extra resources to confirm and localize a potential leak.", "The operations might become costly and false alarms should be avoided.", "On the other hand it is necessary to act on indicators of leaks in order to identify and mitigate impacts.", "Through the certainty quantification offered by the method suggested here it is possible to judge the likelihood of an indication being a false alarm.", "Cost for operation and installation has not been considered in this study.", "Assuming the optimal measurement layout has been chosen, minimizing the travel distance between these locations will save time and cost.", "This will be true for installations requiring ships, operating gliders or other new technologies.", "This is exemplified in Fig. 8 show the shortest travel route for the single measurement methods for twenty locations, using the same start and stop location, blue star, using the genetic algorithm." ]	[ "Seafloor monitoring design to detect leaks from geological CO2 storage.", "Based on site characterization, local environmental statistics and a detection threshold.", "Quantifies the probability of detecting a leak and finds the optimal layout.", "Likelihood of adverse measurements being false alarm is quantified.", "Can be extended to include other constraints, e.g. cost or vulnerable areas." ]
Premixed methane oxycombustion in nitrogen and carbon dioxide atmospheres: Measurement of operating limits, flame location and emissions. Proceedings of the Combustion Institute	The effect of O2 concentration on a premixed swirling flame was studied for changes in discrete levels of CO2 and N2 dilution with application to carbon capture in gas turbine systems. Chemical kinetic models were used to analyze and support generic burner experiments at 37.5 kW with varying O2 mole fraction between 0.21 and 0.70 and cautious measurement of velocity field data via particle image velocimetry representative heat release and flame location. Results showed that under lean N2-diluted operating conditions a minimal change in burner operation for all O2 concentrations was observed. CO2 dilution affected the flame location heat release and operational response more than N2 and is attributed to the primary differences in thermodynamic and transport properties between the two gases. An increase in diluent CO2 in exhaust streams resulted in increased CO concentrations while increased N2 diluent resulted in a rise in NOx emissions.	[ "CENG", "CHEM", "ENGI" ]	[ "Carbon capture", "Oxycombustion", "Swirl combustion" ]	[ "1500", "1606", "2210" ]	[ "The aim of this research is to analyse the characteristics of CO2 and N2 diluted swirl-stabilised combustion of O2 and CH4 mixtures; principally to show how flame location changes in relation to O2 mole fraction and equivalence ratio, in conjunction with the local flow field of the swirl zone.", "Limited combustion research has been published in this field, where premixed or partially premixed natural gas has been burnt in O2–CO2 mixtures [1–10].", "Recent studies on gas turbine burners used exhaust gas recirculation levels up to 30%, and research with oxygen-enhanced natural gas burners has considered molar O2 fractions up to 40% [11,12].", "Other work demonstrated stability limits of tubular burners with various O2 and CO2 concentrations, proving O2–CH4 flames can be balanced with CO2 as a combustion moderator [13].", "Studies on flame front locations demonstrated flame shape is influenced by O2 fraction, but weakly dependent on equivalence ratio [2].", "When comparing diluents, limited studies show that at constant mass flux, CO2 diluted flames are stabilised at higher O2 concentrations compared to N2.", "Such reaction kinetics have been studied with CHEMKIN to show differences in laminar burning velocity of O2–CH4 mixtures with N2 and CO2 diluents; it should be noted also that CO2 contributes to soot oxidization and affects the rate of many chemical reactions compared with N2.", "Under stoichiometric conditions, a pure O2–CH4 flame exhibits flame speeds around 10 times greater than the air–CH4 equivalent [14].", "Moreover, premixed O2–CH4 swirl stabilised burners, also diluted with N2 and CO2, demonstrated dependence of strain rate through chemical kinetics and flow residence times on flame blowoff [15].", "A rapidly mixed tubular burner has shown how oxygen concentration with CO2 and N2 diluents affected stability limits, utilising Damköhler number to describe the flow field in relation to mixing and chemical residence times [16].", "This work also demonstrated that CO2 dissociation in oxygen–methane flames is not significant at temperatures below 2400 K.", "Different velocity, length and chemical timescales lead to a variety of extinction conditions, thus affecting blowoff conditions [17].", "Diluents affect the flame in at least four ways: (1) mixture heat capacity, (2) transport properties, (3) chemical kinetics, (4) radiative heat transfer [15].", "A reduction in burning velocity is noted when N2 is replaced by CO2—primarily attributed to the higher heat capacity of CO2 [18].", "Table 1 shows physical properties for each substance [19].", "As β is variable in the oxidant mixture, care must be taken to identify both equivalence ratio (ϕ) and β when presenting results.", "Since conditions studied herein were designed to evaluate the behaviour of variable O2 content on premixed swirl burners, all data were collected in the region ϕ < 1 and β > 0.21.", "The PREMIX coded laminar flame speed calculator was utilised in CHEMKIN-PRO [20,21] to model the reactant mixtures for ambient conditions of temperature (293 K) and pressure (0.1 MPa) with GRI-Mech 3.0 reaction mechanism [22].", "The simulation employed 53 chemical species and 325 reactions, with solutions based on an adaptive grid of 1000 points, mixture-averaged transport properties, and trace series approximation.", "To examine a hypothesis relating to CO formation via thermal dissociation, the relevant mechanisms were deactivated for specific experiment points to compare this effect in CO2 diluted combustion.", "The high pressure optical chamber (HPOC) at Cardiff University's Gas Turbine Research Centre was used with a high-pressure generic swirl burner (HPGSB) at 37.5 kW thermal power.", "Premixed diluent, fuel, and oxygen entered the burner inlet plenum, travelling through a single radial-tangential swirler and out through the burner exit nozzle.", "Fig. 1 shows a 3-D section view of the HPGSB detail within the HPOC, including detail of the swirler and exit nozzle.", "The HPGSB operates with a cylindrical quartz exhaust confinement, a radial expansion ratio of 3.5 from the burner nozzle exit radius of 20 mm, and a geometric swirl number Sg = 1.04.", "The particle image velocimetry (PIV) system consists of a frequency doubled (532 nm) dual cavity Nd:YAG Litron laser at 5 Hz.", "Laser optics (9080 × 0651) were used to transform the beam into a 1 mm thick sheet.", "A Hi Sense MkII Camera (C8484-52-05CP) was used, with 1.3 megapixel resolution.", "A 60 mm Nikon lens, fitted with a narrow bandpass filter centred at 523 nm, afforded at 90 × 120 mm field of view, with a resolution of 11.27 pixels/mm and a depth of 1.5 mm.", "The flow was seeded with 1.7 µm (based on D3,2) aluminium oxide particles upstream of the burner inlet plenum.", "A frame-to-frame adaptive correlation technique was carried out with a minimum interrogation area of 20 × 20 to 40 × 40 pixels, with adaptability to particle density and velocity gradients.", "A temporally averaged velocity map was created with 189 image pairs, representing approximately 37 s at each test condition.", "PIV velocity maps were utilised for identification of the flame-stabilising shear layer, identified as a line of zero axial velocity.", "This exists between the outward forward flow near the outside edge of the burner exit and inward flow of the central recirculation zone (CRZ).", "Fig. 2(a) shows the processed velocity map for a CO2-diluted oxymethane flame at β = 0.21.", "The arrows represent resultant velocity vectors, providing an indication of flow direction, while vector length corresponds to resultant velocity at each location in space.", "The burner exit at y = 0 mm with the centreline located at r = 0 mm.", "The outward forward flow, shear layer, and CRZ are well-defined, with the latter indicated by areas of high reverse flow.", "This was transformed into a contour plot considering only the axial velocity component, with edge detection utilised to identify the shear layer, Fig. 2(b).", "A Dantec Hi Sense Mk II CCD camera with 1.3 megapixel resolution was coupled to a Hamamatsu C9546-03 L image intensifier for OH* chemiluminescence measurements.", "A 78 mm focal length UV lens was installed on the intensifier, with a narrow band pass filter centred at 315 nm.", "The camera was placed viewing through the top window of the HPOC.", "The image plane is centred on the burner exit giving a view field of ∼100 mm (radial) to 75 mm (axial) at 13.6 pix/mm.", "200 images were captured at 10 Hz for each experiment, with an intensifier gate pulse of 400 μs triggered by the camera aperture.", "To ensure consistency between OH* chemiluminescence measurements, the intensifier gain was held constant throughout.", "A modified open access Abel inversion algorithm [23] was utilised to transform the 3-D OH* chemiluminescence image into a 2-D representation of the OH* intensity distribution, with the underlying assumption that the swirl-stabilised flame is centreline axisymmetric [24].", "Planar laser-induced fluorescence (PLIF) measurements were made at each experiment.", "The PLIF system comprised a Quantel TDL-90 dye laser pumped by the 532 nm output beam of a frequency-doubled Spectra Physics GCR 170-10 Nd:YAG laser at 10 Hz.", "The output beam of the TDL-90 was tuned to 283 nm to excite the (1,0) band of the OH radical, while fluorescence was captured at 315 nm using the intensified CCD camera.", "The output beam was directed through telescopic optics providing a 25 mm wide sheet.", "This produced energies of ∼16 mJ/pulse at 283 nm.", "The gate timing of the image intensifier was set to 1.5 μs and triggered by the Q-switch of the Nd:YAG laser to ensure capture of the OH PLIF signal synchronized with the laser excitation pulse.", "The image intensifier gain was again held constant to ensure comparability, reducing the signal-to-noise ratio.", "For each experiment, 500 images were taken at 10 Hz.", "Results were temporally averaged and corrected for background reflected light and chemiluminescence intensity.", "The field of view was ∼100 mm (radial) by ∼75 mm (axial), with resolution of 13.6 pix/mm, and the camera imaging plane shifted to focus on one half of the burner.", "Previous research in this field examined the relationship between controlling factors of equivalence ratio and molar oxygen concentration (β) with different inert diluents, measuring flame front location and temperature [2].", "Differences in physical and transport properties between N2 and CO2 will affect the flame in terms of heat transfer, and discrete velocities of gases in the recirculation zone, which affect mixing, flame location and heat release.", "Experiments were conducted at constant total molar flow rate for changes in β [4].", "The experimental test points are shown in Tables 2 and 3 for N2 and CO2 respectively.", "The stable operating points of 0.654 and 0.543 mol/s (N2 and CO2 diluted respectively) were selected as exactly half way between the flame flashback and blowoff points at β = 0.21.", "It would not be possible to select a single molar flowrate that would work with both diluents due to the differences in the chemical kinetics.", "Fig. 3 provides modelled laminar flame speeds and adiabatic flame temperatures (AFT) against change in equivalence ratio for each β, and specified experimental conditions.", "Similar trends in AFT and flame speed are evident for both diluents, with CO2 shown to provide greater change in flame temperature for increasing β; rising with O2 fraction, compared to the smaller opposing influence simulated for N2.", "The results are in agreement with the simulation work of Liu et al. [14].", "Direct comparison could not be made with identical flow rates for both diluents, given that the N2 and CO2 diluted flames had different flame stability envelopes.", "All results in the N2-diluted system were taken at a total premixed molar flow rate of 0.654 moles/s and CO2-diluted measurements at 0.543 moles/s.", "Since these experiments were undertaken at constant molar flow rates, flame and flowfield changes result from changes in:", "Gas density, therefore recirculation zones.", "Kinetic interaction between reactive and passive species [15].", "Radiative absorption and emission.", "Referring to Fig. 3, the initial observations regarding CO2 tendency for blowoff become clear since there is a greater difference in flame speed and temperature over ranges tested for the CO2-diluted system.", "The influence of the two diluents on overall heat release is shown in Fig. 4 using Abel inverted OH* chemiluminescence, indicating the conical flame shape expected in a swirl-stabilised burner such as this.", "This shows the influence of O2 fraction on the two diluted flames with constant molar flow rate.", "There is clear difference between these indicative heat release results at different β values, which correlates with CHEMKIN simulations in Fig. 3.", "Starting at β = 0.21 (air) for the N2-diluted case, increasing the O2 concentration reduces the OH* emission intensity across the flame.", "The flame appears more lifted with increasing β (most notably at β = 0.60) resulting from the reduction in AFT, and overall reaction rate as O2 fraction increases.", "For the case of CO2 dilution, the flame at β = 0.21 was detached from the burner, close to the blowoff point, thus out of the field of view.", "The increase in O2 concentration had a significant effect on flame position, causing OH* emission to increase, allowing the flame to attach in the shear zone, correlating with changes presented for increasing β in Fig. 3.", "At β = 0.70, the CO2-diluted flame was comparable to a low β N2-diluted flame, given the similarity in flame speed, temperature and transport properties, as the mixture was comprised largely of O2 with comparable transport properties to N2.", "Comparing diluent properties for these mixtures, the resultant kinetics of CO2-diluted combustion gives rise to significantly cooler, slower flames.", "Molar heat capacity and density increase with CO2 whereas they decrease with N2; the reverse is true for thermal conductivity.", "This results in the observed downstream migration of the reaction zone as chemical timescales are increased.", "Since volumetric and flow timescales are nominally identical for each set of diluents (as confirmed by PIV) it can be said that flame migration is predominantly due to changes in chemical timescales only.", "Fig. 5 shows OH PLIF data for the N2-diluted flame operating from β = 0.21 to 0.70.", "OH PLIF intensity was normalised with respect to the highest measured value across the range.", "Images show half the flame in axial view, hence the r = 0 mm line represents the burner centreline.", "Also included in the image for β = 0.21, 0.40 and 0.60 are PIV measured axial shear lines.", "The flame front doesn't appear to shift significantly over the conditions, although OH intensity near the burner exit does appear to weaken as β increases and flame temperatures are reduced.", "This corresponds to OH* chemiluminescence (Fig. 4) which showed areas of significant heat release moving further downstream from the burner exit whilst diminishing in overall size.", "When the PIV data is superimposed in Fig. 5(a), (c) and (e), it is observed that the flame front is located on the outer edge of the axial shear layer, enclosing the inner recirculation zone.", "As β increases, the interaction between the flame front and the shear zone doesn't appear to change.", "Hence despite the small reduction in flame speed and temperature, the flowfield wasn't significantly affected.", "Fig. 6 shows the OH PLIF data for the CO2-diluted flame operating from β = 0.21 to 0.70.", "It is clear that β has a greater effect on the flame front location in the CO2-diluted case compared to N2 dilution.", "The PLIF laser setup is such that a 25 mm wide laser sheet is introduced into the burner through a side window.", "Thus, the horizontal excitation plane is positioned at the burner exit and covers a distance 25 mm downstream, equal to the width of the laser sheet.", "Included in the image for β = 0.21, 0.40 and 0.60 are the measured axial shear lines.", "The images show that for highest values of β = 0.60 and 0.70, where the flame speed of the mixture is highest, flame location is similarly located to that of the N2-diluted flame (stabilised on the outer edge of the axial shear layer enclosing the inner shear zone at the burner exit).", "In this CO2-diluted case, when β is decreased the flame speed decreases sharply (Fig. 3) causing the flame front to move downstream and ultimately into the recirculation zone itself.", "This is visible in the β = 0.21 image in Fig. 6(a), where the flame has migrated out of the swirling shear layer and stabilised inside the CRZ.", "Coupled with the fact that the CO2-diluted flame visually appeared to be in a near blow-off condition at β = 0.21, OH radicals in Fig. 6(f) are the result of significant change in mixture reactivity at high CO2 dilution.", "Under these conditions, flame speed and AFT reduce significantly, the flame moves further downstream and the retarded reactions then occur within the CRZ, as OH radicals are recirculated towards the burner exit.", "For β = 0.70, the amount of CO2 is relatively low, increasing the flame temperature, and thus yielding a flame shape and location similar to that of a N2-diluted flame at β = 0.21.", "From β = 0.40 (Fig. 6(c)) to β = 0.21 (Fig. 6(a)), the increasing OH PLIF intensity indicates a transition in flame stabilisation location, as the OH PLIF intensity increases in the CRZ, marking significant changes from shear layer stabilisation at β = 0.70.", "In Fig. 6(d), at β = 0.50, the flame is still stabilised in the shear layer but the flame front has moved out of excitation plane of the PLIF laser.", "Referring to Eq. (1), where the relationship between chemical timescales and hydrodynamic behaviour was introduced, it can be seen that the flowfield shape has remained largely unaffected by the heat release location (Fig. 4) for both diluents.", "Hence although there is a difference in transport properties between N2 and CO2, this doesn't appear to significantly alter the turbulence structure in the flame zone.", "As a result of this it can be expected that the Da through the N2 diluted cases has changed relatively little, whereas the change through the CO2 cases is significant.", "Fig. 7 provides example 1-D OH spatial concentration profiles generated for the extreme N2 and CO2 diluted cases (N1, N6, C1, and C6) using the PREMIX solver.", "This suggests an increase in OH fraction, as would be expected, with increased levels of O2.", "Note that OH fractions in the N1 and N6 case are similar which correlates with the OH PLIF results (Fig. 5).", "The OH profile in the C6 case is closer to all N2 diluted cases in addition to having similar AFT and flame speed (Fig. 3), again demonstrating a similar correlation with the PLIF results (Figs. 5 and 6).", "This suggests the change in overall reaction rate and AFT is significantly more influential for flame stabilisation when compared to transport, and hence diffusive properties.", "The exhaust stream was sampled using a water-conditioned averaging probe (9 holes) located approximately 10 diameters downstream of the combustor exit.", "The probe and sample line were maintained at 433 K, with a heated pump used to deliver products to the analysis suite.", "Hot NOx concentrations were quantified using a heated vacuum chemiluminescence analyser (Signal Instruments 4000VM), calibrated in the range 0–39 ppmV.", "A chiller was also employed to reduce the molar water concentration below 1%, with downstream CO concentrations measured using a nondispersive infrared analyser (Signal instruments 9000MGA), calibrated in the range 0–904 ppmV.", "Fig. 8 shows the resultant CO and NOX concentrations.", "Data have not been normalised to account for effective dilution in the product stream, because equivalent volumetric flow rates are maintained for all tests.", "Raw CO concentrations are presented cold/dry, and NOx, hot/wet.", "This was to remove the uncertainty associated with normalising data to an assumed water concentration in the product stream, therefore only qualitative trends in the plots are considered.", "Results demonstrate an expected absence of NOX chemistry in the CO2 diluted flame, plus the unaffected emission of CO across the range of β = 0.21–0.70 exhibited by the N2-diluted flame.", "This was to be expected considering the small different in AFT, and limited weakening of the flame with the N2 experiments.", "NOx production rises with an increase in initial N2, and hotter temperatures.", "There is an increase in CO production as CO2 concentrations rise to β = 0.21 in the CO2-diluted flame, whilst a corresponding change in N2 leads to increased NOX formation.", "Whilst it could be suggested that CO emissions are a product of CO2 thermal dissociation, it is more likely that the CO is a product of incomplete methane combustion owing to the reduced AFT and increased reaction times of the mixture.", "Further CHEMKIN simulations were undertaken to determine whether CO2 diluent could have an effect on CO production via thermal dissociation.", "This showed the difference between the base mechanism (normal GRI-MECH) and the mechanism without thermal dissociation is barely noticeable.", "The results agree with historical work [25] undertaken at various temperatures ranging from 1300 to 3320 °C, showing that below 1500 °C CO2 dissociation to CO was extremely low compared to cases above 2000 °C.", "Since average temperatures of the flame are below this peak value, it was recognized that CO was mostly present as a consequence of incomplete combustion." ]	[]
Genetic Code Analysis Toolkit: A novel tool to explore the coding properties of the genetic code and DNA sequences	The genetic code is degenerated and it is assumed that redundancy provides error detection and correction mechanisms in the translation process. However, the biological meaning of the code's structure is still under current research. This paper presents a Genetic Code Analysis Toolkit (GCAT) which provides workflows and algorithms for the analysis of the structure of nucleotide sequences. In particular, sets or sequences of codons can be transformed and tested for circularity, comma-freeness, dichotomic partitions and others. GCAT comes with a fertile editor custom-built to work with the genetic code and a batch mode for multi-sequence processing. With the ability to read FASTA files or load sequences from GenBank, the tool can be used for the mathematical and statistical analysis of existing sequence data. GCAT is Java-based and provides a plug-in concept for extensibility. Availability: Open source Homepage: http://www.gcat.bio/	[ "COMP" ]	[ "Analysis", "Binary dichotomic algorithm", "Comma free", "Genetic code" ]	[ "1706", "1712" ]	[ "The genetic code is one of nature’s major tools for the transmission of information.", "By assigning amino acids to triplets of nucleic bases, so-called codons, it serves as a kind of dictionary between the two worlds of nucleic acids and proteins.", "One of its most conserved properties is degeneracy respectively redundancy, i.e. different codons may encode the same amino acid.", "Degeneracy is essential in any system that allows error detection or even error correction, and the chance that genetic information is preserved without any error detecting or error correcting mechanisms is very low.", "Why the genetic code has not changed for so long, and how it evolved from a long evolutionary process are still open questions.", "Several approaches from biology, communication theory, mathematics and physics have been made to explain the structure and functionality of the genetic code table.", "One approach is based on the finding of so-called circular codes in large genes of prokaryotes and eukaryotes [1].", "Such codes are subsets of the set of codons that seem to be used by nature in order to eventually detect frame shift errors in the translation process.", "Circular codes are weaker versions of comma-free codes which can detect frame shift errors immediately [2].", "A related approach is based on so-called Binary Dichotomic Algorithms (BDAs) where codons are partitioned according to the chemical properties of their bases [3].", "A short range correlation between the codons has been detected in coding sequences using these algorithms [4].", "Investigations of the structure of circular and comma-free codes and their use in nature led to the motivation to develop the Genetic Code Analysis Toolkit (GCAT) — especially as such features had not been covered by other tools.", "GCAT is developed in Java 8 and partially in Scala –another Java-Virtual-Machine-based language.", "It runs on every major platform (Mac, Linux, Windows etc.).", "GCAT was implemented in a feature driven approach.", "Due to its general-purpose object-oriented nature, Java 8 provides a good and extendable basis for an open toolkit like GCAT.", "Dependencies, e. g. to other open library functions such as BioJava [5] or JGraphX, as well as the build process are managed by Apache Maven.", "Maven allows an easy extensibility and the integration into other projects.", "The implementation was divided into three independent parts: 1.", "A domain model for nucleic compounds – compatible to the compound classes of BioJava.", "2.", "An extensible set of operations, to be performed on sequences or sets of nucleotides.", "3.", "The Swing user interface.", "Each of these parts can be used and enhanced independently.", "Nucleic bases are the atomic elements of the implemented model.", "Multiple bases can be joined to tuples of arbitrary length, e.g. to codons with three bases.", "Multiple tuples either form a set of tuples, or a nucleotide sequence.", "Operations either modify or analyze collections of tuples.", "They can be performed independently or dependently from each other, as well as sequentially or in a batch for multiple sequences at once.", "Therefore, each operation was implemented self-contained and reentrant.", "The result of an operation, such as a transformed nucleotide sequence, can be used in subsequent processing steps.", "Operations are categorized into four different types:", "The combination and permutation of the toolkit’s various operations allow a multitude of modifications and analyses to be performed on nucleotide sequences.", "GCAT features an open plug-in mechanism.", "Due to its open source nature, by utilizing the dynamic loading capabilities of the Java Class Loader, the Reflections API, and the Java Annotation concept it can easily be extended.", "New input- and display elements, as well as new operations can be added, without any modification to the existing implementation required.", "Instead, with a plug-in-like concept new features can be added and the user interface dynamically adapts to any enhancements made.", "GCAT as a Java-based component could be integrated into other bioinformatic toolkits like Bioconductor [6], R [7] and vice versa.", "This is possible for instance with rJava (R to Java) and JRI (Java to R).", "The technical concept to do so is currently being evaluated.", "Fig. 1 shows several different dialogs of GCAT.", "A central component is a comprehensive sequence editor in the analysis tool (cf. 1(a)).", "Different kinds of input patterns, such as a codon wheel and multiple display elements for visualization, such as a graph display, support a substantial analysis.", "The editor can also be used to edit a set of codons or tuples, i. e. no duplicates are allowed.", "A sequence can also be loaded from a FASTA file or from GenBank (incl. web access).", "Within the batch tool (cf. 1(c)), a script of operations, introduced in the previous section, can be maintained and executed on multiple sequences.", "The results will be stored and visualized for each sequence separately.", "The BDA tool (cf. 1(d)) allows the partitioning of codons according to BDAs.", "A well-known partition, for instance, is the Rumer classification.", "In almost all fields of sciences, modern research is based on interdisciplinary approaches.", "In particular, Life sciences, Biology, and Medicine frequently use techniques and methods from Mathematics and Computer Sciences in order to handle, visualize and interpret the continuously increasing amount of experimental data that is available.", "A prominent example is the genetic information of thousands of species that has been sequenced over the last decades and has been stored in huge data banks like GenBank.", "Several tools are available to investigate RNA sequences but they mostly require statistical, mathematical or programming knowledge.", "Our tool is designed to provide a user-friendly open source software that has an intuitive fertile editor.", "GCAT is mainly targeted to biologists and offers several graphical visualizations of data from GenBank or FASTA files as well as the executed routines.", "This is a big advantage over other existing software tools like, for instance, bioconductor [6] since GCAT addresses a much broader audience, allows to see problems on the genetic information from a new, graphical point of view and to pursue new research questions uncovered by the visualization.", "In times of big data and visual analytics our tool makes statistical routines available to biologists (and other researchers) to perform their investigations but also helps them to follow a new graphical approach to classical and modern questions.", "The search for patterns and correlations is significantly simplified by our tool.", "Currently, GCAT focuses on questions around circularity of codes but it is feature-rich and easy extensible.", "GCAT is a novel tool for the analysis of the structure of the genetic code.", "It was designed to be user-friendly, feature-rich, extensible and freely available to the whole scientific community.", "GCATs purpose is to be used as a biological workbench for (theoretical) biologists, mathematicians and physicists, offering unique features that are not available in any other tool and to be easily adaptable to current research needs." ]	[]
Modelling stiffness of furniture manufactured from honeycomb panels depending on changing climate conditions	Furniture users as a rule consider low prices, lower product weight and easy transport of furniture items to the intended location as obvious advantages. However, practically no studies in available literature are found to assess the effect of variable climate conditions on the stiffness of shelves and furniture made from honeycomb panels. The aim of this study was to determine the effect of these changes on the value of Young's modulus and on the stiffness of whole furniture units subjected to torsional loads. Tests were performed on furniture units, which elements were manufactured in five structural design variants. It was shown that the applied materials retain high quality of the furniture units only at the temperature T = 26 °C and humidity H = 40%. An increase in the values of these climate parameters to T = 28 °C and H = 85% results in a drastic deterioration of furniture quality.	[ "ENGI" ]	[ "Climate", "Experiment", "FEM", "Furniture", "Honeycomb", "Stiffness" ]	[ "2205", "2210", "2215" ]	[ "Rational raw material use manifested in the increased utilisation of light materials may ensure economical management of natural resources and reduced greenhouse gas emissions [1].", "Another important justification for the use of lighter materials is connected with attempts to reduce production costs aiming at the increase in profitability.", "In this context, the new cores, as lightweight biomimetic functional-structural materials BEPs are developed.", "This structures characterising by strengthening mechanism of high energy absorption [2].", "Zhang et al. [3] performed experiments on BEPs and honeycomb plates (HPs) to investigate the characteristics of compression, buffering and energy dissipation in beetle elytron plates (BEPs).", "Furthermore, these findings may suggest a biological model for the development of fully integrated honeycomb-structured materials [4].", "For the end user the benefits will always include lower prices, lower weight of the product and easy transport to the intended location.", "Wood as well as commonly used wood-based materials, e.g. particle board (PB), medium density fibreboard (MDF), high density fibreboard (HDF), plywood (PW) or blockboard (BB) are used thanks to the efficient utilisation of their capacity to transfer axial, bending and torsional loads.", "Other reasons for the selection of these materials are connected with their common availability, sustainability, processability, esthetic value and an advantageous weight-to-strength ratio [5–7].", "These are the primary reasons why wood and the above-mentioned wood-based materials are commonly used in furniture design.", "Layered wood-based boards are characterised by highly satisfactory values of Young's modulus and the modulus of elasticity in shear, technologically advantageous isotropy or orthotropy as well as low density [8–11].", "In this respect layered honeycomb panels prove to be attractive materials.", "Low weight is their greatest advantage [12,13], particularly in the manufacture of light furniture for elderly users, disabled users and children [9,14,15].", "At the same time in comparison to other wood materials wood-based honeycomb panels are exposed to greater changes under climate conditions in locations of their use [11,16].", "These results are not only from the functions of these rooms, such as the kitchen, bathroom or the living room, but also climate zones worldwide.", "For this reason in order to efficiently use honeycomb panels of a specific structure their deformability and susceptibility to damage need to be taken into consideration [17,18].", "It would be particularly important to be able to predict these problems in order to more reliably design furniture to be used in various climate zones.", "To date stiffness and strength of entire furniture units have been tested under conditions simulating actual use conditions, i.e. at the humidity and air temperature of approx. 65% and 21 °C, respectively [19–22].", "Study [11] described the effect of changes in humidity and air temperature on stiffness and strength of L-type joints made from honeycomb panels.", "No studies have been conducted to assess the effect of variable climate conditions on the stiffness of shelves and whole furniture produced from framed honeycomb panels.", "The aim of this study was to provide insight into the effect of changes in climate conditions, relative humidity and air temperature, on deflection of shelves made from honeycomb panels differing in the core structure.", "Another objective was to determine the effect of these changes on the value of Young's modulus of these shelves and on stiffness of furniture subjected to torsional loads.", "This knowledge may be used to optimise design processes of light furniture, meeting the requirements imposed in view of their intended use.", "Tests were conducted on particle board (PB) (P2 – furniture boards for interior use), beech plywood (PW), as well as high density fiber board (HDF).", "These boards were used to produce structural elements of bookshelves, including side walls, top elements, bottom elements, shelves and back walls.", "Dimensions of these elements and structural design types are given in Fig. 1 and in Table 1.", "The dimensions of the shelves to be tested are based on the overall dimensions of the furniture.", "Hence L = a-2t, and W = b-16 mm, where a and b respectively the width and depth of the furniture.", "Shelves were manufactured in four structural design variants.", "The board core was composed of a frame of particle board (A, B) or plywood (C, D), filled with hexagonal celled paper core (Fig. 2).", "The top and bottom elements (TE) were manufactured similarly as shelves, with the difference in the width of the rails forming the frame, which was 50 mm in this case.", "Side walls were manufactured similarly as the top and bottom elements (TE).", "In case of facings, HDF boards thicknesses TF = 2.5 mm and TF = 3 mm were used.", "HDF boards with a smaller thickness are characterised by a higher density and a higher modulus of linear elasticity (Table 2).", "Regarding to frames, the rails and paper cores had a thickness of 16 mm and 17 mm for facings 3 mm and 2.5 mm thick respectively.", "The rails were made of particle board (PB) and beech plywood (PW).", "These wood based composites are characterised by a different structure, density, modulus of elasticity and susceptibility to moisture (Table 2).", "It was recognized that this may effect on the stiffness of panels and furniture in different climatic conditions.", "Total thickness of all elements was t = 22 mm.", "The hexagonal core and rails were glued to the facing panels using FOLCO® LIT D 1932-MOD adhesive.", "It is a PVAc adhesive with viscosity of 12000 mPa s, pH = 3 and density of 1080 kg/m3.", "Adhesive was applied onto HDF surface at 120 g/m2.", "The manufactured units were pressed for 10 min at 70 °C and pressure of 0.4 MPa.", "All the elements were edgebanded with ABS bands of 1.5 mm in thickness using Purmelt RS G 270/7 hot-melt glue with viscosity of 30,000 mPa s. Wide panel surfaces were painted with white acrylic paint applied at 80 g/m2.", "The back wall was manufactured from HDF panels of 3.0 mm in thickness, attached to the box with housed joints.", "The side walls, the top and bottom elements TE were assembled using the semi rigid eccentric joints (Fig. 2).", "Shelves A, B, C and D were placed inside the furniture and supported on dowels.", "They are a withdrawable elements, not joined to the side walls (Fig. 3a).", "From this reason, shelves, top and bottom elements were subjected to bending and the furniture without the shelves were subjected to torsion.", "Dimensions of furniture, arrangement and symbols of the outside elements and shelves are shown on Fig. 3.", "It was decided to conduct tests under two extreme climate condition variants, analogously as it was in study [11].", "Obviously the recommendations of the binding standard were considered first [23].", "However, in view of the fact that in countries of the tropical climate zone more demanding climate conditions are found, it was decided to select two climate condition variants.", "One variant with air temperature and humidity of T = 26 °C and H = 40% reflected the climate conditions of the manufacturing plant, in which furniture was manufactured.", "The other variant with air temperature and humidity of T = 28 °C and H = 85% corresponded to climate conditions of countries from the tropical climate zone.", "First physico-mechanical properties of the materials were tested under climate conditions reflecting those of honeycomb board manufacture (T = 26 °C and H = 40%) and at T = 28 °C and H = 85% after 21-day conditioning.", "Humidity was determined in accordance to the standard [24], density according to [25], while strength and the modulus of elasticity in bending was determined according to [26].", "Ten samples were used in each test type, i.e. a total of 60 samples were tested.", "Testing results are given in Table 2.", "Furniture was tested under identical conditions.", "Ten bookshelf units of identical design were manufactured.", "Shelves (A,B,C,D) and bottom element TE in 5 bookshelves were loaded uniformly over their entire surface.", "The value of that load was Qu2 = 1476.3 N/m2 (38 kg).", "The top elements TE were loaded with the force Qu1= 630.6 N/m2 (18 kg) (Fig. 3a).", "Over a period of 21 days 10 bookshelves and 60 samples of selected wood-based materials were conditioned in the production hall, at T = 26 °C and H = 40%.", "In the case of 5 bookshelves every day at the same hour deflections of loaded elements were measured accurate to 0.01 mm.", "At the completion of the first 21-day conditioning cycle 5 bookshelves and 30 samples of wood-based materials were kept for further testing.", "Bookshelves and samples were placed in the conditioning chamber with controlled constant air temperature and relative humidity parameters (T = 28 °C and H = 85%).", "Shelves (A,B,C,D) including bottom element TE and top element TE of bookshelves were uniformly loaded over the entire surface applying Qu2 = 1476.3 N/m2 and Qu1 = 630.6 N/m2, respectively.", "For the next 21 days every day at the same hour deflections of loaded elements were measured accurate to 0.01 mm.", "At the completion of that period samples of wood-based materials were used in testing of physico-mechanical properties.", "In turn, bookshelves were placed in the production hall and board deflection was recorded for the next 14 days.", "Furniture was tested in the shop floor and at a laboratory of a leading furniture manufacturer in Poland.", "That manufacturer supplies their products to Asian countries.", "For this reason the number of days for conditioning under individual climate condition variants was based on the experience of that manufacturer.", "Thus shelf deflection recorded on the last day of each measurement cycle was used to calculate their MOE.", "Moreover, changes in MOE of individual board structures were presented in the function of the number of days in the measurement cycle.", "An analogous procedure was applied for bookcases conditioned at T = 28 °C and H = 85%.", "Measurement results were averaged and compared with the results of analytical and numerical calculations.", "Defining displacement of the i-th board in the general form as:", "Stiffness K was calculated for variable values of MOE of the top element TE, dependent on the assumed climate conditions.", "It was also decided in the calculations not to include shelf stiffness, thus n = 5.", "This is because shelves are the elements, which are not joined with the furniture unit.", "As such they are not subjected to torsion during furniture twisting.", "Thus they have no effect on its stiffness.", "For this reason stiffness of the side walls, the top and bottom elements and the back wall were summed up.", "At the same time it was decided that in each analysed structure all elements will have an identical structural design.", "It was also decided to calculate stiffness of furniture subjected to twisting under variable climate conditions using the finite element method (FEM).", "The numerical model was constructed applying the scheme of support and loading of furniture as in Fig. 3b.", "The force of external load F = 391.9 N was calculated from Eq. (5).", "The bottom element of the furniture unit was attached to four legs of 50 mm in diameter each.", "Three legs were fixed.", "The fourth leg was not fixed (Fig. 3b).", "The side walls and the top and bottom elements were joined flexibly, facilitating contact between their surfaces.", "Stiffness Ks in Nm was also ascribed to each contacting surface (Fig. 5).", "For this purpose furniture joint testing results presented in study [11] were used.", "For compressing joints Ks = Mc was assumed, while for tensing joints it was Ks = MT, for T = 26 °C and H = 40%, and T = 28 °C and H = 85%, respectively.", "The back wall was joined with an articulated joint with the furniture box.", "A total of 3.56·106 nodes and 2.357·106 isotropic solid elements were generated.", "The horizontal and vertical elements were ascribed MOE values established based on bending of the TE panel under varying climate conditions (Table 3).", "The back wall was ascribed a MOE value corresponding to an HDF panel of 3.0 mm in thickness (Table 2).", "Calculations concerning linear elasticity were performed applying the finite element method with the use of the Nastran inCAD®.", "Results of MOE calculations were subjected to the two-way analysis of variance ANOVA using the Statistica v.13 computer software.", "The analysis consisted in the breaking of the variation into additive components.", "Individual variances resulting from the effects of changes in air temperature and humidity as well as board structure types were compared.", "The variance measuring the random error was also analysed.", "This analysis was expected to verify whether the applied factors play a significant role in the modification of MOE values.", "Table 2 presents physico-mechanical properties of wood-based materials used in the manufacture of tested furniture.", "It shows that 21-day conditioning at T = 28 °C and H = 85% resulted in a considerable increase in sample moisture contents.", "For HDF panels of 2.5 mm and 3 mm in thickness this increase amounted to 153% and 132%, respectively.", "In the case of particle boards and plywood of 16 mm and 17 mm, respectively, it was 146%, 143%, 105% and 123%.", "At the same time a slight 1% increase in density of these materials was recorded.", "In contrast, a marked deterioration was observed in the case of mechanical properties.", "For HDF panels of 2.5 mm and 3.0 mm MOE decreased 3.37- and 2.18-fold, respectively.", "For particle boards and plywood of 16 mm and 17 mm MOE decreased 1.98-, 2.00-, 1.31- and 0.74-fold.", "Comparable values were also recorded for the decrease in strength of wood-based materials in bending tests.", "This leads to a conclusion that wood-based materials produced from wood fibres or particles exhibit the greatest sensitivity to changes in climate conditions.", "They may even result in the deterioration of mechanical properties of furniture boards produced from these raw materials.", "Fig. 6 presents the effect of conditioning of bookshelves under varying air temperature and humidity conditions on the deflections of shelves and the top element.", "It results from Fig. 6a that after 21 days of conditioning under shop floor conditions (T = 26 °C; H = 40%), the greatest deflection values were recorded for shelves type A, while they were smallest for shelves type D.", "The top element TE due to the support method exhibited the greatest stiffness.", "Board deflections were increasing gradually with the passing days; however, this increment was not proportional.", "Board conditioning for 21 days at elevated air temperature and humidity (T = 28 °C; H = 85%) resulted in a further, although this time more rapid increase in deflections (Fig. 6b).", "It also needs to be observed here that also under these conditions the increment in deflections was not proportional.", "The greatest deflection was recorded for shelves type A, while it was smallest for shelves type D.", "The top element TE due to its support on three edges was characterised by the greatest stiffness.", "As a result of conditioning of the same furniture bodies for the next 14 days under the shop floor climate conditions (T = 26 °C; H = 40%) board deflections remained almost constant or increased slightly.", "Fig. 7 presents changes in Young's modulus MOE of boards, calculated based on deflections at individual days of measurements.", "It results from that figure and from Table 3 that at the beginning of the conditioning process at T = 26 °C and H = 40%, the modulus of elasticity of boards A, B, C, D and TE amounted to 2254 MPa, 3024 MPa, 3246 MPa, 3503 MPa and 3353 MPa, respectively.", "After 21 days these values decreased by 43%, 62%, 50%, 37% and 56%, respectively.", "As a result of conditioning at air temperature T = 28 °C and humidity H = 85% after the next 21 days of conditioning the modulus of elasticity of boards A, B, C, D and TE decreased 6.6-, 6.2-, 4.9-, 3.8- and 4.6-fold in relation to the values recorded at the end of the previous conditioning period.", "It also results from Table 3 that for most types of tested boards MOE was decreasing further.", "Considering the changes in mechanical properties of wood-based materials caused by changes in climate conditions, as it was presented above, this confirms the regularity that they also affect changes in MOE of furniture panels.", "Thus the greatest stiffness was recorded for shelves D, which internal frames were manufactured from plywood of 16 mm in thickness, while facing was made from 3 mm HDF panels.", "The use of 17 mm plywood and 2.5 mm HDF facing (shelves C) reduces the value of Young's modulus.", "However, its value is much greater in comparison to MOE of boards A and B. Particle boards may be used to manufacture frames only in the case of top elements TE.", "The results of statistical calculations (Table 4) clearly show a significant (p = 0.00) effect of the structure type TYPE, climate conditions TH and interactions of these factors on the value of MOE.", "In turn, Fig. 8 presents a graphic interpretation.", "The obtained graph confirms the significance of the effect of the structure on the increase in the values of MOE and the significance of the effect of elevated air temperature and humidity on the reduction of MOE values.", "At the same time a significant effect of interactions was shown.", "The greatest values of MOE are found for structures of D and TE under the shop floor climate conditions.", "The lowest MOE was obtained for structure A at the climate conditions corresponding to the tropical climate.", "The effect of the described changes in MOE on flexural stiffness K of bookshelves is presented in Fig. 9.", "It shows that furniture bodies manufactured from TE boards are characterised by high initial stiffness.", "Depending on the adopted calculation method: testing, FEM or analytical calculations, the value of index K amounts to 23.5 N/mm, 23.4 N/mm and 22.3 N/mm.", "Assuming the elastic properties of boards, which were established at the end of the conditioning period at T = 26 °C and H = 40%, stiffness of furniture bodies decreased to 15.3 N/mm, 14.4 N/mm and 14.4 N/mm, respectively.", "After the conditioning period at T = 28 °C and H = 85% furniture stiffness should be 3.6 N/mm, 3.2 N/mm and 3.2 N/mm.", "The lowest admissible stiffness of cabinet furniture should be min.", "10 N/mm [10].", "This results from the fact that conditioning of furniture under tropical climate conditions has an adverse effect on furniture quality.", "None of the proposed board structures (A, B, C D, TE) may ensure adequate furniture stiffness after the conditioning period under these conditions.", "This results from the fact that these boards are characterised by low MOE values in comparison to board TE (Table 3).", "Presented results of analytical and numerical calculations do not differ markedly from the results of laboratory measurements.", "Fig. 9 presents slight differences in stiffness between individual variants of the analysed structures.", "Based on the analysis of collected testing results and performed calculations the following conclusions and observations may be formulated.", "Conditioning of furniture at the air temperature T = 26 °C and relative humidity H = 40% results in a significant reduction of stiffness of shelves manufactured from honeycomb panels.", "Under these conditions the most advantageous performance properties are found for lightweight layered boards with the core produced from beech plywood slats of 16 mm in thickness.", "In turn, the use of particle board slats of 17 mm in thickness and facing of 2.5 mm HDF panels is definitely disadvantageous.", "At the climate conditions resembling the tropical climate none of the proposed structures showed sufficient stiffness.", "Thus it would be advantageous to increase facing thickness and the resulting Young's modulus.", "Satisfactory stiffness of bookshelves at twisting was obtained only at the air temperature T = 26 °C and relative humidity H = 40%.", "At elevated air temperature and humidity furniture stiffness is below the critical value of 10 N/mm.", "Analytical and numerical calculations confirmed by the results of laboratory measurements emphasise the effectiveness of modelling the stiffness of furniture structures based on properties of elastic properties of materials.", "Thus the obtained results may be used to optimise the design of light furniture items, meeting the requirements for their respective intended uses." ]	[ "The effect of humidity and air temperature on the quality of furniture manufactured from honeycomb panels was studied.", "Changes in stiffness of horizontal furniture elements were determined.", "Changes in stiffness of furniture bodies were determined.", "Results of calculations based on the analytical and numerical models were compared." ]
The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality	With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me2SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me2SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me2SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me2SO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.	[ "AGRI", "BIOC" ]	[ "Bioprocessing", "Cryopreservation", "Dimethylsulfoxide", "HOS TE85", "Human mesenchymal stem cells", "Toxicity" ]	[ "1100", "1300" ]	[ "Cell therapies hold the potential to revolutionise healthcare as regenerative medicines, replicating the success of the human therapeutic protein industry.", "However, cell therapies are more complex than protein therapeutics, which to the makes the preservation, long-term storage and shipment of cellular therapies a challenging prospect.", "Cryopreservation methods for long-term storage are described only in brief when reported in cell therapy clinical trial protocols.", "For example, while a cooling rate may be provided, processing times before freezing or after thawing are rarely given [13].", "Components commonly used in lab-based cryopreservation protocols, including animal serum and dimethylsulfoxide introduce commercial, safety and regulatory risks for new therapies [38].", "Animal-component free cryopreservation solutions are already available, such as the CryoStor® range from BioLife Solutions and PrimeXV®-FreezIS from Irvine Scientific.", "However, with cell-based therapeutics approaching the critical Phase III stage of development, there is a clear need to develop cryopreservation processes that operate at meaningful scale, and which integrate with other stages of an overall bioprocess.", "A well-integrated cryopreservation process enables the decoupling of commercially scaled cell therapy manufacture from final delivery and administration to patients.", "This in turn allows more cost-effective supply chain strategies for initial cell banking of isolated donor material, as well as the final product.", "The ideal result is an inventory of well-stored and consistent cell therapy product, which can be globally distributed and administered at an affordable cost.", "Human mesenchymal stem cells (hMSCs) are the most likely candidates for early successful human stem cell therapies, with hundreds of clinical trials evaluating their therapeutic potential [39].", "MSCs are multipotent; relatively easy to isolate; have the capacity for self-renewal and proliferation and are undergoing assessment for a number of therapeutic indications including immunological, cardiac and musculoskeletal conditions [17,28].", "From a manufacturing perspective, clear definition of critical quality attributes and a link to a therapeutic mechanism of action (MoA) is necessary.", "Unfortunately, the complexity of MSCs and diversity of patients and individual disease progression has resulted in a lack of clarity around MoA for these therapies [4].", "A variety of potency assays have been developed for some therapeutic indications, but at present there is no consensus on which should be used for a given cell type and therapeutic indication.", "The International Society for Cellular Therapy (ISCT) recommends a minimal hMSC specification as being adherent to culture plastic; positive (≥95%) for the expression of CD73, CD90 and CD105, negative (≤2%) for the expression of CD14, CD19, CD34, CD45 and HLA-DR, and possessing the capacity to differentiate towards the chondrogenic, adipogenic and osteogenic lineages in vitro [8].", "These guidelines continue to be widely used to specify identity assays for MSCs.", "Using cryopreservation to decouple production and delivery of hMSC-based therapies is a popular strategy, as demonstrated by a 2013 survey highlighting that over 80% of MSC-based regulatory submissions used cryopreservation to store and transport the final product [23].", "For example, Prochymal® (Mesoblast, USA), which has been conditionally approved for use in children with graft-versus-host disease in New Zealand and Canada, is stored in 15 mL Cryocyte bags with 10% (v/v) Me2SO [14].", "However, recent reports have suggested that cryopreserved cells do not perform as well therapeutically compared with freshly cultured cells [10,25].", "Cryopreservation may be one of the reasons, as well as in vitro cell age, why several recent hMSC clinical trials have failed to reach their primary end-points, in contrast to earlier academic-led trials using freshly cultured cells.", "Given the technical nuances of cryopreservation, it is important to determine whether transient loss of cell function is an unavoidable feature of cryopreservation, or whether it is caused by aspects of the process that differ with process scale.", "Me2SO has been used as a cryoprotectant since the 1950s [21], but there is some evidence of rare but significant adverse effects in patients, including strokes, heart attacks and haemorrhages after infusion with bone marrow cryopreserved in 10% (v/v) Me2SO [6].", "Notably, not all adverse reactions can be directly attributed to Me2SO [34] and the FDA Guidance for Human Somatic Cell Therapy and Gene Therapy [37] does not prohibit the use of Me2SO as a CPA.", "However, there is a perception that minimizing exposure of cells to Me2SO will mitigate infusion-related effects in patient and importantly, Me2SO is also cytotoxic to human cells above 0 °C and has been linked to changes in differentiation capacity of stem cells [1,9,36,43,44].", "The mechanisms of Me2SO cytotoxicity are not well defined, but may be related to the enhanced diffusion of other molecules, including toxins, across cell membranes [42], or through destabilisation of normal ionic homeostasis.", "The use of Me2SO and other permeating cryoprotectants must be managed carefully to minimise the transplantation of poorly functioning cells; dead cells; cell debris or potential cytotoxins into patients.", "Cells are often processed in small batches for routine research using passive slow cooling devices (e.g.", "Mr. Frosty™ or CoolCell®), with modest cell densities around 1 × 106 cell/mL and cells being kept on ice until transfer into their cooling device.", "However, working cell banks for manufacturing can contains hundreds of vials and allogeneic therapies are expected to require lot sizes of 1 × 109 or 1 × 1010 cells to meet market demands at affordable cost, with each dose containing 5 × 107 - 2.5 × 108 cells [31].", "Cryopreservation processes will therefore need to be in place to enable preservation of large numbers of small cryovials for banking and cryobags or larger vials (e.g. Crystal® or Daikyo Crystal Zenith® vials) for cell product packaging.", "In both instances, processing times will increase compared to lab scale and maintaining chilled temperatures during vial filling and the particulate testing required by USP <790> (Visible particulates in injections) for product vials is an operational burden.", "This means that the detrimental effects on cell quality of exposure to cryoprotectants like Me2SO will likely impose limits to the achievable process scales for hMSC therapy manufacturing.", "For example, it has been reported that for recombinant baby hamster kidney (rBHK) cells, cryopreservation bags must be filled within a 2 h window to avoid adverse effects from Me2SO exposure [15].", "Similarly, Hunt et al. [16] showed that the “recovery index”, a measure of viability from membrane integrity and colony-forming-ability after thaw, of CD34 positive cells can be reduced by as much as 50% when exposed to 25% (v/v) Me2SO for up to an hour at 20 °C.", "Katkov et al. [18] showed that human embryonic stem cells lose around 50% expression of the vital Oct-4 marker after exposure to Me2SO using standard protocols.", "These examples indicate that exposure of MSCs to Me2SO will need to be limited in order to maintain therapeutically desirable cell characteristics.", "Furthermore, these tolerances will need to be defined and built into not just manufacturing but also clinical practice.", "For example, with product thawing and preparation for infusion or injection the implications of failures in a thawing protocol or device (e.g. long-term maintenance of the vial at > ambient temperature) and delays in preparing the patient for treatment must be considered in order to develop preparation guidelines for the product.", "To that end, we investigated the effect of Me2SO on cell quality by exposing bone marrow derived hMSCs to this cryoprotectant for varying amounts of time i) without, ii) prior to, and iii) after employing standard freezing protocols.", "A maximum exposure time of 2 h at ambient temperatures prior to freezing or at 37 °C after freezing was chosen to represent worst case scenarios.", "Results are discussed in terms of the risk of product failure with cells consider to be ‘overexposed’ to Me2SO once cell characteristics such as attachment capability and phenotype fall below pre-defined standards such as those set by the ISCT or FDA.", "Furthermore, the sensitivity of hMSCs to Me2SO was compared to that of an osteosarcoma-derived cell line, HOS TE85, to establish whether hMSCs are more or less susceptible to Me2SO-induced effects than similarly sized human cells.", "Osteosarcoma cells of the line HOS TE85 were acquired at passage 51 (ATCC, USA) and hMSCs derived from bone marrow at two passages post extraction were cultured as adherent monolayers in 25 cm2 T-flasks (Nunc, UK).", "hMSCs had been extracted from ethically sourced fresh bone marrow aspirate obtained from Lonza (Lonza, Cologne AG) from a healthy donor after the patient provided informed consent.", "HOS TE85 cells were incubated in MEM medium (α-MEM, 1 g/l glucose with 10% (v/v) US origin FBS (Performance Plus) and 2 mM l-glutamine, (Thermo-Fisher, UK)), and hMSCs in DMEM medium (made up in the same way with 1 g/L glucose (Thermo-Fisher, UK) in place of α-MEM) in a humidified incubator at 37 °C, 5% CO2.", "Passaging was done every 2–3 days (HOS) or 7 days (hMSC).", "A 100% medium exchange was done for hMSCs on day 4.", "Spent-medium was collected before passaging for metabolite analysis.", "Cells were passaged with 0.25% (w/v) trypsin/EDTA.", "Samples were taken for cell counting and viability assessment, then the remaining cells were centrifuged at 220 g for 5 min.", "After aspirating the supernatant, the cell pellet was gently suspended in fresh medium.", "Cells were then used to seed new flasks at 2 × 104 cells/cm2 (HOS) and 5 × 103 cells/cm2 (hMSC).", "For HOS cells, 50 μL samples were taken and mixed with 50 μL of trypan blue stain (Sigma Aldrich, UK) then cells were counted manually using a haemocytometer and light microscope.", "A Nucleocounter NC-100 (Chemomotec, Denmark) was used to count hMSCs, which measures membrane integrity using propidium iodide (PI).", "These two counting methods have been previously found to be statistically similar [32].", "For cryopreservation experiments, HOS cells were harvested after 2 days in culture and hMSC cells were harvested after 7 days in culture, using trypsin as described above in Section 2.1.", "After centrifugation, trypsin containing medium was aspirated and the resulting cell pellet was then suspended in freeze medium (10% (v/v) Me2SO, 90% (v/v) FBS) at room temperature.", "1 mL aliquots were loaded into 1.8 mL cryovials (Nunc, UK).", "Cryovials were placed into a CoolCell (BioCision, USA) and quickly transferred into a −80 °C freezer.", "This passively cooled the vials at approximately −1 °C/minute according to the manufacturer [2].", "The total processing time from harvesting to loading of the CoolCell into the freezer was no more than one hour.", "After 4 h and no later than 24 h, cryovials were stored in the vapour phase of a liquid nitrogen cryostorage unit for at least 1 week.", "Cryovials were retrieved from cryostorage then thawed in a 37 °C water bath for 4 min.", "Thawed cells were then diluted 1:1 in the vial, followed by 1:4 in 37 °C pre-warmed medium.", "Diluted cells were then centrifuged, counted and seeded into 25 cm2 tissue culture flasks.", "Cells were exposed to Me2SO following three different schemes.", "For the ’just exposed’ group, cells were suspended in a freeze medium at room temperature for 15, 30, 60 or 120 min, and then washed and cultured with fresh culture medium, followed by analysis for viability and attachment.", "In the ‘exposure + freeze’ group, cells were exposed to Me2SO as per the “Just Exposed” group, transferred to cryovials and then frozen with a passive freezing device (1 °C/min) (CoolCell, BioCision), stored in the vapour phase of liquid nitrogen for one week and then thawed as normal.", "For the ‘freeze + exposure’ group, cells were frozen and stored as described in Section 2.3, but after thawing were left at 37 °C for 15, 30, 60 and 120 min before being washed in fresh medium, cultured and analysed.", "After one passage, all groups were analysed for growth, phenotype and metabolic activity, and then cultured for four further serial passages and their growth, phenotypic and metabolic activity was compared.", "Cells at a density of 2.5 × 106/mL were lysed with two freeze/thaw cycles.", "In each cycle, cells were suspended in dH2O, transferred to a −20 °C freezer for 24 h then warmed in a 37 °C water bath.", "100 μL of cell lysate was transferred to a 48 well plate.", "Then, 300 μL of a working solution (1 (p-nitrophenyl phosphate substrate) pNPP tablet for every 5 mL, 1 part 20 mM MgCl2, 1 part 1.5 M Alkaline buffer solution and 7 parts dH2O) was added to the lysate.", "This solution was incubated for one hour in the dark at room temperature, before being quenched with 100 μL of 3M NaOH.", "100 μL of the sample was transferred to a 96-well plate and measured with a plate reader at 405 nm absorbance.", "A standard curve of p-nitrophenol in the working solution without a pNPP tablet was used.", "All reagents were from Sigma-Aldrich, UK.", "This assay was carried out using a previously described protocol [5].", "Harvested cells were suspended at 5 × 105 cells/mL in medium and 200 μL of cell suspension was added per well of a V bottomed 96-well plate (Corning, UK).", "The plate was centrifuged at 300 g for 5 min, the supernatant aspirated, and then the cells were re-suspended in 200 μL of flow cytometry staining buffer (R&D Systems, UK).", "The plate was centrifuged and the supernatant removed.", "As per Table 1, 5 μL of each antibody (BD Biosciences, UK) was then added to the cell pellets.", "The plate was then incubated in the dark at room temperature for 30 min, before being centrifuged.", "Stained cells were washed twice with staining buffer, then suspended in 200 μL of staining buffer.", "Data were obtained using a Guava® easyCyte 8HT flow cytometer (Merck Milipore, UK) equipped with excitation lasers at 488 nm and 640 nm and running guavaSoft™ InCyte™ (v2.5) acquisition software.", "For each sample, a minimum of 5000 (gated on forward/side scatter) events were recorded.", "Spectral overlap compensation was determined using fluorophore-conjugated anti-mouse Ig,κ antibodies.", "Analysis after acquisition and compensation was done with FlowJo v7.6.5.", "(Treestar, Inc, USA) software.", "To analyse for co-expression of all five markers, serial gating was used [5].", "Gates were set firstly on the CD73+ve/CD105+ve population, subsequently on the HLA-DR-ve/CD90+ve and CD34−ve/HLA-DR-ve population (see Fig. 3E).", "After exposure/thawing an additional flask was seeded to evaluate attachment efficiency.", "After one hours' incubation, cells were enzymatically detached from the culture vessel and counted.", "Cell counts were normalised to the known seeding density to determine attachment efficiency.", "All statistical data analysis was performed using the SPSS version 21 (IBM).", "Significance was determined using a One Way ANOVA test, followed by Dunnett's test for comparison with controls.", "Values of p < 0.05 were considered to be statistically significant.", "All data quoted represent the mean of two duplicates from three independent experiments, ± standard deviation (SD), unless otherwise stated.", "HOS TE85s and early passage hMSCs were exposed to Me2SO as described in Section 2.4 and membrane integrity as a proxy for viability was subsequently measured either immediately after exposure (just exposed group and freeze + exposure groups) or post-thaw (exposure + freeze).", "The viability of the HOS TE85 and hMSC cell populations decreased in a time-dependent manner under all three conditions of Me2SO exposure (Fig. 1A and C).", "Viability fell to ∼70% when cells were exposed to Me2SO for 120 min once thawed after freezing (i.e. the ‘freeze + exposure’ group).", "It is noteworthy that the viability range exceeded the US FDA recommended lower limit for membrane for therapeutic applications [23,38].", "These data indicate the importance of timing when carrying out the viability testing as part of release criteria as close to point of administration as possible, rather than at the immediate point of thaw (if these are to be different).", "These results are similar to those reported by Hunt et al [16] who found that CD34+ cells exposed to 25% (v/v) Me2SO for up to 20 min at 2 °C showed negligible drop in viability, with loss of viability observed only with increasing time and temperature of exposure.", "However, the study is limited to Me2SO exposure only, rather than the combination of exposure and freezing as shown in this work.", "Here we show that none of the just exposure groups (without cryopreservation) were statistically different from the non-exposed controls.", "This suggests that it is the combination of Me2SO exposure and the cumulative complications of freezing and thawing, such as changes in osmolality during slow cooling and rewarming, which results in the loss of membrane integrity, rather than exposure to CPA per se.", "It has been established that, although cells may appear viable upon thaw based on simple measures of viability like trypan blue exclusion, they may undergo delayed onset apoptosis [40].", "For example, Aye et al. [1], showed that after 3 h CHO cells exposed to 20% Me2SO still exhibit viability of over 90%, but after 24 h this was reduced to just 10%.", "Therefore, cell attachment one hour after seeding was also investigated as a potential indicator of cell survival.", "hMSC homing and engraftment in vivo might be necessary for sustained clinical function [19].", "Normal attachment efficiency one hour after seeding is >85%, but both freezing and Me2SO exposure time had impact on this response.", "The Frozen + Exposure group had the lowest attachment efficiency after two hours (Fig. 1C and D), with just 65% attachment for HOS TE85 cells, and 53% for hMSCs, a significant reductions of 21% and 46% respectively.", "Significant drops in attachment from the un-exposed controls were observed in the HOS TE85 120 min Freeze + Exposure group, and in hMSC 60 and 120 min Exposure + Freeze and Freeze + Exposure group.", "Cell attachment has not been used extensively to assess cell quality, but it is one of the three key criteria for MSC identity proposed by Dominici et al., in 2006 [8], and perhaps the simplest to observe and quantify.", "Pal et al. [27] have previously noted unquantified abnormal cell attachment of stem cells after Me2SO exposure for 24–48 h at low concentrations, 0.01% (v/v), 0.1% (v/v) and 1% (v/v) [27].", "The data here also show that cell populations have an observed viability based on membrane integrity after thawing, but that this does not correlate to the number of cells that had adhered to tissue culture plastic one hour after seeding.", "The latter should be a more stringent metric of viability, as cell adhesion and spreading are reliant on active processes including actin filament formation [26] and energy metabolism.", "Furthermore, the proteolysis of cytoskeletal components, which maintain cell shape and are crucial for cell-surface attachment, is one of the first stages of delayed-onset apoptotic cell death commonly seen after cryopreservation [35].", "Using attachment efficiency might therefore be a better candidate benchmark for release from the manufacturing site, as it relates better to MSC health and identity after recovery from cryopreservation.", "It was clear that using the same 70% threshold shows that cells reach this level of acceptability after around 1 h of exposure to Me2SO and indicates that any longer than this would constitute overexposure as cell function is affected.", "Me2SO has previously been shown to affect cell proliferation both negatively [35] and positively [30].", "Therefore, to assess the potential for longer-term impact of Me2SO exposure on cell quality, hMSC and HOS TE85 cell proliferation and basic metabolic profiles (Section 3.3) were assessed over multiple passages post-exposure.", "When growth over one passage post-thaw was investigated, there was no statistically significant effect of any Me2SO treatments on HOS TE85 cells (Fig. 2A and B).", "The lack of a significant impact on growth rate indicates that in addition to the cells' quick recovery on thaw, Me2SO has a limited effect on growth capability of osteosarcoma cells once washed away.", "The hMSCs also showed a downward trend in growth with increasing exposure time (Fig. 2C), across all three treatment groups.", "However, only the Freeze + Exposure 60 and 120 min groups were statistically different from the non-exposed control.", "This lag in growth rate over the first passage post-thaw may be an important consideration for the therapeutic use of cryopreserved hMSCs, as engraftment and expansion in-vivo have direct bearing on the potential mechanisms of action clinically.", "Delayed outgrowth may also be an issue when hMSCs are banked in readiness to seed subsequent culture vessels for expansion as part of a cell therapy manufacturing process.", "The increased time required to achieve a desired cell yield directly increases costs.", "Furthermore, the loss of cells early in the process lowers the potential expansion headroom per banked vial, restricting the scale of manufacturing.", "It is also important to consider growth over several passages in order to assess whether Me2SO exposure has any sustained long-term impact on cells.", "This is important for cell therapies if the cells need to persist in the body for several days, or integrate into the host tissue and differentiate for their therapeutic effect.", "The HOS TE85 cells were cultured for five serial passages to see what effect Me2SO had on long-term growth of these cells.", "Fig. 2C shows that over the 5 passages (at least 12 doublings), there was no significant difference between any of the treatment groups.", "Baseline experiments on HOS TE85 cells (data not shown) demonstrated that the exposed cells behaved in a similar fashion to the non-exposed groups.", "Consequently, we saw no long-term impact on HOS TE85 proliferative capacity or culture growth rate.", "Human MSCs from the Just Exposure group were also cultured over 5 passages and as shown in Fig. 2D significant differences from the non-exposed control were observed in the 120 min group, further confirming that exposure of this time frame to Me2SO causes a significantly detrimental effect on hMSC growth.", "Notably, a correlation of attachment to long term growth of 0.9704 was observed (Fig. 2E), indicating that a 1 h attachment efficiency assay reflected the longer-term growth potential for hMSCs which obviates the need for protracted outgrowth studies in future.", "Indeed, a short-term attachment efficiency assay has previously been used to successfully predict proliferative potential with human embryonic stem cells (hESCs) [24].", "We therefore propose that cell attachment after 1 h is strongly correlated (Fig. 2E) with long-term expansion potential of the cell types cultured herein.", "This suggests that the impact of any delayed onset cell death is minimal if one wishes to culture cells in vitro.", "Clearly, there is potential for donor and processing variability, and this correlation should be explored during early product development to confirm its predictive value where cell expansion is a key part of a cell therapy bioprocess.", "However, it is not clear whether a correlation between in-vitro adhesion efficiency and expansion can be predictive of cell adhesion and retention in-vivo during cell therapy delivery.", "Mechanistically, this correlation is explained by the simple fact that these cells require adhesion to a surface before they can replicate.", "Fewer adherent cells mean a smaller effective seed population, with subsequent impact on long-term expansion potential in the absence of changes to the mean cell proliferation rate.", "This allows for a much quicker analysis process-weeks compared to 1 h for determining the long-term effect growth on cells (via colony forming unit counts).", "During addition and removal of cryoprotectant solutions, slow cooling and rewarming, cells are subjected to changes in osmolality and volume, with the latter being linked to changes in metabolism [20].", "Furthermore, mitochondrial and metabolic changes have been shown to occur during stem cell differentiation [41], highlighting that process conditions that alter MSC metabolism could influence their function.", "As such, offline metabolic sampling during post-thaw expansion could provide early warning of detrimental changes.", "These could then be evaluated more closely with more laborious assays focusing on specific aspects of cell state.", "Accordingly, spent medium was analysed for glucose and lactate at each passage, and in the case of hMSCs at each medium exchange as well, to assess whether cells exposed to Me2SO exhibited a change in their metabolic profile.", "From these data, the metabolic yield of lactate from glucose can be calculated and monitored.", "The maximum theoretical yield of lactate from glucose is 2 mol/mol.", "Values lower than this indicate glucose is being diverted elsewhere whereas values > 2 mol/mol indicate lactate is being generated from a non-glucose source [11].", "For the HOS TE85 cells, a control value of glucose yield from lactate was shown to be 1.403 mol/mol (Fig. 3A) based on cells grown in continuous culture.", "None of the exposed groups were significantly different from the non-exposed control, indicating that there was no substantial change in glucose or lactate cell metabolism after freezing or Me2SO exposure.", "However, there was variation across the hMSC groups (Fig. 3B).", "The hMSCs exposed after thaw for 60 min (Freeze + Exposure), and all three 120 min exposure groups were statistically significantly different from the non-exposed control.", "This indicates that 1 h or more of exposure to Me2SO can influence the subsequent hMSCs metabolic profile during in-vitro expansion.", "All exposed groups except the 15 min “Just Exposure” groups are above the 2 mol/mol theoretical yield limit, indicating the cells are converting an additional energy source to lactate, which is likely to be glutamine in the growth medium.", "This has been previously noted and proposed by Rafiq et al. [29] and perhaps indicates a less severe metabolic change than that experienced in the 60 min and 120 min groups.", "Furthermore, any change in metabolic profile could indicate that the cells are stressed or differentiating, and therefore cannot grow as efficiently, hence phenotype was assessed.", "Both cell types were cultured for one passage before phenotype analysis occurred, allowing any long-term phenotypic effect to be revealed.", "HOS TE85 phenotype was analysed by using an alkaline phosphatase (ALP) assay.", "ALP activity is indicative of a cells ability to mineralise bone, as it increases the local concentration of inorganic phosphate and decreases the concentration of extracellular pyrophosphate, which is an inhibitor of mineralisation [12].", "While HOS TE85s are osteocarcoma cells and not true osteoblasts, baseline experiments (data not shown) showed that these cells retained some ALP activity in culture.", "Longer exposure to Me2SO lowered ALP activity per cell, indicating a detrimental change in the osteoblastic phenotype (Fig. 3C).", "A reduced expression of ALP has previously been reported in pancreatic cells after exposure to Me2SO [22].", "When we consider the hMSCs, the expression of five extracellular markers was investigated; the co-expression of these is shown in Fig. 3D.", "There was a change in the phenotype away from the ISCT criteria [8] across all three groups as Me2SO exposure time increases, with significant loss of phenotypic marker expression in the three most extreme conditions.", "Therefore, exposure to Me2SO beyond an hour has a substantial negative effect on the hMSC phenotype after the cryopreservation process and thus constitutes overexposure.", "This may also indicate that Me2SO is promoting differentiation of hMSCs.", "As Me2SO has been used in conjunction with retinoic acid and 5-azacytidine to promote cardiac differentiation of human foetal liver-derived MSCs and can induce differentiation of human embryonic stem cells [7,18,27].", "Although not statistically significant, the data also shows that the effect on phenotype was enhanced when cells were frozen.", "This indicates that factors such as osmotic stress and the changes in metabolism noted earlier in these groups may also affect phenotype.", "This is consistent with the fact that metabolic changes have previously been shown to occur during stem cell differentiation [41].", "However, that phenotypic expression significantly differed from the hMSC standard after 120 min Me2SO exposure, but cell viability on thaw was above the FDA's recommended minimum 70% viability criteria [4] raises the question whether on thaw viability is a reasonable release criteria in the absence of other indicators.", "The successful cryopreservation of human cells is necessary to enable the production of off-the-shelf cell therapies, but challenges remain to retain cell functionality at commercial scale.", "Preventing changes to phenotype and metabolism is vital for cell therapeutics including hMSCs, which have been described by Caplan and Correa [3] as a “drugstore” for injury with the ability to establish a regenerative microenvironment through secreted protein factors, which regulate the local immune response at the injury site.", "If the cells are not what the physician or patient expects and cells have lost their therapeutic function or are unable to engraft, then patients will have been given at best an expensive placebo with added risk of harm.", "However, working cell banks for manufacturing can contains hundreds of vials and with cellular therapeutics requiring >109 cells per dose [31] and with these increased volumes the cell-cryoprotectant exposure time will increase as the process scales from a laboratory to manufacturing environment.", "Limits on cryoprotectant exposure time, which will vary depending on the cell source as well as the upstream processing, may drastically reduce the scale of manufacture per batch.", "Hence we looked at two worst case scenarios using hMSCs and have shown that overexposure to Me2SO, found here to occur with >1 h exposure prior to or after freezing, had a significant effect on cell metabolism and altered phenotype.", "We believe that these limits need to be accounted for when designing a larger scale manufacturing process and when training clinical staff in the thawing and use of cells preserved in Me2SO to minimise the risk of product failure.", "For example, according to USP <790> (Visible Particulates in Injections) all vials of injectables need to be visually inspected for particulates and this a time consuming element of production, with chilling of vials prior to inspection adding to the burden due to condensation on the outside of the vials.", "Our data indicates that for products packaged in 1.5–5 mL cryovials, if a semi-automated vial filling systems such as the CellSeal® system from Cook RegenTec was being used to fill vials at a speed of 10 vials per 1–2 min, these would potentially not need to be chilled prior to particulate testing, especially if filling and testing could be co-located to ensure the vials were processed within this hour timeframe.", "Notably, similar trends were also seen with an osteoblast (HOS TE85) cell line indicating these results are not unique to MSCs.", "Defining the cell sensitivity to cryoprotectant exposure will be crucial to allow sensible decision-making with regard to larger-scale process design, including batch sizing and selection of equipment to meet processing requirements within a well-defined processing window.", "As there must be some exposure of cells to cryoprotectant post-thaw, controlling pre-freeze exposure is also essential to provide a safety-margin for exposure time during processing and removal of cryoprotectant after thawing, as the effects of cryoprotectant exposure will be cumulative.", "As such, tackling the issue of Me2SO toxicity, by providing alternatives such as non-permeable sugars like Trehalose, in combination with permeabilising polymers [33], or negating the issue will be important for the commercialization of cell therapies." ]	[]
The Progress and Grand Challenge of Urbanization in China		[ "CENG", "COMP", "ENER", "ENGI", "ENVI", "MATE" ]	[]	[ "1500", "1700", "2102", "2200", "2305", "2501" ]	[ "Over the past few decades, China's economy has maintained rapid growth, with an average annual gross domestic product (GDP) growth rate of more than 10% (Fig. 1 [1]).", "In recent years, the Chinese economy has entered a “new normal,” in which the GDP growth rate has slowed but remained stable at around 7%.", "Rapid development and urbanization in China supplement and promote each other, enabling 400 million people to live in cities and enjoy the modern urban life.", "For most of its long history, China has been a traditional agricultural country, with 90% of people living and farming in rural areas and only 10% living in cities.", "Until the 1970s, this proportion remained almost unchanged, and at present, the majority of middle-aged people, their parents, and their grandparents still live in rural areas.", "After 1978, however, due to the demand for labor for economic development, the labor force began to flow from rural to urban areas.", "Rough estimates put the growth rate of the urban population at almost 1% per year, which means that 10 million people move from the countryside to cities every year (Fig. 2 [2]).", "By the end of 2012, China's urbanization rate had reached 52.57%, or a population of 0.71 billion.", "In the process of rapid urbanization, logistics and traffic surge have led to massive urban infrastructure construction.", "For example, prior to 1979, China's highway networks were not developed; however, after over 30 years of development, China's total expressway mileage reached 104 000 km by the end of 2013, exceeding the US mileage of 92 000 km and ranking first in the world.", "China's railway construction has also witnessed rapid growth.", "By the end of 2014, China's operated railroad reached 112 000 km, ranking second in the world.", "China's high-speed railway, operating at more than 300 km·h−1, will cover 16 000 km and rank first in the world (Fig. 3).", "With my own family living in Shanghai, I am especially impressed with the convenience brought about by the high-speed railway.", "In the past, it took two hours to travel from Beijing to Shanghai by plane.", "However, due to weather conditions, regulatory control, and other reasons, flights were frequently delayed.", "It now takes only four hours to travel from Beijing to Shanghai by high-speed railway; and the trip is very convenient and efficient, with no delays.", "China has become the world's largest producer and consumer of cars.", "In 2014, China produced 23 722 900 cars and sold 23 491 900, ranking first worldwide for six consecutive years.", "The growth in this industry has led to both industrial development and improved travel within China.", "Looking back, most people in Beijing 25 years ago used bicycles as a means of transport.", "At that time, bicycles made up 70% of urban transportation vehicles.", "Even the former US President George H. W. Bush and his wife Madam Barbara Bush rode bicycles in Hutong (old lane) areas in Beijing when he worked as the Chief of the US Liaison Office in Beijing in 1970s.", "Foreign friends of China joked that it was “a kingdom of bicycles.”", "Of course, the rapid development of the automotive industry has also introduced significant problems.", "Every year, during National Day and the Spring Festival, there are seven days of holidays.", "Many people choose to travel by car during this vacation, and traffic becomes extremely heavy, leaving people waiting for long periods of time at the entrances of highways (Fig. 4 shows the situation in Shanghai).", "Even the overpasses are usually blocked by excessive numbers of cars.", "Thus, the question of how to ease the pressure on urban traffic is a major challenge for city leaders and managers.", "China possesses 20% of the world's population but only 7% of the global arable land; and China's water resources per capita are only 28% of the global average (Fig. 5).", "According to the 2009 United Nations statistics, more than 400 cities in China have insufficient municipal water supplies, and 110 cities suffer water shortages.", "Resources such as timber, minerals, oil, and natural gas are also scarce.", "It is impossible for China to use rich resources to support its many people as well as supporting societal development, so urbanization must be accomplished in an environmentally sustainable manner.", "Industrial and social developments have resulted in a certain degree of air pollution.", "In recent years, large-scale air pollution incidents have occurred at times in East China, especially in regions near Beijing and regions with relatively rapid economic development.", "In 2014, large-scale smog incidents occurred repeatedly in such regions.", "In January of 2015, the number of severe smog days (Class VI pollution) in Beijing reached 14 consecutive days (Fig. 6).", "In the past 20 years, China's urbanization has been oriented by the construction of iconic facilities such as central business districts (CBDs) and industrial parks in order to attract investment.", "However, the construction of urban infrastructure has often lagged behind.", "Although the investment in infrastructure in China's major cities is enormous, the construction of underground drainage systems has often been neglected, with serious consequences.", "For example, Beijing witnessed a rainstorm event on July 21, 2012 that flooded many areas of the city, causing 79 deaths and economic losses of 11.64 billion CNY (Fig. 7).", "Many small Chinese towns and villages have maintained a pleasant natural landscape and cultural characteristics of their ethnic regions, remaining beautiful and quiet (Fig. 8).", "At the same time, however, many of the metropolitan regions resemble each other.", "For example, large cities such as Beijing, Shanghai, and Hong Kong look very similar and lack major differences.", "Culture is the spirit and soul of a nation.", "Protecting and developing the national culture and promoting cultural revival should become an important responsibility of urban construction.", "Therefore, we should retain some of the characteristics of the national culture in city designs, and large cities should include buildings with Chinese characteristics and a cultural heritage.", "China faces many other challenges with urbanization that have not been discussed here.", "How do we deal with these challenges?", "Speakers at the Global Grand Challenges Summit in Beijing in September 2015 shared a wide range of new ideas and methods to address these and other issues.", "As city planners, engineers, architects, governments, and other actors work on urbanization in China, new answers will have to be found through both innovation and cooperation." ]	[]
Highly efficient multipurpose graphene oxide embedded with copper oxide nanohybrid for electrochemical sensors and biomedical applications	A novel graphene oxide/copper oxide (GO@CuO) nanocomposite (NCS) was successfully synthesized via hydrothermal method. The NCS was characterized by XRD, SEM, TEM and BET surface area analysis. The NCS modified electrode was applied to detect the dopamine (DA) and paracetamol. The voltammograms obtained during the oxidation studies revealed that the as-synthesized GO@CuO NCS sensor shows a highly senstive catalytic activity. The oxidation peak potential (Epa) of DA at the bare carbon paste electrode (BCPE) and modified carbon paste electrode (MCPE) were observed at 0.1115 V and 0.1127 V, respectively. This electrode exhibits good and satisfactory results in the determination of DA in a commercial injection. In addition, the NCSs show enhanced antimicrobial and anticancer activities thanks to the combined effect of GO and CuO.	[ "MATE" ]	[ "Cyclic voltammetry", "Dopamine", "GO@CuO nanocomposite", "Modified carbon paste electrode" ]	[ "2501", "2502", "2503", "2504" ]	[ "Dopamine (DA) belongs to a catecholamine family, which plays an important role in the functions of the central nervous system.", "In the brain, DA functions as a neurotransmitter and shortage of DA, particularly the death of DA neurons in the nigrostriatal pathway, causes Parkinson's disease [1].", "Likewise, paracetamol (PC) is a non-steroidal anti-inflammatory drug that finds widespread application for its strong analgesic and antipyretic action.", "Practically, it is widely applied for patients with headache, backache, arthritis, migraines, neuralgia, menstrual cramps, and postoperative pain.", "However, it does not show any harmful side effects [2].", "Nevertheless, the biomolecules of PC lead to hypersensitivity or overdose causes damage to the liver and kidney leading to hepatoxicity and nephrotoxicity.", "Recently, many analytical methods have been employed for the determination of biomolecules such as chemiluminescence, spectrophotometry, titrimetric and electrochemistry.", "Among them, electrochemical sensors have attracted much attention due to their excellent properties viz., low-cost, simplicity, high sensitivity, and handing convenience [3].", "Nevertheless, the high cost of noble metal electrodes limits their usage in many applications.", "Hence, the development of a highly sensitive and selective electrode without an enzyme or noble metal is necessary.", "In recent times, nanomaterials research has gained greater momentum owing to their possession of thermo electric, optic, catalytic, mechanical properties.", "The surface coating of the electrode with nanoparticles is an attractive approach for enhancing the scope of electrochemically modified electrodes [4].", "Graphene oxide (GO) stands out amongst the most significant substituent of graphene and it is a trusted material for different innovative fields such as optoelectronics, catalysis, nano-electronic compounds, gas sensors, supercapacitors, and medical field [5].", "Numerous composites comprising of graphene oxide and metal oxides viz., NiO, MnO2, CuO, Fe2O3, TiO2, ZnO, SnO2, In2O3 and Ce2O3 have been studied for many diverse applications [6–8].", "Similarly, CuO is an assured composite because of its minimal effort, eco-richness, non-poisonous quality, and effortless preparation in different states of nanosized measurements [9].", "Since ancient times, Cu and its oxides are known to apply for various biomedical applications like wound healing ointments, dental work, food packaging, coating on clinical equipment etc., due to its inherent antimicrobial and anticancer activity [10–12].", "In order to get an enhanced biological efficiency and also to meet some particular requirements, the composite nanomaterials are in demand.", "In this way, GO can render the suitable platform to host or functionalize with CuO nanoparticles [13].", "The combination of GO and CuO could be a productive integration of the properties of two components that can head to the novel series of hybrid materials bearing new features.", "This hybridization of GO and CuO is known to enhance the active sites including superior functioning and very good intrinsic properties.", "Thus, in the quest for materials with enhanced biological activity (antimicrobial and anticancer activity), we found these hybrid materials worth exploring.", "However, there are still rather few studies on the biological activity of carbon-based materials hybridized with metal-based nanoparticles (silver, copper etc.) [14,15].", "To the best of our knowledge, researches on the biological activity of graphene oxide embedded with copper oxide (GO@CuO) nanocomposites (NCSs) have not been reported yet.", "Thus, it is clinically necessary to identify new therapeutic molecules that may significantly enhance the biological efficacy.", "These aspects of nanomedicines remain a subject of particular interest.", "In this article, NCSs were synthesized by adjusting the pH of the GO dispersion followed by mixing of copper sulfate solution.", "The synthesized material was characterized by various analytical and spectroscopic techniques and then used to modify the carbon paste electrode.", "The electrochemical effect of biomolecules on this GO@CuO modified electrode was studied.", "At the same time, the novel NCS material was used for selective determination of different biomolecules in the presence of different interfering analytes at biological pH. Their antimicrobial and anticancer activities are reported here.", "All chemicals were purchased from S.D. Fine-Chem Mumbai, India, until and unless stated otherwise.", "Analytical Reagent (AR) grade chemicals without any additional purification were used in the experiments.", "Silicone oil, graphite powder, hydrogen peroxide (30 wt %), sodium nitrate (98%), dopamine hydrochloride, sulfuric acid (98 wt%), sodium di-hydrogen orthophosphate (NaH2PO4), potassium permanganate, copper(II) nitrate tri-hydrate, disodium hydrogen phosphate (Na2HPO4), sodium hydroxide and all of the stock solutions for the preparation of composites were prepared by using double distilled water.", "GO was prepared by utilizing a modified Hummers' method [16].", "Briefly, 15 g of graphite powder was added into 250 mL of cooled sulfuric acid in an ice bath.", "At that point, 25 g of KMnO4 and 6 g of NaNO3 were added continuously with mixing and cooled so that the temperature of the solution was kept at 15–20 °C.", "The solution was then mixed at 35 °C for 25 min and the temperature was raised to 80 °C.", "After that, 250 mL of doubly distilled water was gradually mixed at 80 °C for 30 min.", "To prevent the oxidation, 50 mL of 30% H2O2 solution and an extra 500 mL of deionized water were added consecutively to decrease the effect of KMnO4.", "Further, the sample was filtered, washed with 100 mL of deionized water and took after by ultrasonic treatment for 15 min.", "The precipitates was isolated by centrifugation and after that dried in a vacuum stove at 50 °C for 18 h. NCS was prepared by fabrication of anchored CuO nanoparticles on to GO.", "In the first stage, 20 mL of 0.2 mol/L NaOH solution was gradually added into a 20 mL of 0.1 mol/L copper(II) nitrate trihydrate solution containing 0.005 mol/L of Triton X-100 with steady mixing.", "At that point, 65 mL of deionized (DI) water was added gradually into the above solution with mixing to get Cu(OH)2.", "In the second step, a certain amount of GO (1:2) was diffused in 20 mL of DI water through ultrasonication.", "To this solution, 1.2 mL of Cu(OH)2 was added and the pH was adjusted to 10.0 by adding an amount of NaOH.", "The subsequent dark solution was cooled normally to room temperature and washed three times with DI water and ethanol.", "At last, the compound was dried in an autoclave at 60 °C for 8 h.", "The powder X-Ray diffraction (XRD) patterns of NCS were obtained by using the Bruker D2 Phaser X-Ray diffractometer equipped with graphite monochromatized Cu Kα radiation and a Ni-filter.", "The structural morphology of NCS were observed by Field Emission Scanning Electron Microscope (FESEM) (JEOL, JSM-840) operated at 15 kV and Transmission Electron Microscope (TEM) (JEOL, JSM 1230) images were carried out by microscope at an accelerating voltage of 200 kV.", "Thermogravimetric analysis (TGA) was performed on TA instruments Q50.", "The heating rate was maintained at 10 °C/min in an inert atmosphere.", "Fourier transform infrared (FTIR) analysis was used to determine the surface functional groups (Bruker ATR) where the spectra were recorded from 400 to 4000 cm−1.", "Moreover, the electrochemical experiments were carried out in a three electrode cell system, which contained a bare carbon paste electrode (BCPE), CPE/GO@CuO nanocomposites (MCPE) as the working electrode.", "The BCPE was prepared by hand mixing of 80% graphite powder with 20% silicon oil in an agate mortar to produce a homogenous paste and the carbon paste was packed into the cavity of electrode of 4 mm in diameter.", "Then, the surface of BCPE was smoothed on a weighing paper and the electrical contact was provided by a copper wire connected to the carbon paste in the end of the tube.", "The MCPE was prepared by adding 2,4,6,8 and 10 mg NCS to above mentioned graphite powder and silicone oil mixture.", "The electrochemical workstation (CHI 608E) was utilized to assess the electrochemical properties of the NCS in 0.2 M phosphate buffer (pH 7.2) as the electrolyte in a three-electrode configuration utilizing cyclic voltammetry (CV).", "This contains a three-electrode cell system, a MCPE as the working electrode, an aqueous saturated calomel electrode (SCE) as the reference electrode and a Pt wire as the auxiliary electrode.", "The mass loading of the active material for each modified carbon paste electrode was about 4 mg of NCS.", "The in vitro antimicrobial activity of as-synthesized NCS was evaluated against different human pathogens namely Staphylococcus aureus (S. aureus, NCIM 5021), Bacillus subtilis (B. subtilis, NCIM 2999), Escherichia coli (E. coli, NCIM 2574), Pseudomonas aeruginosa (P. aeruginosa, NCIM 5029), Aspergillus flavus (A. flavus, NCIM 524) and Candida albicans (C. albicans, NCIM 3471).", "The microbial strains were cultured overnight at 37 °C in nutrient broth and potato dextrose agar medium.", "The broth cultures were compared to the turbidity with that of the standard 0.5 McFarland solution.", "All the Micro-organisms were maintained at 4 °C for further use.", "All the pure microbial strains were obtained from National Chemical Laboratory (NCL), Pune, India.", "The newly synthesized compounds were tested in vitro using the agar disc diffusion method by taking streptomycin and fluconazole as standard drugs for bacteria and fungi, respectively.", "The antimicrobial potentialities of the NCS were estimated by using pre-sterilized filter paper disks (6 mm in diameter) impregnated with NCS (dissolved in 100 μg/mL), which was placed on the inoculated agar.", "The plates were incubated for about 24 h at 37 °C in the case of bacteria and 48 h at 28 °C in the case of fungi.", "The zone of inhibition around the well in each plate was measured.", "The statistical analyses of the above results were performed using IBM SPSS version 20 (2011).", "One way ANOVA (analysis of variance) at value p < 0.001 followed by Tukey's Post Hoc test with p ≤ 0.05 was used to determine the significant differences between the results obtained in each experiment.", "The minimum inhibitory concentration of the NCS was determined by a dilution method.", "In this method, the NCS was dissolved and diluted to give two-fold serial concentrations.", "The NCS concentration was made from 5 to 75 μg/mL.", "The MIC value was determined as the lowest concentration of the NCS inhibiting the visual growth of the microorganism on the agar plate.", "The normal cells (Vero-ATCC® CCL-81™) and human cancer cells (HeLa-S3-,d.d2s″ > ATCC® CCL-2.2™) and (MDA-MB-231-,d.d2s″ > ATCC® HTB-26™) were maintained in Modified Eagles Medium (MEM) supplemented with 10% FCS, 2% essential amino acids, 1% each of glutamine, non-essential amino acids, vitamins and 100 U/mL Penicillin–Streptomycin.", "Cells were subcultured at 80–90% confluence and incubated at 37 °C in a humidified incubator supplied with 5% CO2.", "The stock cells were maintained in 75 cm2 tissue culture flask.", "A possible mechanism for the formation of NCS is explained as follows.", "GO is a layered material bearing oxygen-containing functional groups on their basal planes and edges.", "These functional groups can act as the anchor sites and, consequently, make nanoparticles formed in situ attack on the surfaces and on the edges of GO sheets.", "Accordingly, in the early stages, the positive Cu2+ ions formed in the presence of solvent easily adsorb onto these negative GO sheets via the electrostatic force.", "A large amount of nuclei was formed in a short time owing to the hydrolysis reaction of Cu(NO3)2.", "Then the oxygen atoms of the crystal growth units might form bonds with the functional groups via intermolecular hydrogen bonds or coordination bonds, acting as the anchor sites for the crystallites to grow further [17].", "The biocompatibility of nanoparticles is an important issue in therapeutic applications.", "Therefore, the biocompatibility and cytotoxicity of the NCS were evaluated by a colorimetric assay.", "The as-synthesized NCS was tested against different cell-lines, namely Vero-ATCC® CCL-81™, HeLa-S3-,d.d2s″ > ATCC® CCL-2.2™, and MDA-MB-231-,d.d2s″ > ATCC® HTB-26™.", "The cell viability results reveal that the different cells treated with NCS exhibited dosage-dependent and time-dependent behavior.", "However, the as-obtained NCS showed no obvious cytotoxic effect on normal cells, indicating an excellent biocompatibility of the prepared NCS.", "This lower cytotoxicity of the NCS against normal cell lines suggests its potential biological applications.", "For instance, the survivability of cells are found to be 78% for normal cells and 35% for cancer cells at a higher dose (100 μg/mL) of NCS, which is generally considered as a high toxicity for cancer cells.", "For all the cell lines with the mentioned NCS concentration, the mean and standard errors were found to be within an acceptable limit.", "This statistical data indicates the repeatability and consistency of the experiments.", "The biocompatibility for normal cells is perhaps due to the impact of targeting agents.", "However, further studies are required to understand the precise mechanism for cell interaction.", "The phase composition and structures of NCS were examined by XRD and the corresponding pattern is shown in Fig. 1c.", "The diffraction peaks observed at 2θ values of 32.5°, 35.52°, 38.78°,46.3°, 48.76°, 53.76°, 58.36°, 61.76°, 66.15° and 67.94° correspond to the (110), (111), (200), (112), (202), (020), (021), (113), (311) and (220) planes, respectively.", "They are similar to the characteristic diffractions of monoclinic phase of CuO (JCPDS 48-1548), where the (001) reflection peak of the layered GO (Fig. 1b) almost disappeared.", "It was suggested that the diffraction peak would not be prominent when GO was exfoliated.", "In this composite, the CuO dominated the GO layer, as revealed by SEM studies [18,19].", "Fig. 2 shows the surface morphology of NCS at different magnifications.", "A typical SEM image exhibits non-uniform CuO nanoparticles with the sizes ranging from 100 to 200 nm.", "After combinating with GO to form the GO@CuO composite, the CuO nanoparticles are decorated and firmly anchored on the GO layers with a rather high density.", "GO may favor the hindrance of CuO from agglomeration and enable their good distribution, whereas the CuO serves as a stabilizer to separate GO sheets against the aggregation.", "In addition, the GO@CuO composite was observed to have the specific surface area of 21.9 m2/g from the Brunauer–Emmett–Teller (BET) examination and to be porous in nature [20].", "The TEM image of the NCS shown in Fig. 3 reveals the existence of a large quantity of CuO nanoparticles with sizes ranging from 100 to 200 nm.", "It can be seen that the GO shows an ultrathin wrinkled paper-like structure and the CuO nanoparticles tend to aggregate like a needle.", "Based on the Energy-dispersive X-ray spectroscopy (EDX) results, the atomic weight ratio of C, Cu and O is 5.03%, 71.25% and 23.72%, respectively.", "Thermogravimetric analysis (TGA) is also a significant analytical technique.", "The decomposition behavior of the GO@CuO NCS was studied by TGA.", "A large weight loss can be observed at 250 °C, which is caused by the combustion of the carbon.", "After that, the prominent loss in mass was not observed till 600 °C, but a sudden drop in the mass appeared around 640 °C.", "In order to understand the nature of functional groups on their surface, FTIR measurements were conducted.", "Fig. 4 illustrates the FTIR spectra of GO@CuO.", "For GO, the peak at 3438 cm−1 corresponds to the O–H stretching vibration.", "The vibration of C–OH was observed at 1262.21 cm−1.", "The peak 1634.9 cm−1 is attributed to the C–C stretching vibration.", "The absorptions peaks at 2856.29 cm−1 and 2926.3 cm−1 represent the symmetric and anti-symmetric stretching vibrations of CH2.", "The absorption peaks at 1390.67 cm−1 and 1107 cm−1 are corresponding to the stretching vibration of C–O of the carboxylic acid and C–OH of alcohol, respectively.", "The adsorptions at 506 cm−1 and 622.83 cm−1 are the characteristic stretching vibrations of CuO bond in the monoclinic CuO [20].", "The other adsorption peaks may be due to OH bending vibrations of some constitutional water incorporated in the CuO structure.", "From the spectrum of the composite material, the characteristic peaks of both components can be seen.", "Thus, the FTIR results confirm the anchoring of CuO nanoparticles on the surface of GO sheets.", "The MCPE was found to be stable, even after 20 cyclic voltammetric scans.", "The prepared electrode could be used for more than 60 days if preserved in a closed container.", "Relative standard deviation (RSD) was calculated for anodic current and potential of 1 mM [K4Fe(CN)6] in 1 M KCl respectively.", "The electrochemical response of the GO@CuO NCS of a MCPE was studied by the standard 1 mM [K4Fe(CN)6] in 1 M KCl as a supporting electrolyte with a scan rate 50 mVs−1.", "A comparison of the corresponding peak potential differences ΔEp of different modified electrodes is given in Table 1.", "At the BCPE, the anodic peak potential (Epa) 0.1473 V peak currents significantly increased at the MCPE with the anodic peak potential.", "Peak currents Ipc and Ipa of [K4Fe(CN)6] at MCPE increased as compared to those at the BCPE.", "Possibly a large pore volume of the NCS provided a large surface area, leading to the enhancement in the peak current, and these results confirmed that the presence of NCS in the BCPE matrix improved the sensitivity by enhancing the electron transfer process.", "Therefore, the NCS played an important role in improving the electrochemical performance of the MCPE.", "The influence of the modifier GO@CuO NCS concentration in the carbon paste matrix on the electrochemical behavior of PC and DA was also investigated (Fig. 5) in order to optimize the conditions in a 0.2 M phosphate buffer (pH 7.2) at a scan rate of 50 mV s−1.", "Note that, the 4 mg MCPE responses to the maximum current as compared with the 2, 4, 6, 8 and 10 mg of NCS and the voltammograms of DA and PC in the same buffer solution were recorded separately.", "This optimized concentration was maintained during further investigations of the biomolecules.", "The cyclic voltammograms obtained for the electrochemical responses of 5 × 10−5 M DA and 1.0 × 10−6 M PC were recorded in a 0.2 M phosphate buffer as the supporting electrolyte at pH 7.2.", "The results showed well-defined redox peaks at MCPE.", "The corresponding peak potential differences ΔEp = 0.0802 V and ΔEp = 0.0998 V for the DA and PC at the MCPE are shown in Figs. 6 and 7.", "The oxidation peak potential (Epa) of DA at BCPE and MCPE were observed at 0.1115 V and 0.1127 V, respectively.", "PC peak currents significantly increased at the MCPE with the Epa and Peak currents (Ipa) increased compared to those at BCPE (Table 2).", "These results confirmed that the presence of NCS in the CPE matrix improved the sensitivity and the large pore volume NCS provided a large specific area, leading to the enhancement in the peak current.", "The effect of a scan rate for DA and PC in a phosphate buffer solution at pH 7.2 was studied by the CV at the MCPE.", "Fig. 8 shows an increase in the redox peak current with increasing the scan rate (0.05 to 0.200 V s−1).", "This result proposes the direct electron transfer in the modified electrode surface of DA.", "Indeed, a good linearity between the scan rate (v) and the redox peak current was found (Fig. 9) for the MCPE with correlation coefficients of R2 = 0.99, which indicates that the electron transfer reaction was diffusion-controlled process.", "Similar behaviors were observed for PC (Fig. 10).", "This, however, supports the electron transfer reaction in adsorption-controlled process.", "In order to verify the reliability of the method for the analysis of DA as a pharmaceutical product, the proposed MCPE was applied to the dopamine hydrochloride injection (DHI).", "5 mL of DHI solution (40 mg/mL) was diluted to 25 mL of double distilled water and then 0.2 mL of this diluted solution was filled in a 10 mL volumetric flask.", "The DHI solution in 0.2 M phosphate buffer solution of pH 7.2 at the BCPE and the MCPE was measured at the scan rate of 50 mV s−1.", "The results confirmed that the proposed method could be effectively used for the determination of DA in commercial samples and the MCPE proposed could be efficiently used for the determination of DA in injections.", "The influences of various foreign species as interfering compounds on the determination of DA, DHI solution and on the selectivity of the NCS sensor were investigated under the optimum conditions 40 mg/mL at the 0.2 M phosphate buffer solution of pH = 7.2.", "Tolerance limit was defined as the maximum concentration of interfering foreign species that caused an approximate relative error of ±5% for the determination of neurotransmitter.", "Here, it was found that no significant interference for the detection of DA was observed from the selected compounds such as KCl 5000 μM and CaCl2 4000 μM.", "These results confirmed the highly sensitive catalytic activity for the DA analysis in the presence of other interfering substances.", "The electrochemical response as the peaks remains unchanged after successive 20 cyclic voltammetric scans, which confirms the good stability of MCPE.", "The NCS was evaluated for antimicrobial activities by means of an agar disc diffusion method whereas the minimum inhibitory concentration (MIC) was determined by a dilution method.", "The NCS demonstrated in vitro antimicrobial activity against four bacterial strains belonging to the Gram-positive (S. aureus, B. subtilis), Gram-negative (E. coli, P. aeruginosa) and two strains of fungi namely A. flavus, C. albicans.", "The results are presented in Table 3.", "The MIC is defined as the lowest concentration of nanoparticles that inhibits the growth of a microorganism.", "The NCS showed MIC at 28 and 31 μg/mL for E. coli and P. aeruginosa, respectively.", "According to MIC, E. coli and P. aeruginosa exhibited the highest sensitivity toward NCS while B. subtilis, C. albicans and A. flavus showed the least sensitivity among the tested microbes.", "The antimicrobial activity of the tested NCS was compared to the positive control drugs, streptomycin and fluconazole.", "The antibacterial properties of NCS are mainly attributed to adhesion with bacteria because of their opposite electric charges, resulting in a reduction at the bacterial cell wall.", "It was previously reported that the interaction between Gram-negative bacteria and NCS was stronger than that of Gram-positive bacteria because of the difference in cell walls, cell structure, physiology, metabolism, or degree of contact of organisms with nanoparticles.", "Gram-positive bacteria has thicker peptidoglycan cell membranes compared to the Gram-negative bacteria.", "So, it is harder for NCS to penetrate, resulting in a low antibacterial response [21].", "The GO/CuO NCS was synthesized by the modified Hummers method followed by the hydrothermal treatment.", "The abundant porous architectures of NCS exhibited a high selectivity and a good reproducibility of the voltammetric response.", "The prepared MCPE is considered to be very useful in the construction of simple devices in the field of medicine for the diagnosis of dopamine deficiency.", "The oxidation peak potential (Epa) of DA at BCPE and MCPE was observed at 0.1115 V and 0.1127 V, respectively.", "The electrochemical response remains unchanged after successive 20 cyclic voltammetric scans.", "Further, the NCS hybrid nanomaterials have shown very good biocide activity against tested microorganisms (S. aureus, B. subtilis, E. coli, P. aeruginosa, A. flavus and C. albicans).", "In addition, the NCS was found to be non-toxic for normal cells (Vero-ATCC® CCL-81™), while it is highly toxic for human cancer cells (HeLa-S3-,d.d2s″ > ATCC® CCL-2.2™) and (MDA-MB-231-,d.d2s″ > ATCC® HTB-26™).", "Therefore, this new class of the NCS hybrid nanomaterials is highly beneficial for biomedical applications." ]	[]
Cost-effectiveness and resource implications of aggressive action on tuberculosis in China, India, and South Africa: a combined analysis of nine models	Background The post-2015 End TB Strategy sets global targets of reducing tuberculosis incidence by 50% and mortality by 75% by 2025. We aimed to assess resource requirements and cost-effectiveness of strategies to achieve these targets in China, India, and South Africa. Methods We examined intervention scenarios developed in consultation with country stakeholders, which scaled up existing interventions to high but feasible coverage by 2025. Nine independent modelling groups collaborated to estimate policy outcomes, and we estimated the cost of each scenario by synthesising service use estimates, empirical cost data, and expert opinion on implementation strategies. We estimated health effects (ie, disability-adjusted life-years averted) and resource implications for 2016-35, including patient-incurred costs. To assess resource requirements and cost-effectiveness, we compared scenarios with a base case representing continued current practice. Findings Incremental tuberculosis service costs differed by scenario and country, and in some cases they more than doubled existing funding needs. In general, expansion of tuberculosis services substantially reduced patient-incurred costs and, in India and China, produced net cost savings for most interventions under a societal perspective. In all three countries, expansion of access to care produced substantial health gains. Compared with current practice and conventional cost-effectiveness thresholds, most intervention approaches seemed highly cost-effective. Interpretation Expansion of tuberculosis services seems cost-effective for high-burden countries and could generate substantial health and economic benefits for patients, although substantial new funding would be required. Further work to determine the optimal intervention mix for each country is necessary. Funding Bill & Melinda Gates Foundation.	[ "MEDI" ]	[]	[ "2700" ]	[ "The World Health Assembly's post-2015 End TB Strategy formalises goals for aggressive action against tuberculosis, including reductions in global incidence by 50% and mortality by 75% by 2025.1", "To meet these targets, major advances are needed in high-burden countries.", "The TB Modelling and Analysis Consortium conducted a multimodel evaluation to assess the goals' feasibility,2 finding that aggressive but feasible scale-up of existing approaches could achieve the reductions described by the global targets in South Africa but not in India or China.", "If targets can be met, understanding whether doing so represents the best use of funding or is even affordable is crucial.", "Conversely, if targets cannot be met, expansion of tuberculosis services is not without value.", "Although the End TB Strategy provides an important consensus to invigorate the fight against tuberculosis and attract funding, Ministries of Health also need to consider local priorities and programmatic constraints.", "In this context, an understanding of the resources required for scale-up and a comparison of the performance of competing intervention approaches are crucial.", "In this analysis, we aimed to describe the costs and health outcomes of aggressive intervention against tuberculosis, and to assess cost-effectiveness, financial implications, and patient economic burden of these interventions.", "Although previous studies3–5 have assessed the cost-effectiveness of various interventions in high-burden settings, few have compared multiple interventions simultaneously and assessed affordability.", "Quantification of the effect of these interventions on patient-incurred costs is also important, in view of the high disease burden in low-resource settings6,7 and the growing policy interest in catastrophic health-care spending,7,8 a concern addressed explicitly in the End TB Strategy.1", "Evidence before this study", "The World Health Assembly's post-2015 End TB Strategy proposes aggressive action to reduce tuberculosis incidence and mortality worldwide.", "Major reductions in high-burden countries will be essential for achieving these targets.", "Before this study, little quantitative evidence existed on the feasibility of efforts to reach these targets in high-burden settings and on the cost of implementing aggressive service expansion.", "We reviewed published work (English-language articles identified on PubMed, supplemented by the authors' familiarity of the relevant literature) on the cost-effectiveness of tuberculosis interventions relevant to China, India, and South Africa.", "Most studies addressed only one intervention area, and few investigated issues of affordability.", "Although the economic burden of tuberculosis on individuals and households is known to be high, few studies have estimated patient-incurred costs as part of cost-effectiveness analysis.", "None of the studies reviewed were designed to address the issues raised by the global End TB Strategy targets.", "Collaborative analysis with multiple models to assess intervention cost-effectiveness has previously been done for HIV policy changes but not for tuberculosis policy.", "Added value of this study", "In this study, we found that substantial improvements in the reach and quality of tuberculosis care might be cost-effective according to conventional criteria, despite requiring substantially increased funding compared with current practice.", "By estimating patient-incurred costs at the same time as health service costs, we were able to understand the relative magnitude of these effects—for some interventions, patient cost savings are larger than the additional costs borne by health services, leading to net cost savings under a societal perspective.", "By comparing multiple intervention approaches, our findings reveal the relative efficiency of each approach in the generation of health benefits.", "From these comparisons, efforts to improve access to care seemed the most beneficial and cost-effective in each setting.", "Our findings also show substantial variation in results predicted by different models, pointing to important uncertainties in the evidence base for predicting long-term costs and health outcomes.", "Implications of all the available evidence", "Combined with evidence from previous cost-effectiveness studies, the results of our analysis lend support to efforts to scale up tuberculosis services, motivated by the End TB targets.", "Our findings also reveal wide differences in the effect and efficiency of different approaches, implying that countries will need to carefully consider the approaches taken to service expansion.", "The variation in results also shows a clear need for further empirical research to strengthen the evidence base used for tuberculosis policy modelling, and thereby improve the reliability of future analyses.", "DPC and PD are employees of the Bill & Melinda Gates Foundation, which funded the study.", "These authors were acting as subject matter experts rather than agency representatives, and did not have veto power over any study decision.", "The corresponding author had full access to all data and final responsibility for the decision to submit for publication.", "The models for China and South Africa had fairly consistent results in terms of incremental costs (figure 1).", "In China, introduction of Xpert MTB/RIF seemed more expensive than expansion of access to care and improvement of treatment quality, because of high diagnostics costs and increased volume for MDR tuberculosis treatment.", "In South Africa, expansion of access to care had higher costs than other single-intervention scenarios, because of the high costs of expanding screening in primary care.", "Results for India showed clear differences between models.", "In particular, the results for expansion of access to care differed, with some models predicting cost savings over the 20 year period.", "This diversity of results points to the uncertain consequences of private sector intervention—central to several scenarios modelled for India—and different assumptions about the effect of shifting patients from low-quality care to high-quality care.", "For incremental patient-incurred costs, most intervention scenarios showed cost savings compared with the base case (figure 2).", "These cost savings resulted from reduced disease burden and from the inclusion of social protection or incentives paid for attending care, or both, in many intervention scenarios.", "By contrast, incremental patient-incurred costs were positive for the scenario of introducing Xpert in China, because of improved diagnosis of MDR tuberculosis and high costs that patients incur at present to receive second-line treatment.", "For India and South Africa, expansion of access to care generated the greatest patient cost savings among the single-intervention scenarios.", "Figure 3 presents incremental costs, health benefits (ie, DALYs averted), and cost-effectiveness ratios from both health service and societal perspectives for each intervention scenario compared with the base case (see appendix pp 13–15 for results of individual models).", "Results for South Africa included any costs or cost savings from changes in ART service volume.", "Exclusion of these costs reduced cost-effectiveness ratios by 10–20% (appendix p 16).", "In China, Xpert introduction averted fewer DALYs at higher cost than expansion of access to care and improvement in treatment, the results for which largely overlap.", "This finding might be related to the relatively minor increase in sensitivity of the Xpert algorithm over current practice and the low success rates for MDR tuberculosis treatment at present, both of which were features of the modelled scenarios.", "In India, expansion of access to care seemed the most attractive among the single-intervention strategies.", "On average, the scenario of improving treatment averted more DALYs at lower cost than Xpert introduction or active case finding, although results for this strategy vary widely.", "Active case finding in the general population generated minimal health benefits and comparatively high costs per DALY averted.", "In South Africa, tuberculosis services are predominantly publicly provided, and thus patient-incurred costs are lower relative to health service costs.", "Consequently, cost-effectiveness ratios differed little between health service and societal perspectives.", "Cost-effectiveness ratios were similar across scenarios, although the magnitude of effects was substantially greater for expansion of access to care than for other single-intervention scenarios.", "Figure 4 presents average annual tuberculosis service costs for each scenario, showing the budgetary implications of aggressive scale-up.", "In all three countries, the combination scenario required substantially increased funding.", "In China and South Africa, resource requirements peaked at about three times existing tuberculosis service costs.", "In China and India, cost increases were projected to decline over time, whereas for South Africa high spending levels were expected to persist, which is attributable to high ongoing costs of expanding access to care.", "Major sources of uncertainty relating to costs that affected our results included, for China, additional programme investments needed to support scale-up, costs of Xpert introduction, and costs of providing second-line treatment.", "For India, the cost of implementing public–private partnerships to expand access was the major source of uncertainty, followed by uncertainty around programme costs and Xpert introduction costs.", "For South Africa, the costs of implementing mobile health services to increase access was the major source of uncertainty, followed by the costs of implementing symptom screening in primary care.", "Full results of the sensitivity analysis are given in appendix pp 31–33.", "The post-2015 End TB Strategy aims to reinvigorate action on tuberculosis control and achieve substantial and rapid reductions in incidence and mortality.", "In this study, we assessed the costs and cost-effectiveness of aggressive expansion of tuberculosis services with existing technology and interventions.", "Compared with current practice, all intervention scenarios in India and South Africa—and all but the scenario of Xpert introduction in China—had a cost per DALY averted that fell below the country's gross domestic product (GDP) per person, even before patient cost savings were considered.", "GDP per person is a conventional threshold for identifying highly cost-effective interventions,23,24 yet many potential public health interventions meet this criterion.25", "Recent work has highlighted that this threshold might not adequately reflect opportunity costs in many settings.26", "However, in view of the very low costs per DALY averted for many interventions and the substantial reductions in economic burden for patients, the results suggest that some form of expansion of tuberculosis services is likely to be cost-effective for each country, and this finding is robust because it is supported by the results of every participating model.", "Involvement of experts from each country was crucial for the development of realistic policy scenarios.", "The process of developing these scenarios revealed the importance of local epidemiology and care patterns in determining the relevant interventions for a particular setting, such that important interventions for one country (eg, private sector intervention in India and isoniazid preventive therapy for HIV-positive individuals in South Africa) were thought irrelevant or to have minimal benefit for the other countries.", "Therefore, the activities suggested for each scenario differed between countries.", "Despite this heterogeneity, a consistent finding across countries was the importance of expanding access to care, thereby reducing the duration of infectiousness and mortality risks for individuals with active infection.", "The combination scenario produced the largest average reduction in DALYs in each country, followed by expansion of access to care, although with a higher cost per DALY averted than other interventions in South Africa.", "In India and South Africa, expanding access to care had a substantially greater effect than improving treatment, whereas in China this difference was only marginal, reflecting China's past success in improving case detection.", "For both India and China, Xpert introduction had a smaller health effect and higher costs than improving treatment or expanding access, despite Xpert producing major improvements in the detection of MDR tuberculosis.", "This finding is related to low treatment quality for MDR tuberculosis in these two countries at present, characterised by poor retention and cure rates, and by high treatment costs.", "If the major consequence of Xpert introduction is to expand access to MDR tuberculosis treatment (as assumed in this study), then cost-effectiveness ratios are likely to remain high while the treatment costs of MDR tuberculosis remain high.", "Despite our broadly positive cost-effectiveness findings, our results raise concerns regarding affordability.", "In all three countries, the annual tuberculosis service costs of the combination intervention were more than double the base case levels and remained elevated throughout the 20 year projection (figure 4).", "For India and South Africa, the average annual increase in spending required in the first 5 years of the combination scenario (compared with the base case) represented 3–4% of present government health sector funding, compared with only 0·15% in China.", "Only in India was the effect on tuberculosis burden sufficient to return the budget to the same level as the base case after 20 years.", "Although human resource needs and other health system constraints were not modelled explicitly, they might also present major challenges to scale-up.", "Although affordability and health system constraints remain a challenge, a key finding of this study is that, across all three countries, more aggressive tuberculosis policy would substantially reduce the economic burden of tuberculosis on patients and their families.", "These results reflect reductions in direct costs and reductions in income loss from early identification and effective treatment and prevention.", "For India and China, several interventions directly targeted the costs borne by patients, using such reimbursements and monetary incentives to the patient to improve patient adherence to effective diagnosis and treatment.", "Factoring the effects on patient-incurred costs into the cost-effectiveness ratio will substantially strengthen the investment case for expanding tuberculosis services, with both expansion of access to care and improvement in treatment producing net savings in societal costs in the evaluation period.", "Despite this study's strengths and breadth, care should be taken in drawing conclusions about the cost-effectiveness of any one intervention approach.", "Although we investigated a range of interventions, because of the complexity of using multiple models we considered only a restricted set of options.", "In reality, each intervention could be implemented at different scale and in various combinations with one another, and other interventions might also be considered.", "By comparing our set of intervention scenarios with the base case, we provide an initial scoping of the effects that might be achieved with each approach and highlight broad priority areas.", "However, ideally a full set of mutually exclusive strategies would be compared simultaneously, to identify the optimal set of services for a given budget or cost-effectiveness threshold.", "A consequence of our more limited approach is that even though the combination scenario seemed cost-effective compared with current practice, it is possible that another combination—potentially involving a subset of interventions or components of the intervention scenarios, more or less aggressive scale-up, or interventions not be considered in this analysis—might be optimal.", "To our knowledge, we are the first to use multiple independently developed models to assess the cost-effectiveness and affordability of tuberculosis intervention options.", "An important benefit of this approach was the opportunity to compare model results and in so doing understand the variation in predictions across models.", "Research in HIV has highlighted the variation in results possible when modelling complex disease and health system dynamics,25,27 and by using multiple models we were able to identify major uncertainties that would remain undetected by single-model analyses.", "We found substantial variation in the net health and economic consequences predicted for several intervention scenarios, and in the rankings of interventions implied by these results.", "Since we applied a standard cost model, this variation is primarily due to uncertainty in the processes of tuberculosis epidemiology and interventions in high-burden settings—realised as differences in model structure and parameterisation—as well as uncertainty about how specific programme actions will affect tuberculosis epidemiology and outcomes.28", "For costs, we had few empirical data for several interventions, and for the programme costs of supporting scaled-up service provision and addressing health system constraints and increasing use.", "Our results are sensitive to these uncertainties.", "Because of the complexity of using multiple models, we were unable to systematically investigate uncertainty in individual epidemiological parameters, yet these uncertainties are also likely to be consequential.", "These uncertainties are related to the nature of the scenarios we examined, which were designed to extend the reach and quality of services far above current levels.", "Although the use of multiple models provides some indication of the uncertainty, these projections will not include unanticipated (and therefore unmodelled) factors that will limit the health effect of interventions or increase the costs.", "The major coverage expansions described by the intervention scenarios are unprecedented for tuberculosis control, and the possibility of unanticipated challenges might be higher than those for more conventional policy options.", "In this study, we examined the cost-effectiveness of a set of tuberculosis interventions using multiple models, bringing together a community of country experts, modellers, and economists.", "In view of the limitations described previously, this study is a crucial first step in supporting resource allocation to and within tuberculosis control programmes.", "We found that a wide range of context-sensitive interventions are likely to be cost-effective and alleviate financial burden, but at substantial cost.", "Further work is needed to inform tuberculosis policy.", "In the future, policy decisions will ideally involve country-led planning processes—exemplified by South Africa's investment case analysis in 2016—which can more fully examine the range of candidate policies and attendant implementation challenges, validate modelling assumptions, and evaluate budget needs against options for increasing funding.", "In collaboration with national tuberculosis programme representatives for each country, we defined scenarios for scaling-up existing interventions to high yet feasible coverage by 2025.", "We projected long-term outcomes using multiple independently developed models of tuberculosis epidemiology and health services, and estimated costs by synthesising model outputs with empirical cost data and expert opinion on implementation approaches.", "We estimated health effects (disability-adjusted life-years [DALYs] averted) and resource implications for the 20 year period (2016–35), and calculated costs from multiple perspectives.", "For each country we compared intervention scenarios with a base case in which present intervention coverage is maintained.", "We undertook this study for China, India, and South Africa because of their substantial tuberculosis burden and their contrasting HIV and tuberculosis epidemiology and organisation of tuberculosis services.", "In China, tuberculosis incidence is 68 cases per 100 000 population and mortality is three per 100 000 population8 (equivalent to 3% of global tuberculosis mortality), and the country has achieved progressive reductions in tuberculosis burden in the past three decades.9", "In India, tuberculosis incidence is 167 per 100 000 population and mortality is 19 per 100 000 population (accounting for 17% of global tuberculosis mortality), and a large private sector provides roughly half of all tuberculosis care.10", "In South Africa, tuberculosis incidence is 834 per 100 000 population and mortality is 178 per 100 000 population (equivalent to 6% of global tuberculosis mortality), with both incidence and case fatality driven by the HIV epidemic.", "With input from national tuberculosis programme representatives for each country, we defined scenarios describing scale-up of specific interventions to high coverage by 2025 (table 1), making use of currently available intervention options and considering local policy preferences and capacity.", "Although intervention areas were defined in advance, country experts determined whether additional scale-up of an intervention was appropriate for their country, and the anticipated scale and pace of coverage improvements.", "Scenario descriptions included the activities required to produce coverage changes: for example, in South Africa, improving access to tuberculosis diagnosis could be achieved through outreach to underserved areas and symptom screening for individuals attending primary care.", "Specification of activities helped to define the feasible extent of scale-up, and allowed the costs of each scenario to be estimated (see appendix p 3 for a description of the disease course, treatment, and final outcomes).", "We assessed interventions separately and also considered a scenario representing the combination of all interventions for each country.", "A base case scenario represented continuation of current practice, with service coverage held at existing levels.", "Services not specifically addressed in a scenario were held at current coverage levels, with two exceptions: for South Africa, we assumed that antiretroviral therapy (ART) coverage would increase to 77% by 2025; and for India, we assumed all retreatment tuberculosis cases would receive drug susceptibility testing by 2019.", "Both policies have high-level commitment in their respective countries.", "We projected policy outcomes using multiple independently developed Mycobacterium tuberculosis transmission models.3,11–19", "Models had to represent the major mechanisms determining tuberculosis outcomes in each setting, be consistent with existing evidence on epidemiology and service provision, and simulate outcomes needed to estimate summary health and economic effects.", "Of 11 models in the modelling exercise, nine fulfilled these requirements and contributed inputs for the economic analysis (table 2; appendix pp 4–7), and additional details are provided by Houben and colleagues.2", "Models produced standardised outputs describing service use under each scenario, and outputs to estimate the economic burden of tuberculosis on patients and households.", "We also did a systematic review20 to collate and synthesise unit costs for each country.", "Empirical tuberculosis cost data are limited;20 where local values were unavailable we adapted estimates from other settings using local prices, holding country consultations to ensure face validity.", "We developed country-specific cost models to combine service use estimates with unit costs.", "We divided cost categories into diagnosis (ie, active or passive screening, tuberculosis diagnosis, and drug resistance testing), first-line treatment for active disease, multidrug-resistant (MDR) tuberculosis treatment, treatment of latent infection for HIV-positive individuals (ie, isoniazid preventive therapy; South Africa only), and programme overheads (ie, high-level overheads supporting service delivery).", "We estimated direct intervention costs and changes in the costs of core services (eg, passive detection and treatment) indirectly affected by policy change.", "To estimate programme overheads, we examined past programme expenditures and consulted with WHO and country experts.", "Patient-incurred costs were calculated as the sum of direct medical costs (ie, fees paid to providers to receive care [eg, consultation fees] and to purchase drugs), direct non-medical costs (ie, expenses incurred to receive care, such as travel costs, excluding fees paid to providers), and indirect productivity costs (ie, income loss due to tuberculosis symptoms or treatment, and opportunity cost of productive activity forgone due to untreated active disease or time taken to receive treatment).", "Several intervention strategies used reimbursements or incentives paid for attending care to offset patient-incurred costs.", "Additional details on the costing approach and input values are shown in the appendix pp 17–31.", "We report economic costs from several perspectives: a tuberculosis service perspective, representing costs borne by national tuberculosis programmes and associated service providers; a health service perspective, summing tuberculosis service costs (for South Africa, costs or cost savings associated with ART were also taken into account); and a patient perspective, including costs or cost savings realised by individuals with active tuberculosis or receiving tuberculosis care; as well as a societal perspective combining patient and health service costs.", "We did sensitivity analyses for unit costs and programme overheads.", "We measured health benefits using DALYs.", "Models produced standardised outputs for calculating DALYs averted compared with the base case.", "Outputs included deaths by age and year, and the yearly population distribution across tuberculosis-related and HIV-related health states (appendix pp 8–10).", "Disability weights (appendix p 11) were derived from a multicountry valuation study,21 and remaining life expectancy (appendix p 12) from country-specific life tables22 (values not truncated at the analytic horizon).", "In all scenarios, the assumptions were that interventions would reach peak coverage before 2025 and that they would be extended to 2035 maintaining 2025 coverage levels.", "This 20 year evaluation period balanced conflicting concerns: that longer-term projections would be increasingly unreliable, but that a short evaluation period would exclude important policy consequences, as health benefits are lagged relative to implementation costs.", "We summed costs and DALYs over the 20 year period (2016–35).", "For cost-effectiveness analyses, we discounted these outcomes at 3% per year.", "Costs represent 2014 US$ (equal to 6·1 Chinese Yuan, 60·9 Indian Rupees, and 10·8 South African Rand).", "Model results are presented individually and averaged across models.", "Cost-effectiveness ratios were calculated as mean incremental cost divided by mean incremental health benefits for each scenario, as compared with base case.", "To describe affordability, we compared annual undiscounted costs for each scenario, averaged across models with equal weights.", "As we estimated economic costs, annual results are a smoothed version of actual financial needs." ]	[]
Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells	Regulatory B cells (Bregs) play a critical role in the control of autoimmunity and inflammation. IL-10 production is the hallmark for the identification of Bregs. However, the molecular determinants that regulate the transcription of IL-10 and control the Breg developmental program remain unknown. Here, we demonstrate that aryl hydrocarbon receptor (AhR) regulates the differentiation and function of IL-10-producing CD19+CD21hiCD24hiBregs and limits their differentiation into B cells that contribute to inflammation. Chromatin profiling and transcriptome analyses show that loss of AhR in B cells reduces expression of IL-10 by skewing the differentiation of CD19+CD21hiCD24hiB cells into a pro-inflammatory program, under Breg-inducing conditions. B cell AhR-deficient mice develop exacerbated arthritis, show significant reductions in IL-10-producing Bregs and regulatory T cells, and show an increase in T helper (Th) 1 and Th17 cells compared with B cell AhR-sufficient mice. Thus, we identify AhR as a relevant contributor to the transcriptional regulation of Breg differentiation.	[ "BIOC" ]	[]	[ "1300" ]	[ "B cells with immunosuppressive capacity, known as regulatory B cells (Bregs), play an important role in restraining inflammation.", "In mice, regulatory function has been ascribed to IL-10-producing transitional type 2 marginal zone precursors (T2-MZPs) (Evans et al., 2007), marginal zone (MZ) (Gray et al., 2007) and CD1dhiCD5+B cells (Yanaba et al., 2008), plasmablast (Matsumoto et al., 2014), and plasma cell (Lino et al., 2018) populations.", "Bregs suppress inflammatory cytokine production by T cells and promote the differentiation of Foxp3+ regulatory T cells (Tregs), primarily via the secretion of interleukin-10 (IL-10) (Carter et al., 2011; Rosser et al., 2014).", "Mice lacking IL-10-expressing B cells develop exacerbated autoimmune arthritis and experimental autoimmune encephalitis (EAE) (Carter et al., 2011; Fillatreau et al., 2002), and adoptive transfer of IL-10-deficient B cells to arthritic mice fails to suppress inflammation (Carter et al., 2011).", "B cell receptor (BCR) engagement, Toll-like receptor (TLR) agonists lipopolysaccharide (LPS; TLR2 and TLR4) (Lampropoulou et al., 2008; Tian et al., 2001), CpG oligo-deoxynucleotides (TLR9), and inflammatory cytokines such as IL-1β and IL-6 or IFN-α are all potent inducers of B cell-derived IL-10, suggesting an important role for Bregs in the restoration of tolerance after infection or inflammation (Menon et al., 2016; Rosser et al., 2014).", "Cell-derived signals arising from B and T lymphocyte cross-talk, including T cell-derived CD40L and IL-21, further support the expansion of Bregs (Mauri et al., 2000; Yoshizaki et al., 2012).", "Bregs, in turn, suppress inflammatory cytokine production by T cells and promote the differentiation of Foxp3+ regulatory T cells (Treg) (Carter et al., 2011; Oleinika et al., 2018; Rosser et al., 2014).", "Unlike in murine T cells, in which it is well established that IL-10 expression is controlled by several transcription factors, including c-Maf and the aryl hydrocarbon receptor (AhR) (Apetoh et al., 2010), there is limited knowledge of the transcriptional control of IL-10 production by Bregs.", "To date, studies of the molecular control of B cell IL-10 production have been limited to the examination of NFAT downstream of STIM1/STIM2 calcium sensors (Matsumoto et al., 2011) and Myd88 (Liu et al., 2019) in mice and ERK (Li et al., 2012) and STAT3 (Blair et al., 2010) in humans.", "More recently, Blimp1 and IRF4 (Matsumoto et al., 2014) have been linked to IL-10+ plasmablasts, but at least for Blimp1 and IRF4, neither transcription factor appeared to directly control the production of IL-10 by splenic B cells (Matsumoto et al., 2014).", "To explore the molecular mechanisms regulating the differentiation of B cells into IL-10-producing Bregs, we used an IL-10-eGFP reporter mouse (Madan et al., 2009).", "We compared the gene expression profiles of IL-10eGFP+CD19+CD21hiCD24hiBregs, IL-10eGFP−CD19+CD21hiCD24hiB cells, and IL-10eGFP−CD19+CD21intCD24int follicular (FO) B cells isolated from arthritic mice (FO B cells do not produce IL-10 and do not suppress arthritis after adoptive transfer; Evans et al., 2007).", "We chose to study IL-10+CD19+CD21hiCD24hiBregs because this population contains both T2-MZP and MZ B cells, which together have been shown to contain the vast majority of splenic IL-10-producing Bregs (Evans et al., 2007; Gray et al., 2007).", "IL-10eGFP+CD19+CD21hiCD24hiBregs have a unique transcriptome, characterized by a highly restricted cytokine/chemokine profile that distinguishes them from the IL-10eGFP−B cell subsets.", "AhR was among the most significant differentially expressed transcription factors in IL-10eGFP+CD19+CD21hiCD24hiBregs, compared with both IL-10eGFP−CD19+CD21hiCD24hiB cells and IL-10eGFP−FO B cells.", "We show that AhR binds upstream to the transcription start site (TSS) of the Il10 locus in IL-10eGFP+B cells but not in IL-10eGFP−B cells.", "LPS and anti-IgM, stimuli previously shown and confirmed here to induce the expression of AhR (Vaidyanathan et al., 2017; Villa et al., 2017), promote the differentiation of CD19+CD21hiCD24hiB cells (a population poised to become Bregs; Evans et al., 2007; Matsumoto et al., 2011) into IL-10+CD19+CD21hiCD24hiBregs.", "Taking advantage of high-throughput sequencing, we demonstrated that activation of AhR under this Breg-polarizing condition results in the suppression of several pro-inflammatory cytokine and chemokine genes and in a highly restricted phenotype in CD19+CD21hiCD24hiB cells congruous with an immunosuppressive population.", "In vivo, B cell-specific deletion of AhR caused exacerbated arthritis, reduced IL-10 production by CD19+CD21hiCD24hiBregs, and reduced the frequency of Foxp3+Tregs and expansion of Th1 and Th17 cells.", "These results support a role of AhR in the differentiation of IL-10-producing Bregs and in the control of their immunosuppressive phenotype.", "To identify candidate genes that regulate the transcription of IL-10 in Bregs, arthritis was induced in IL-10eGFP reporter mice (Vert-x) (Madan et al., 2009).", "We sorted splenic IL-10eGFP+CD19+CD21hiCD24hiBregs, IL-10eGFP−CD19+CD21hiCD24hiB cells, and IL-10eGFP−FO B cells (the two GFP− populations are hereafter referred to as IL-10eGFP−B cell subsets) and profiled these cells using gene expression microarray (Figures 1A and 1B).", "Of note, very few IL-10-producing Bregs were present in the joint or draining lymph nodes (DLNs) of arthritic mice (Figure S1A).", "This sorting strategy was chosen to capture the majority of described Breg subsets, including IL-10+T2-MZP, IL-10+MZ, and IL-10+CD1dhiCD5+, which have been shown to exert suppressive capacity via IL-10 in this model of arthritis and in other models of autoimmunity (Brummel and Lenert, 2005; Evans et al., 2007; Tian et al., 2001; Yanaba et al., 2009).", "Virtually no IL-10-expressing plasma cells or plasmablasts (LAG-3+ plasma cells [Lino et al., 2018] and CD138+CD44+ plasmablasts [Matsumoto et al., 2014]) were detected in the spleen or DLNs following the induction of arthritis (Figures S1B–S1H).", "The purity of sorted IL-10eGFP+ and IL-10eGFP− subsets isolated for the microarray was more than 98% (Figure S1I).", "Principal-component analysis (PCA) revealed three distinct groups along the first dimension, with the IL-10eGFP+CD19+CD21hiCD24hiBreg population clustered separately away from both IL-10eGFP−B cell subsets (Figure 1C).", "Analysis of gene expression revealed 1,073 differentially expressed genes (DEGs) between IL-10eGFP+CD19+CD21hiCD24hiBregs and IL-10eGFP−CD19+CD21hiCD24hiB cells and 1,267 genes that were differentially expressed between IL-10eGFP+CD19+CD21hiCD24hiBregs and IL-10eGFP−FO B cells (fold change > 1.5 and adjusted p value < 0.05) (Figure S1J; Figure 1D).", "In the context of arthritis, splenic Bregs have been shown to produce mainly IL-10 (Rosser and Mauri, 2015).", "Of the cytokine genes upregulated in IL-10eGFP+CD19+CD21hiCD24hiBregs, only Il10 and Ebi3 reached an adjusted p value of < 0.05, compared with both IL-10eGFP− B cell subsets.", "However, because Il12a was not found to be upregulated in IL-10eGFP+CD19+CD21hiCD24hiBregs, we excluded a role of IL-35 in these cells.", "Although a trend in the increase of transcripts for pro-inflammatory genes such as Il1a, Il12b, Il15, and Il18 was noted, the expression of these genes was not significantly different from the IL-10eGFP−B cell subsets (Figure 1E).", "IL-10eGFP−FO B cells, which unlike CD19+CD21hiCD24hiB cells do not suppress inflammation on adoptive transfer (Evans et al., 2007), express a different transcriptional profile, characterized by higher expression of cytokines and cytokine receptors known to mediate inflammatory responses, compared with IL-10eGFP+CD19+CD21hiCD24hiBregs (Figures 1E and 1F).", "The transcripts that were significantly differentially expressed in IL-10eGFP+CD19+CD21hiCD24hiBregs versus IL-10eGFP−B cell subsets are summarized in (Figure 1G).", "In keeping with the anti-inflammatory role of Bregs, we found that the Th2-attracting chemokines Ccl17 and Ccl22 (Imai et al., 1999; Nakayama et al., 2004), and the chemokine receptor Cxcr3, important for the trafficking of lymphocytes to the synovium in arthritis (Mohan and Issekutz, 2007), were upregulated in IL-10eGFP+CD19+CD21hiCD24hiBregs compared with IL-10eGFP−B cell subsets (Figures 1E–1G).", "These results showed that in the context of arthritis, splenic Bregs displayed a distinct anti-inflammatory transcriptional profile compared with IL-10eGFP−B cell subsets.", "To screen for molecules involved in IL-10 transcription and Breg function, genes concordant for comparison 1 (between IL-10eGFP+CD19+CD21hiCD24hiBregs and IL-10eGFP−FO B cells) and comparison 2 (between IL-10eGFP+CD19+CD21hiCD24hiBregs and IL-10eGFP−CD19+CD21hiCD24hiB cells) (Figure S1K) were filtered on the basis of the transcription factor Gene Ontology term, resulting in 23 candidates (Figure 2A).", "Pathway analyses showed that AhR represented a central network hub (Figure 2B) and was the most significantly enriched candidate in IL-10eGFP+CD19+CD21hiCD24hiBregs (adjusted p value < 3.34 × 10−5; Table S1; Figure S1L).", "Analysis of transcription factors previously shown to be associated with IL-10 transcriptional regulation in other lymphocyte subsets, including Tregs (Iyer and Cheng, 2012), confirmed that AhR was the most significantly upregulated IL-10-related transcription factors in IL-10eGFP+CD19+CD21hiCD24hiBregs, in comparison with IL-10eGFP−B cell subsets (Figure 2C).", "Microarray signal intensities for Il10 and Ahr were higher in IL-10eGFP+CD19+CD21hiCD24hiBregs compared with IL-10eGFP−B cell subsets (Figure 2D).", "qPCR analysis confirmed that Il10 and Ahr mRNA expression was higher in IL-10eGFP+CD19+CD21hiCD24hiBregs than in both IL-10eGFP−B cell subsets (Figures 2E and 2F).", "Corroborating the results in Figures 2E and 2F, assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed increased accessibility in both the Il10 and Ahr loci in IL-10eGFP+CD19+CD21hiCD24hiBregs, in comparison with both IL-10eGFP−B cell subsets (Figure 2G).", "We and others have recently shown that BCR signals combined with TLR stimulation induce a substantial upregulation of AhR in B cells (Vaidyanathan et al., 2017; Villa et al., 2017).", "Because BCR signals together with TLR engagement are known to be pivotal in the generation of Bregs (Rosser and Mauri, 2015), next we investigated whether the differentiation of Bregs “marked” by the induction of IL-10 expression by these stimuli was AhR dependent.", "For this purpose, CD19+CD21hiCD24hiB cells were sorted from the spleens of arthritic mice and stimulated with LPS ± anti-IgM.", "An 11-fold increase in the frequency of IL-10+CD19+CD21hiCD24hiBregs and a 200-fold increase in the production of IL-10 by CD19+CD21hiCD24hiBregs were observed upon LPS+anti-IgM stimulation compared with unstimulated cells (Figures 3A–3C).", "Increased levels of AhR expression in IL-10+CD19+CD21hiCD24hi Bregs compared with IL-10−FO and IL-10−CD19+CD21hiCD24hiB cells were confirmed using flow cytometry (Figures S2A and S2B).", "We established the kinetics of AhR and AhR pathway-associated gene expression in relation to Il10 transcription, after activation with Breg-polarizing stimuli.", "We observed a peak in the expression of Ahr and Cyp1a1 (the gene encoding the AhR-dependent cytochrome P4501A1) at 6 h post-stimulation with LPS+anti-IgM, followed by an upregulation of AhR repressor (Ahrr) and Il10 at 24 h. No significant changes in the expression of the AhR binding partner AhR nuclear translocator (Arnt) were observed (Figures 3D–3H).", "Of note, ex vivo CD19+CD21hiCD24hiB cells display higher expression of Ahr (confirmed at the protein level using flow cytometry and western blotting), Il10, Cyp1a1, Ahrr, and Arnt compared with FO B cells (Figures S2C–S2J).", "Together these data show that IL-10+CD19+CD21hiCD24hiBregs express the highest levels of AhR compared with the IL-10−CD19+CD21hiCD24hi and IL-10−FO B cells and that in CD19+CD21hiCD24hiB cells, AhR upregulation precedes the production of IL-10.", "To understand the role of AhR in the regulation and function of IL-10 expression in CD19+CD21hiCD24hiBregs, we isolated CD19+CD21hiCD24hiB cells and FO B cells from Ahr+/− and Ahr−/− mice and stimulated them in vitro with LPS+anti-IgM.", "Lack of AhR significantly reduced the ability of CD19+CD21hiCD24hiB cells to differentiate into IL-10-producing CD19+CD21hiCD24hiBregs compared with AhR competent CD19+CD21hiCD24hiB cells (Figure 3I).", "In addition, Ahr−/− CD19+CD21hiCD24hiB cells presented a reduced capacity to inhibit IFN-γ production by CD4+T cells in vitro compared with Ahr+/− CD19+CD21hiCD24hiB cells (Figures 3J and 3K).", "FO B cells failed to produce IL-10 and to suppress IFN-γ by CD4+ T cells, irrespective of AhR expression (Figures 3I–3K).", "To assess if activation of AhR directly with endogenous ligands promotes the differentiation of CD19+CD21hiCD24hiB cells into IL-10+CD19+CD21hiCD24hiBregs, we stimulated sorted CD19+CD21hiCD24hiB cells or FO B cells with the AhR ligand 6-formylindolo(3,2-b) carbazole (FICZ).", "AhR activation significantly upregulated the expression of Cyp1a1 and Il10 in the CD19+CD21hiCD24hiB cell subset but not in FO B cells compared with the vehicle control (Figure 3L).", "Secretion of IL-10 was further enhanced by the addition of FICZ to LPS+anti-IgM-stimulated CD19+CD21hiCD24hiB cells, compared with LPS+anti-IgM alone (Figure S2K).", "An increase in Breg IL-10 expression was observed when CD19+CD21hiCD24hiB cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; enriched in aromatic amino acids that give rise to AhR ligands; Veldhoen et al., 2009) (Heath-Pagliuso et al., 1998) compared with RPMI media (Figure S2L).", "We next investigated whether AhR regulates IL-10 expression in IL-10eGFP+CD19+CD21hiCD24hiBregs by directly binding the Il10 locus.", "To address this, we took advantage of the JASPAR tool (Khan et al., 2018) and identified putative AhR binding sites in 500 bp regions up to −5 kb upstream and +5 kb downstream of the Il10 TSS and designed primer probes to span these regions (Figure 3M).", "We sorted IL-10eGFP+ and IL-10eGFP−B cells, after stimulation with LPS+anti-IgM+FICZ (the combination of stimuli was used to maximize AhR activation and translocation to the nucleus) and performed chromatin immunoprecipitation (ChIP) qPCR on AhR-bound DNA (Figure 3N).", "Significantly enriched AhR binding was observed at −3.5 kb upstream of the Il10 TSS in the IL-10eGFP+ population.", "Minimal binding of AhR was observed in other regions of the Il10 locus.", "As a positive control, we confirmed that under these experimental conditions, there was enriched binding of AhR to the promoter of Cyp1a1 but no binding to Gapdh, an AhR-independent housekeeping gene (Figure 3O).", "These results show that AhR controls IL-10 transcription and the function of Bregs.", "To examine the role of AhR in controlling the differentiation of CD19+CD21hiCD24hiB cells into Bregs, and to ascertain the relative contribution of AhR in establishing the restricted Breg phenotype identified in the microarray (Figures 1E–1G) in addition to IL-10 production, we took advantage of mice with a B cell-specific deficiency of AhR (Ahrfl/−Mb1cre/+) (Villa et al., 2017)) (Figures S3A and S3B).", "The use of these mice avoids any cell extrinsic effects of AhR that could indirectly influence Breg differentiation (Stockinger et al., 2014).", "CD19+CD21hiCD24hiB cells were sorted from immunized Mb1cre/+ and Ahrfl/−Mb1cre/+ mice, and the transcriptional profile of sorted CD19+CD21hiCD24hiB cells was compared before and after stimulation under Breg-polarizing conditions (LPS+anti-IgM).", "Both the normalized counts for Ahr and the accessibility of the Ahr locus measured by ATAC-seq increased in LPS+anti-IgM-stimulated Mb1cre/+ CD19+CD21hiCD24hiB cells compared with ex vivo Mb1cre/+ CD19+CD21hiCD24hiB cells (Figures S4A and S4B).", "Signaling pathway impact analysis (SPIA) of DEGs revealed overrepresented pathways relating to cytokine-cytokine receptor interactions and chemokine signaling in stimulated Mb1cre/+ CD19+CD21hiCD24hiB cells versus ex vivo Mb1cre/+ CD19+CD21hiCD24hiB cells (Figure 4A).", "Analysis of the genes differentially expressed within this pathway confirmed that under Breg-polarizing conditions, several genes identified in the IL-10eGFP+ signature (as shown in Figure 1G), including Il10, Ccl22, and Il2ra, were upregulated, while those associated with the IL-10eGFP− signature, including Il12a, Il10ra, and Ltb, were downregulated under Breg-polarizing conditions (Figure 4B; Figure S4C).", "A signature of genes related to cytokine-cytokine receptor interaction was upregulated in Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hi B cells under LPS+anti-IgM stimulation compared with Mb1cre/+ (Figure 4C).", "Forty-four of 102 genes in this pathway were significantly differentially expressed between Ahrfl/−Mb1cre/+ and Mb1cre/+ CD19+CD21hiCD24hiB cells (Figures 4C and 4D).", "Of the genes that were differentially expressed under Breg-polarizing conditions (Figure 4B), pro-inflammatory cytokines including Il6, Tnf, Il2, and chemokines such as Ccl3, Ccl5, and Cxcl16, known to recruit lymphocytes to the inflamed synovia in models of arthritis (Kasama et al., 1995; Ruth et al., 2006; Szekanecz et al., 2000; Thornton et al., 1999), were upregulated in the absence of AhR (Figure 4D).", "The absence of AhR expression led to downregulation of the Il5ra gene, which had previously been associated with Breg function (Klinker et al., 2013), Ccl22, and Il18, which we have identified as Breg-associated genes in the microarray.", "Testing key arthritogenic pro-inflammatory transcripts (Feige et al., 2000; Ohshima et al., 1998; Thornton et al., 2000) using qPCR confirmed that Il2, Il6, and Tnf were increased in Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells, and Ccl22 and Il5ra were decreased compared with Mb1cre/+ CD19+CD21hiCD24hiB cells.", "The increase in pro-inflammatory cytokines in Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells was confirmed using ELISA (Figures 4E and 4F).", "Specific interrogation of differentially accessible regions (DARs) in loci encoding genes from the cytokine-cytokine receptor interaction pathway revealed an overall decrease in chromatin accessibility in these genes in Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells.", "Seventy-eight DARs (p < 0.05) were identified among the genes regulated by AhR at the transcriptional level, including Il2, Il6, Ccl3, Ccl5, Il5ra, Ccl22, and Il10 between LPS+anti-IgM polarized Ahrfl/−Mb1cre/+ and Mb1cre/+ CD19+CD21hiCD24hiB cells (Figure 4G).", "To confirm that AhR suppresses pro-inflammatory gene expression during the development of Bregs, we blocked AhR signaling in vitro with CH-223191.", "Concordant with the results identified with RNA sequencing (RNA-seq), blocking AhR signaling resulted in upregulation of Il6 and Tnf and downregulation of Il10 and Ccl22 mRNA (Figure S4D).", "AhR can repress gene expression indirectly by controlling the activity of additional molecules involved in gene regulation, including genes related to the NF-κB pathway.", "Because we have previously shown that the NF-κB pathway is linked to IL-10 production in Bregs (Rosser et al., 2014), next we analyzed the effect that the lack of AhR in B cells had on the NF-κB pathway in LPS+anti-IgM-stimulated cells.", "A reduction in NF-κB pathway-related genes, including Cd40 and Myd88, required for IL-10 production, was observed in Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells.", "This was mirrored by upregulation of genes relating to inflammation (Tnf, Tnfaip3, Ccl4, and Il1r1) in Ahrfl/−Mb1cre/+ compared with Mb1cre/+ CD19+CD21hiCD24hiB cells (Figure S4E).", "Our data suggest that AhR contributes the Breg transcriptional program by suppressing pro-inflammatory gene expression.", "To rule out whether this effect is secondary to the decrease in IL-10, we cultured wild-type (WT) and Il10r−/− CD19+CD21hiCD24hiB cells with LPS+anti-IgM in the presence or absence of the selective AhR antagonist CH-223191.", "The expression of Il6 and Tnf was significantly increased in both WT and IL-10R−/− CD19+CD21hiCD24hiB cells cultured with the AhR antagonist, suggesting a direct effect of AhR in the suppression of pro-inflammatory gene expression (Figure S4F).", "Collectively, these data show that under Breg-polarizing conditions, AhR acts as a molecular switch that “turns off” a number of pro-inflammatory cytokines and chemokines in CD19+CD21hiCD24hiB cells, while promoting the expression of IL-10+CD19+CD21hiCD24hiBreg-associated cytokines and receptors.", "Having confirmed the contribution of AhR in the programming of the IL-10+CD19+CD21hiCD24hiBreg transcriptional profile, we explored the impact of AhR deficiency specifically in B cells on the immune response associated with arthritis.", "Ahrfl/−Mb1cre/+ mice developed exacerbated arthritis compared with control Mb1cre/+ mice (Figure 5A).", "Histological analysis of joint tissue showed an increase in immune cell infiltration in the synovia and hyper-vascularization in Ahrfl/−Mb1cre/+ compared with control Mb1cre/+ mice (Figure 5B).", "The enhanced inflammation was associated with a significant increase in the frequency and number of IFN-γ- and IL-17-expressing CD4+ T cells in the spleen and DLN (Figures 5C–5F).", "Ahrfl/−Mb1cre/+ mice had an increased frequency and number of IL-17+CD4+ cells and a reduction in the frequency and total number of Foxp3+Tregs in the inguinal DLN compared with the control mice (Figures 5F–5H).", "Increased levels of IL-17 were observed in the synovium of inflamed joints of Ahrfl/−Mb1cre/+ compared with the control group, whereas IFN-γ levels were undetectable (Figure 5I).", "Adoptive transfer of Mb1cre/+ or Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells into syngeneic mice showed that only CD19+CD21hiCD24hiB cells from Mb1cre/+, but not from Ahrfl/−Mb1cre/+ mice, significantly inhibited disease and Th17/Th1 differentiation in the recipient mice (Figures 5J–5L), confirming that CD19+CD21hiCD24hiB cells were less effective at suppressing inflammation in the absence of AhR.", "To establish that the increase in disease severity and the impact on the T cell compartment was due to the lack of AhR-expressing Bregs, rather than a consequence of abnormal B cell development, we next compared the frequencies of pro, pro-pre, pre, immature, transitional (T), early, and late mature B cells in the bone marrow or T1 or FO B cells in the spleens of Ahrfl/−Mb1cre/+ and Mb1cre/+ mice.", "We observed no differences in these populations, suggesting that the increase in arthritis severity and in pro-inflammatory T cells was indeed due to a reduction of CD19+CD21hiCD24hiBregs rather than a consequence of abnormal B cell development (Figures 6A and 6B; Figures S5A–S5H).", "In line with previous findings showing that AhR represses differentiation of B cells into plasma cells (Tucker et al., 1986), we found increased frequencies of splenic plasma cells (Figures S5I and S5J) and increased Prdm1 mRNA expression in Ahrfl/−Mb1cre/+ B cells relative to Mb1cre/+ B cells (Figure S5K).", "Despite changes in the frequency of plasma cells in the spleens, there were no differences in the amounts of secreted IgG, IgM, and IgA in the serum of arthritic Ahrfl/−Mb1cre/+ versus Mb1cre/+ mice (Figure S5L).", "Although there were no differences in the number of splenic CD19+CD21hiCD24hiB cells between the two groups, Ahrfl/−Mb1cre/+CD21hiCD24hiB cells secreted significantly less IL-10 than Mb1cre/+CD19+CD21hiCD24hiB cells, following stimulation with LPS+anti-IgM (Figure 6C).", "IL-10 production by Ahrfl/−Mb1cre/+ CD19+B cells was significantly reduced in response to TLR9 stimulation (Figures S6A–S6C).", "Comparable levels of Ebi3 and Il12a were present in B cells from Ahrfl/−Mb1cre/+ mice and Mb1cre/+ mice (Figures S6D and S6E).", "These results, together with our previous findings showing a redundant effect of IL-35 in Bregs in this model (Vasconcellos et al., 2011), excluded that Bregs were non-functional because of a lack of IL-35 production in the absence of AhR.", "The observed IL-10 reduction was not due to impaired B cell proliferation, as an equivalent expression of Ki-67 in Mb1cre/+ and Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells was observed both directly ex vivo after antigen-induced arthritis (AIA) and after stimulation with LPS+anti-IgM (Figures S6F–S6I).", "No difference in genes related to the cell cycle was observed, including Ccno, previously been shown to be regulated by AhR in splenic B cells (Villa et al., 2017), between Mb1cre/+ and Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells (Figure S6J), suggesting that there was no impairment in BCR-driven regulation of B cell entry into the cell cycle (Richards et al., 2008).", "Mesenteric lymph nodes (MLNs) are important sites for the licensing of Breg development, through the exposure to microbiota-derived pro-inflammatory signals IL-1β and IL-6 (Rosser et al., 2014).", "Fewer CD19+CD21hiCD24hiB cells, and a decreased amount of B cell-derived IL-10 after in vitro polarization with LPS+anti-IgM, were observed in the MLN of Ahrfl/−Mb1cre/+ mice compared with control Mb1cre/+ mice (Figures 6D–6F).", "Of interest, no difference in gut-homing integrin α4β7 expression was observed in splenic or MLN-derived CD19+CD21hiCD24hiB cells between Ahrfl/−Mb1cre/+ and Mb1cre/+ mice, suggesting that the reduction of Bregs observed in the MLN of Ahrfl/−Mb1cre/+ mice is α4β7 independent (Figures S6K–S6M).", "We can exclude that the decreased frequency of Bregs was the consequence of a reduction in monocyte-derived lL-1β and IL-6 produced in the spleens or in the MLNs, as equivalent amounts of these cytokines were produced by these cells in both Ahrfl/−Mb1cre/+ and Mb1cre/+ mice (Figures S7A–S7D).", "Thus, our results collectively show that mice lacking AhR expression in B cells phenocopy the results we have previously observed in mice with IL-10−/−B cells (Carter et al., 2011) and influence IL-10+CD19+CD21hiCD24hiBreg differentiation in a cell-intrinsic manner.", "Bregs are generated in the periphery in response to a variety of “homeostatic” inflammatory stimuli including activation through TLRs and by pro-inflammatory cytokines (Rosser and Mauri, 2015).", "The transcriptional program that governs the differentiation and function of IL-10+CD19+CD21hiCD24hiBregs remains virtually unknown.", "Here, we show that AhR contributes to the differentiation of CD19+CD21hiCD24hiB cells into functionally suppressive IL-10+CD19+CD21hiCD24hiBregs by regulating their IL-10 production and by repressing the transcription of pro-inflammatory mediators.", "The importance of IL-10 in mediating the suppressive effect of Bregs is well established, and its role is corroborated by in vivo results showing that mice lacking IL-10-producing B cells develop exacerbated autoimmunity (Carter et al., 2011).", "Similarly, AhR deficiency restricted to B cells impairs IL-10+CD19+CD21hiCD24hiBreg differentiation and function, resulting in an increase of IFNγ and IL-17-expressing CD4+ T cells, a decrease in Tregs, and the development of an exacerbated arthritis.", "AhR plays a pleiotropic role in the regulation of several immune responses (Stockinger et al., 2014), most notably in the differentiation of CD4+ T cells, in which AhR influences both the differentiation and activation of Th17 cells, which are known to play a major role in the pathogenesis of several autoimmune diseases (Stockinger and Omenetti, 2017) and the differentiation of CD4+ T cells into Tr1 cells (Apetoh et al., 2010).", "Immune suppression was one of the earliest known observations of AhR function.", "2,3,7,8-Tetrachlorodibenzo-ρ-dioxin (TCDD), an environmental contaminant and a potent AhR agonist, was found to suppress delayed hypersensitivity (DTH) responses to tuberculin (Vos et al., 1973).", "More recently, it has become apparent that AhR has a conserved role in the regulation of IL-10 across the innate and adaptive immune system, controlling IL-10 production in natural killer cells (Wagage et al., 2014), peritoneal (Kimura et al., 2009) and bone marrow-derived macrophages (Shinde et al., 2018), and Tr1 cells, in which AhR binding to the Il10 promoter region has been described (Apetoh et al., 2010).", "We demonstrate that in IL-10+B cells, AhR binds upstream of the Il10 TSS to a different genomic region than in Tr1 cells, suggesting that there are cell context- and cell signal-specific epigenetic differences in the regulation of Il10 (Saraiva et al., 2005).", "An interesting finding in our study was the discovery that AhR partly controls the differentiation of CD19+CD21hiCD24hiB cells into a polarized IL-10+CD19+CD21hiCD24hiBreg population that produces only IL-10 by contributing to IL-10 induction and by suppressing the transcription of several pro-inflammatory cytokines, such as Il2, Il6, and Tnf.", "AhR has been previously shown to inhibit pro-inflammatory IL-17 and IFN-γ cytokine production in T cells and to induce Tr1 cell differentiation in the gut (Ye et al., 2017).", "In addition, in the absence of AhR in macrophages, mice are more susceptible to LPS-induced endotoxic shock and present with an increase in pro-inflammatory IL-6 expression (Kimura et al., 2009).", "AhR deletion in microglial cells led to the upregulation of Ccl2, Il1b, Nos2, and Vegfb gene expression, factors known to be involved in inflammation and neurodegeneration (Rothhammer et al., 2018).", "Our data reveal that AhR preserves the immunosuppressive function of splenic IL-10+CD19+CD21hiCD24hiBregs by silencing a pro-inflammatory transcriptional program.", "Whether AhR complexes bind to XRE on the loci of pro-inflammatory cytokines and directly inhibit their expression in B cells, or if AhR coordinates the suppression of pro-inflammatory immune responses through interaction with other transcription factors in B cells, warrants further study.", "Our adoptive transfer results suggest that the predominant effect of AhR loss in B cells on AIA is through the loss of IL-10.", "We have previously shown that adoptive transfer of IL-10−/− B cells is unable to suppress arthritis (Carter et al., 2011; Evans et al., 2007).", "Here we show that WT mice do not get worse disease than the PBS control-injected mice upon adoptive transfer of AhR−CD19+CD21hiCD24hi B cells.", "Equally, AhR−CD19+CD21hiCD24hi B cells do not suppress disease onset in the recipient mice, unlike the transfer of control AhR+CD19+CD21hiCD24hi B cells.", "These data therefore suggest that the deleterious effects observed in AIA is the consequence of the reduced amount of IL-10.", "However, we cannot rule out the possibility that in vivo, AhR-deficient B cells contribute to overall inflammation through the upregulation of pro-inflammatory cytokines and chemokines or indirectly through the recruitment of other cell types.", "We have recently reported that AhR deletion, rather than impairing the capacity of B cells to proliferate, instead compromised their ability to commence the cell cycle (Villa et al., 2017).", "Indeed, there was reduction in Ccno mRNA expression in splenic B cells isolated from naive Ahrfl/−Mb1cre/+ compared with Mb1cre/+ mice.", "Here we show that this defect is overcome during an arthritogenic response, as no change in Ki-67 expression or in genes regulating the cell cycle, including Ccno, were observed between Mb1cre/+ and Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells taken directly from arthritic mice.", "Furthermore, no difference was observed in Ki-67 expression between Mb1cre/+ and Ahrfl/−Mb1cre/+ CD19+CD21hiCD24hiB cells after restimulation with LPS+anti-IgM.", "Although, in the latter, we used purified CD19+CD21hiCD24hi B cells instead of total splenic B cells and used different stimuli to activate this population, which together might account for the differences observed in this study.", "Therefore, these data confirmed that the phenotype observed in the absence of AhR expression in B cells is not due to impaired B cell proliferation but instead is due to the reduced ability of B cells to differentiate into Bregs and reduction in the production of IL-10.", "Our results show that although the loss of B cell AhR expression reduces IL-10+CD19+CD21hiCD24hiBreg frequency and leads to an expansion of plasma cells, it does not affect the frequencies or absolute numbers of B cell populations up to a mature naive B cell stage.", "Taken together with previous findings showing that AhR is expressed by B cells from the immature B cell stage in the bone marrow (Villa et al., 2017; Yamaguchi et al., 1997a, 1997b), we suggest that B cell AhR expression is important primarily for the control of IL-10+CD19+CD21hiCD24hiBreg immune-regulatory transcriptional programming and restricting plasma cell development but is dispensable for homeostatic B cell development.", "We report that Ahrfl/−Mb1cre/+ mice have increased frequencies of splenic plasma cells compared with control Mb1cre/+ mice, and these results are in line with those showing that both prototypic AhR agonists (polycyclic aromatic and planar halogenated hydrocarbons) affect terminal differentiation of B cells and humoral immune responses by inhibiting plasma cell differentiation and reducing the production of IgM (Schneider et al., 2009; Tucker et al., 1986; Vaidyanathan et al., 2017).", "We find increased levels of Prdm1 (Blimp-1) mRNA expression in B cells lacking AhR, consistent with previous findings showing that the suppression of terminal differentiation is mediated through AhR increases Bach2 expression (De Abrew et al., 2011); Bach2 in turn represses the expression of Blimp-1, a key transcription factor that controls B cell differentiation into immunoglobulin-producing plasma cells (Turner et al., 1994).", "We have extended the significance of these results to an inflammatory model and shown that early B cell development and maturation of B cells is not affected by B cell AhR expression but that AhR is required for the differentiation of IL-10+CD19+CD21hiCD24hiBregs.", "It is tantalizing to propose that the increase in plasma cells observed in mice lacking AhR+IL-10+CD19+CD21hiCD24hiBregs is due to the impaired function of Bregs.", "We have previously shown that at least in humans, IL-10 produced by Bregs directly inhibits T helper cell differentiation, which prevents plasma cell differentiation (Menon et al., 2016).", "Here we have shown that AhR, in response to inflammatory signals, plays an important role in the homeostatic maintenance of Breg function by acting as a molecular brake, preventing the differentiation of Bregs into effector B cells producing pro-inflammatory mediators.", "In addition to identifying that AhR regulates IL-10 expression in Bregs, our data highlight an additional mechanism by which AhR restrains inflammatory responses.", "These results add to a growing body of evidence supporting AhR as a key modulator of immune tolerance and therefore a potential therapeutic target in autoimmunity.", "Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Professor Claudia Mauri ([email protected]).", "This study did not generate new unique reagents.", "IL-10eGFP mice were as described (Madan et al., 2009), courtesy of Prof Karp.", "C57BL/6 mice were from purchased from Envigo, UK.", "Ahr−/−, Ahr+/−, Mb1cre/+ (courtesy of Prof Reth) and Ahrfl/-Mb1cre/+ mice were kindly provided by Prof. Brigitta Stockinger.", "IL-10R−/− mice were kindly given courtesy of Professor Werner Muller.", "Male and female mice were used at 8–12 weeks of age and were bred and maintained at the animal facility, University College London.", "All experiments were approved by the Animal Welfare and Ethical Review Body of University College London and authorized by the United Kingdom Home Office.", "AIA was induced and assessed as previously described (Rosser et al., 2014).", "Briefly, mice were injected subcutaneously at the tail base with 200 μg of methylated BSA (mBSA; Sigma-Aldrich) emulsified in 100 μL Complete Freund’s Adjuvant (CFA).", "CFA was made by mixing 3mg/ml of Mycobacterium tuberculosis (Difco) in Incomplete Freund’s Adjuvant (IFA; Sigma-Aldrich).", "After 7 days, mice received an intra-articular (IA) injection of 10 μL of PBS containing 200 μg mBSA in the right knee and 10 μL PBS alone in the left knee as a control.", "Joint size was measured using callipers (POCO 2T; Kroeplin GmbH) at daily intervals and swelling was calculated as a percentage increase in size between the inflamed and control knee.", "All experiments, unless stated otherwise, were performed at day 7 post-IA injection.", "For splenocyte and lymph node cell preparation, organs were mashed through a 70 μM cell strainer (BD Biosciences), as previously described (Rosser et al., 2014), and erythrocytes from spleens were lysed using Red Cell Lysis Buffer (Sigma-Aldrich).", "B cells were negatively purified by magnetic separation, according to manufacturer’s instructions (Miltenyi Biotec).", "Cells were cultured with either RPMI 1640 (Sigma-Aldrich) containing L-glutamine and NAHCO3 or Iscove’s Modified Dulbecco’s Medium (IMDM; Pan Biotech), enriched in AhR agonists (Veldhoen et al., 2009), supplemented with L-Glutamine and 25mM HEPES.", "Media were supplemented with 10% fetal calf serum (LabTech), 1% penicillin/streptomycin (100U/ml Penicillin+100 μg/ml streptomycin; Sigma-Aldrich) and 50 μM 2-Mercaptoethanol (ThermoFisher Scientific).", "Cells were cultured at 37°C with 5% CO2.", "Total lymphocytes, B cells and B cell subsets were cultured for 48h with CpGb ODN1826 (1 μM; Invivogen), LPS (1 μg/ml; Sigma-Aldrich) ± anti-mouse IgM (10 μg/ml; Jackson ImmunoResearch).", "or anti-CD40 (10 μg/ml; BioXcell).", "In addition, AhR agonist FICZ (100nM; Enzo LifeSciences) was added to culture.", "For 48h culture, anti-IgM ± FICZ were added 24h into culture.", "Splenic CD19+CD21hiCD24hi B cells were isolated from Mb1cre and Ahrfl/-Mb1cre mice in the remission phase of arthritis, at day 7 post-IA injection.", "Sorted cells were either left untouched (ex-vivo) or stimulated with LPS+anti-IgM for 6h in IMDM media.", "Dead cells were excluded using DAPI.", "Total RNA was isolated from these populations using the Picopure RNA isolation kit (ThermoFisher Scientific), according to manufacturer’s instructions.", "60bp single reads were sequenced on 3 lanes of an Illumina hiseq.", "130-500ng of total RNA was fragmented followed by reverse transcription and second strand cDNA synthesis.", "The double strand cDNA was subjected to end repair, A base addition, adaptor ligation and PCR amplification to create libraries.", "Libraries were evaluated by Qubit and TapeStation.", "Sequencing libraries were constructed with barcodes to allow multiplexing of samples in 3 lanes.", "Around 23-43 million single-end 60-bp reads were sequenced per sample on an Illumina HiSeq 2500 V4 instrument.", "Poly-A/T stretches and Illumina adapters were trimmed from the reads using cutadapt.", "Resulting reads < 30bp were discarded.", "Reads were mapped to the Mus musculus GRCm38 reference genome using STAR (Dobin et al., 2013).", "Gene annotations were applied from Ensembl (EndToEnd option and outFilterMismatchNoverLmax was set to 0.04).", "Gene expression levels were quantified using htseq-count (“HTSeq,” n.d.) (Anders et al., 2015), using the gtf above.", "Transcripts per million (TPM) values were estimated independently using Kallisto (Bray et al., 2016).", "Differential expression analysis was performed using the default settings of the edgeR algorithm (Robinson et al., 2010).", "p values reflect two-sided p values obtained using the exact test proposed by Robinson and Smyth (Robinson and Smyth, 2008) for a difference in means, between two groups of negative binomial random variables (implemented in edgeR package).", "Signaling Pathway Impact Analysis (SPIA) (Tarca et al., 2009) was used to detect significantly over-represented pathways, with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways database (Kanehisa et al., 2016) employed as a reference.", "The full mouse genome was used as background for enrichment.", "ATAC-seq was performed on splenic IL-10eGFP+CD19+CD21hiCD23hiCD24hi, IL-10eGFP-CD19+CD21hiCD23hiCD24hi and IL-10eGFP-FO B cells.", "In addition, ATAC-seq was performed on splenic total CD19+CD21hiCD24hi B cells isolated as above for the RNA-seq from Mb1cre/+ and Ahrfl/-Mb1cre/+ mice either left untouched (ex-vivo) or stimulated with LPS+anti-IgM for 6h in IMDM media.", "After sorting, 40,000 were washed with 1xPBS (10% FCS).", "The cell pellet was prepped for sequencing by using the Nextera DNA library preparation kit (Illumina).", "Briefly, 10.5 μL nuclease free water, 12.5 μL 2x Transposase buffer, 2 μL transposase and 0.25 μL digitonin (0.05%) per reaction were added to the cell pellets.", "Cells were incubated at 37°C for 30 minutes.", "DNA was then purified using a MinElute PCR purification kit (QIAGEN), according to manufacturer’s instructions.", "Following DNA purification, 1μl of eluted DNA was used in a qPCR reaction to estimate the optimum number of amplification cycles.", "Library amplification was performed using custom Nextera primers and was followed by solid phase reversible immobilization (SPRI) size selection to exclude fragments larger than 1,200bp.", "DNA concentration was measured with a Qubit fluorometer (Life Technologies).", "The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq4000 platform and the 50bp single-end configuration.", "Bioinformatic analysis was performed as previously described (Rendeiro et al., 2016).", "Briefly, Illumina Casava1.7 software was used for basecalling.", "Sequenced reads were trimmed for adaptor and Nextera sequences and reads were mapped to mm10 reference genome using bowtie2 v2.2.4 with the “–very-sensitive” parameter.", "Duplicate reads were marked and removed with picard tools version 1.118.", "Reads were extended to the average fragment size and bigWig files containing counts of reads per basepair created.", "Peaks for ATAC-seq samples were called with MACS2 version 2.1.1.20160309 (Zhang et al., 2008) using the “–nomodel” and “–extsize 147” parameters.", "Peaks were assigned to genes by proximity.", "If a peak overlapped the gene body or promoter ± 2500bp of the transcription start site (TSS), the peak was assigned to that gene.", "If a peak did not fall into these criteria, the peak was assigned to the closest TSS.", "If the nearest TSS to the peak was further than 100kb away, no gene was assigned.", "qPCR analyses were performed as described (Nistala et al., 2010).", "RNA from isolated B cells/subsets was extracted using Arcturus Picopure RNA isolation kit (ThermoFisher Scientific) and RNA was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad), according to manufacturer’s instructions.", "qPCR was performed on the cDNA samples using iQ SYBR® Green Supermix (Bio-Rad), according to manufacturer’s instructions.", "Primers were used at a concentration of 10 μM.", "Quantitect primers for Arnt, Ahrr and Cyp1a1 were purchased from QIAGEN.", "Primers for β-Actin and Ahr were custom designed with the following sequences: Act Forward (5′-AGATGACCCAGATCATGTTTGAG-3′); Act Reverse (5′- AGGTCCAGACGCAGGATG-3′); Ahr Forward (5′-AGGATCGGGGTACCAGTTCA-3′); Ahr Reverse (5′-CTCCAGCGACTGTGTTTTGC-3′); Il6 Forward (5′-GCCTTCTTGGGACTGATGCT-3′); Il6 Reverse (5′-TGCCATTGCACAACTCTTTTC-3′); Il5ra Forward (5′-GGTCCCGGTATGCAGTTCTA-3′); Il5ra Reverse 5′-AGCCGAATGCTGGAAAAGTG-3′.", "Ccl22 (Hao et al., 2016), Ebi3 (Shen et al., 2014), Il2 (Martins et al., 2008), Il10 (Yanaba et al., 2009), p35 (Shen et al., 2014) Tnf (Denaës et al., 2016) were used as previously described.", "qPCR data were calculated as the ratio of gene to β-Actin expression by the relative quantification method (ΔΔCt; means ± s.e.m. of triplicate determination).", "Supernatants from cell cultures were harvested and analyzed for cytokines using standard sandwich IL-2, IL-6, IL-10, IL-17 and TNFα ELISA Kits (R&D Systems) and performed according to manufacturer’s instructions.", "Serum was collected from Mb1cre/+ and Ahrfl/-Mb1cre/+ day 7 post AIA and was analyzed for total IgA, IgG and IgM (ThermoFisher Scientific).", "Flow cytometry was performed with the following directly conjugated antibodies from Biolegend: CD1d Percp/Cy5.5 (1B1), CD3 BV605 (17A2), CD4 BV711 (RM4-5), CD5 AF647 (53-7.3), CD19 BV785 (6D5), CD21 APC (CR2/CR1), CD23 FITC (B3B4), CD24 PE-Cy7 or BV421 (M1/69), CD43 PECy7 (S7), CD138 BV711 (281-2) and Tim-1 PE (RMT1-4).", "CD249 PE (BP-1) was purchased from BD Biosciences.", "For multi-color flow cytometric surface staining, cells were stained at 4°C for 20 min as previously described (Nistala et al., 2010).", "LIVE/DEAD fixable blue Dead Cell Stain (Life Technologies) was used to exclude dead cells from flow cytometric analysis.", "For measurement of intra-nuclear Blimp-1, Ki-67 and FoxP3 expression, cells were fixed for 25 minutes with FoxP3 Fixation buffer (ThermoFisher Scientific) and Blimp-1 AF647 (5E7), Ki-67 BV421 (16A8; Biolegend) or FoxP3 APC (FJK-16 s; ThermoFisher Scientific) was added in permeabilisation buffer.", "Intracellular cytokine analysis was performed as described previously (Rosser et al., 2014).", "Briefly, for detection of IFN-γ, IL-10 and IL-17, splenocyte or lymph node cells were cultured in complete medium with PMA (50ng/ml; Sigma-Aldrich), Ionomycin (250ng/ml; Sigma-Aldrich) and Brefeldin A (5 μg/ml; Sigma-Aldrich) for 4.5h. Cells were then stained with surface markers followed by intracellular fixation and permeabilisation (ThermoFisher Scientific).", "Cells were incubated with IFN-γ ΑPC (XMG1.2; BD PharMingen), IL-10 PE (JES5-16E3; Biolegend) and IL-17 PE (TC11-18H10.1; BD PharMingen).", "FP635 and eGFP reporter expression were analyzed ex vivo without fixation.", "B cell subsets were sorted using a cell sorter (FACSAria; BD PharMingen) by using CD19 BV785, CD21 APC, CD23 FITC and CD24 PE-Cy7.", "Dead cells were excluded by the use of 4,6-diamidino-2-phenylindole at 1 μg/ml (DAPI; Sigma).", "For cell sorting for RNA-seq and ATAC-seq, addition of BV605 dump channel antibodies against CD3 (17A2), CD4 (RM4-5), CD8a (53-6.7), CD11b (M1/70), CD11c (N418), F4/80 (BM8), Ly6C/G (RB6-8C5), erythroid cells (TER-119) and TCRβ (H57-597) were incorporated (Biolegend).", "Sort purity of B cell subpopulations was routinely > 95%.", "Flow cytometric data were collected on an LSRII or LSR Fortessa (BD PharMingen) using FACS Diva software.", "Data were analyzed using Flowjo (Tree Star).", "Splenic B cell subsets from Ahr−/− and Ahr+/− mice were sorted at day 7 post IA injection and stimulated with CpGb (ODN2006) for 6 hours.", "Cells were washed and then co-cultured with 0.5 μg/ml plate-bound anti-CD3 (145-2C11, BD Biosciences) for 72 hours with CD4+CD25- (bead isolated) T cells from Ahr+/− mice.", "Following stimulation, cells were analyzed for CD4+ IFN-γ expression.", "The percentage suppression of IFN-γ was calculated as a percentage reduction in IFN-γ from CD4+ cells cultured alone, compared to when B cell subsets were added to culture.", "CD19+CD21hiCD24hi B cells were isolated from spleens of Mb1cre/+ and Ahrfl/-Mb1cre/+ mice after remission from AIA and 5x106 were transferred into recipient wild-type mice on the day of intra-articular injection.", "The control group (no transfer) received a PBS injection.", "For histopathological examination, joints from Ahrfl/-Mb1cre/+ and Mb1cre/+ mice were removed post-mortem and fixed in 5% formalin and decalcified in 5% EDTA.", "The fixed joints were embedded in paraffin and 4 μm sections were cut and stained with hematoxylin-eosin.", "Splenic B cell subsets were sorted and RNA extracted using columns (Picopure, Life Technologies) and hybridized to murine mogene 2.0 ST arrays (Affymetrix).", "Raw CEL files were processed using the online GeneProfiler tool (accessible at https://www.beringresearch.com).", "Briefly, the GeneProfiler pipeline consists of present/absent call detection, (McClintick and Edenberg, 2006) Robust Microarray Average (RMA) normalization, and outlier detection (Kauffmann et al., 2009).", "Differential expression analysis was performed using the limma package (Ritchie et al., 2015).", "Total Vert-X splenic B cells were bead cell sorted and cultured for 24h with LPS, followed by addition of anti-IgM (10 μg/ml) and FICZ (100nM) at 24h into culture.", "After 48h total, total B cells were sorted based on eGFP for IL-10+ and IL-10- populations.", "Cells were fixed for 10 minutes with 1% (vol/vol) formaldehyde and quenched with 400mM Tris.", "Fixed cells were lysed with 120 μL lysis buffer (1% (wt/vol) SDS, 10mM EDTA and 50mM Tris-HCl, pH 8.1, 1 × protease inhibitor ‘cocktail’ (Roche), 1mM PMSF) per 5x106 cells.", "Chromatin was sheared to 200-500bp fragments and 10% of the initial chromatin material was kept as input.", "The chromatin was diluted 5-fold in Dilution Buffer (1% (vol/vol) Triton X-100, 2mM EDTA, 150mM NaCl and 20mM Tris-HCl, pH 8.1) and incubated overnight, after preclearing, with 1 μg/106 cells of a polyclonal AhR-specific antibody (BML-SA210; Enzo Life Sciences).", "Immunoprecipitation took place by incubation with protein G Dynal magnetic beads (Invitrogen), held for at least 3 hours at 4°C.", "Immunoprecipitated chromatin complexes were washed with High Salt Wash Buffer (2x), Low Salt Wash Buffer (2x), LiCl Wash Buffer (2x) and TE Buffer (2x).", "Immunoprecipitated chromatin was eluted from the magnetic beads with Proteinase K Digestion Buffer and heated at 65°C for at least 6h for reverse crosslinking.", "DNA fragments were purified with NucleoMag beads kit (MN) and were analyzed by SYBR Green Quantitative Real-time PCR.", "The following primers were used for ChIP qPCR: Il10 −3.5kb forward (5′-AGGGCTTGATAACGTGTGAGT-3′); Il10 −3.5kb reverse (5′-TGAACTTCACACCCAGCTTGAG-3′); Il10 −2kb forward (5′-TAAGAGGTGCTGCTTCTCCTG-3′); Il10 −2kb reverse (5′-TGGCACTGGACAGTTCTATGA-3′); Il10 −0.5kb forward (5′-AGGGAGGAGGAGCCTGAATAA-3′); Il10 −0.5kb reverse (5′-CCTGTTCTTGGTCCCCCTTTT-3′); Il10 +2kb forward (5′-GCCACATGCATCCAGAGACAC-3′); Il10 +2kb reverse (5′-GTGCCTCAAAGTCACTCCCAC-3′); Cyp1a1 −3.6kb forward (5′-GCTCTTTCTCTGCCAGGTTG-3′); Cyp1a1 −3.6kb reverse (5′-GGCTAAGGGTCACAATGGAA-3′); Cyp1a1 promoter forward (5′-AAGCATCACCCTTTGTAGCC-3′); Cyp1a1 promoter reverse (5′-CAGGCAACACAGAGAAGTCG-3′); Gapdh promoter forward (5′-GCGCGAAAGTAAAGAAAGAAGCCC-3′); Gapdh promoter reverse (5′-AGCGGCCCGGAGTCTTAAGTATTAG-3′).", "5x106 cells CD19+CD21hiCD24hi and FO B cells were FACS sorted from arthritic WT mice and lysed for 15 minutes at 4°C with cell lysis buffer (Cell signaling technology) for extraction of whole cell lysate.", "Protein was resolved by SDS-PAGE, transferred to nitrocellulose membranes and blotted using anti-AhR at 1/1000 (Enzo Life sciences) and anti-β-actin at 1/1000 (Cell Signaling Technology).", "Bound antibodies were revealed with a goat-anti rabbit H&L HRP-conjugated secondary antibody (1/1000) and ECL western blotting substrate (ThermoFisher Scientific).", "Heatmap analyses for microarray, RNA-seq and ATAC-seq datasets were performed using Multiple Experiment Viewer (MeV_4_8) software (Saeed et al., 2003).", "Hierarchical clustering was applied to genes using average linking clustering with the Euclidean distance metric.", "All data are expressed as mean ± s.e.m, unless stated otherwise.", "For in vivo studies, power calculations were performed on data showing mean maximum wild-type arthritic knee swelling of 2 mm with a s.d. of 0.39 mm, and an expected test group (transferred T2-MZPs) arthritic knee swelling of 1.4 mm.", "Group sizes of three mice or above were sufficient to reach a statistical power of at least 80% (http://www.statisticalsolutions.net/pss_calc.php).", "Mice were assigned at random to treatment groups for all mouse studies and, where possible, mixed among cages.", "Clinical scoring was performed in a blinded fashion.", "Mice that developed adverse reactions to protocols were excluded from datasets.", "Statistical significance was determined using unpaired t tests (comparison of two groups), using mann-whitney tests (comparison of two groups, non-parametric data), one-way ANOVA (comparison of three or more groups) or two-way ANOVA (comparison of two or more groups with 2 independent variables).", "One and two-way ANOVA were assessed with Bonferonni’s multiple comparison tests.", "All data met the assumption of statistical tests and had a normal distribution and variance was similar between groups that were statistically compared.", "Results were considered significant at p ≤ 0.05.", "Statistical tests were performed using GraphPad Prism (La Jolla, CA, USA) v.6, Software for Apple Mac.", "The microarray, RNA-seq and ATAC-seq datasets generated during this study are available at ArrayExpress: E-MTAB-7345, E-MTAB-7375, E-MTAB-7525 and E-MTAB-8393." ]	[ "IL-10+ Bregs are identified by high expression of AhR", "B cell AhR deficiency leads to exacerbated arthritis and impaired Breg function", "AhR directly binds to and regulates the expression of IL-10 in Bregs", "AhR maintains Breg phenotype by suppressing pro-inflammatory gene expression" ]
Origin of spectacular fields of submarine sediment waves around volcanic islands	Understanding how large eruptions and landslides are recorded by seafloor morphology and deposits on volcanic island flanks is important for reconstruction of volcanic island history and geohazard assessment. Spectacular fields of bedforms have been recognised recently on submerged flanks of volcanic islands at multiple locations worldwide. These fields of bedforms can extend over 50 km, and individual bedforms can be 3 km in length and 150 m in height. The origin of these bedform fields, however, is poorly understood. Here, we show that bedforms result from eruption-fed supercritical density flows (turbidity currents) in some locations, but most likely rotational landslides at other locations. General criteria are provided for distinguishing between submarine bedforms formed by eruptions and landslides, and emphasise a need for high resolution seismic datasets to prevent ambiguity. Bedforms associated with rotational landslides have a narrower source, with a distinct headscarp, they are more laterally confined, and internal bedform structure does not suggest upslope migration of each bedform. Eruption-fed density currents produce wide fields of bedforms, which extend radially from the caldera. Internal layers imaged by detailed seismic data show that these bedforms migrated up-slope, indicating that the flows that produced them were Froude supercritical. Due to the low density contrast between interstitial fluid and sediment, the extent and dimensions of submarine eruption-fed bedforms is much greater than those produced by pyroclastic density currents on land.	[ "EART" ]	[ "eruption-fed turbidity currents", "geohazards", "landslides", "submarine bedforms", "volcanic islands" ]	[ "1901", "1906", "1908", "1912" ]	[ "Silicic caldera-forming eruptions and landslides on volcanic islands represent some of the highest sediment flux events on Earth.", "They can transport tens to hundreds of cubic kilometres of material (Pyle, 1995; Hunt et al., 2013), most likely over a few hours to days.", "Both caldera-forming eruptions and landslides are extremely hazardous for local populations, and can generate far-travelling tsunami that effect more widespread distal coastlines.", "Explosive eruptions produce large amounts of material that can be rapidly transferred from the vent to submarine slopes and basins via volcaniclastic density flows (Cas and Wright, 1991; Allen et al., 2012).", "The landslides that occur on volcanic flanks can be unusually large, and involve both the subaerial and submarine domains of the edifice (White, 2000; Masson et al., 2006; Watt et al., 2012).", "Further, they can also evacuate material to surrounding basins through density flows (Le Friant et al., 2015; Watt et al., 2015).", "It is therefore important to understand how eruptions and landslides are recorded on submerged volcanic island flanks, and to distinguish between caldera-forming density current deposits and large submarine landslides as they pose distinctly different types of hazards.", "Worldwide, recent work has recognised extensive fields of bedforms on submerged volcanic island flanks (Wright et al., 2006; Silver et al., 2009; Gardner, 2010; Leat et al., 2010 and references therein) that are some of the largest amplitude bedforms yet documented on the seafloor (Fig. 1; Symons et al., 2016).", "‘Bedform’ is used here to denote undulations without any reference about their origin.", "These undulating areas of seafloor can extend for over 50 km, and individual bedforms have wavelengths up to 3 km, and wave heights of 10–150 m. However, the origin of these bedforms is uncertain.", "As shown by debate over the origin of such bedforms in non-volcanic settings, they can potentially result from both rotational landslide blocks and remoulding of the seabed by density flows (Lee et al., 2002).", "The origin of these submarine bedforms is also particularly interesting because they are larger than any known bedforms on the subaerial flanks of volcanoes.", "Identified submarine bedform fields are extensive and contain large amplitude bedforms.", "In contrast, subaerial volcanic flank bedform fields are less extensive and contain smaller-scale bedforms associated with terrestrial dilute pyroclastic density currents (e.g. Sigurdsson et al., 1987; Branney and Kokelaar, 2002; Brown and Branney, 2004), or irregular hummocky terrain formed by debris avalanches and landslides (e.g. Crandell et al., 1984; Cas and Wright, 1991).", "We therefore seek to understand what the contrasting dimensions of submarine and subaerial bedform fields can tell us about important general differences between subaerial and submarine volcanic mass flows (Moorhouse and White, 2016).", "Eruption-fed submarine density flows sourced from either subaerial or submarine vents can be initiated by either collapse of eruption columns or disintegration of active lava domes (Cas and Wright, 1991; Kokelaar and Busby, 1992; Head and Wilson, 2003).", "Sediment waves (cyclic steps, anti-dunes, etc.) and scours are thought to be the submarine slope expression of these flows (Kostic and Parker, 2006; Spinewine et al., 2009; Cartigny et al., 2011).", "Failure of submarine volcanic shelves and slopes can result from earthquakes, edifice uplift, magma intrusion and extrusion or the sudden accumulation of large volumes of pyroclasts during volcanic eruptions (Watt et al., 2014).", "These failures commonly affect deep substrate and terminate proximally at steep headwall scars (Masson et al., 2006).", "Landslide deposits include rotated domains, huge individual blocks, and various types of density flow deposits (Masson et al., 2006; Watt et al., 2015).", "Landslide deposits made up of rotated blocks can form an undulating seafloor that shares some surficial similarities to sediment waves emplaced by eruption-fed density flows (Lee et al., 2002; Leat et al., 2010).", "This can make it difficult to distinguish landslide block and sediment flow deposits, as previously noted for bedforms in non-volcanic sites (Lee et al., 2002).", "Our lack of understanding of proximal submarine volcanic slope deposits is a consequence of the lack of attention that they have received.", "Basin records of volcanic deposits are relatively well understood from the rock record (Trofimovs et al., 2006; Allen and McPhie, 2009; Jutzeler et al., 2014a) and ocean drilling (Nishimura et al., 1991; Le Friant et al., 2015; Busby et al., 2017).", "In contrast, scientific drilling has never been successful in proximal environments (ODP135; IODP340), high-resolution geophysical data is limited (Gardner, 2010; Leat et al., 2010; Casalbore et al., 2014a, 2014b), and extensive subaerial exposures of these deposits are rare and often incomplete (Cas and Wright, 1991; Allen and McPhie, 2009).", "Our overall aim is to understand the origin and wider significance of extensive fields of bedforms on volcanic island flanks, whose occurrence has been recently recognised in locations worldwide.", "We first consider bedforms on the flanks of Macauley and Raoul Islands in the Kermadec Arc, using particularly detailed seismic surveys and bathymetric mapping data.", "We provide general criteria for distinguishing between deposits from eruption-fed density flows and landslides (Table 1).", "We show how these general criteria can help to understand the origin of submarine bedforms at other volcanic islands, and emphasise the need for detailed seismic surveys to reduce ambiguity.", "We conclude by discussing why similar extensive and well-developed bedforms are not formed subaerially, and what this may tell us about eruption-fed density flows and landslides in general.", "Macauley and Raoul Islands are located in the intra-oceanic Kermadec Arc (Fig. 2), being the uppermost subaerial edifices of two active, voluminous submarine stratovolcanoes (Wright et al., 2006; Shane and Wright, 2011; Barker et al., 2013).", "Onshore Macauley Island, basal deposits comprise basaltic lavas and phreatomagmatic deposits, overlain by the Sandy Bay Tephra Formation and younger basaltic lavas.", "The 5.7 ka Sandy Bay Tephra is exposed in cliffs 15–100 m high, and is the only confidently known silicic activity related to this volcano (Smith et al., 2003; Shane and Wright, 2011).", "It is made up of >30 flow units from wet pyroclastic density currents, most likely associated with a large magnitude submarine explosive eruption originated from the now-submarine caldera lying to the northwest of the island (Lloyd et al., 1996; Wright et al., 2006).", "In contrast, the last 4 ka on Raoul Island are dominated by ∼15 km3 of deposits from multiple dry or phreatomagmatic silicic explosive eruptions linked to two summit calderas (Rotella et al., 2014).", "Kongsberg EM300 30 kHz multibeam echo-sounder and multichannel seismic data were acquired around Macauley and Raoul Islands during May 2007 onboard RV Tangaroa (TAN0706) and comprises one of the most detailed datasets yet for bedform fields on volcanic island flanks.", "The multibeam data are gridded to a 25 m cell size with a vertical resolution <1% of water depth.", "A total of 5 multichannel seismic lines were collected around the two islands using a GI airgun source and a 48 channel streamer.", "A seismic velocity of 1600 m/s was used to convert two way travel time to depth on all seismic profiles.", "Using the bathymetry data, analysis of each bedform field was carried out by first categorising the morphologies of the bedforms within each bedform field.", "Bedforms were categorised as either convex, concave or linear in planform.", "Second, the wavelength and wave height of each bedform was catalogued.", "As part of this analysis, the upslope limb length and downslope limb length of each bedform was recorded; combined with the seismic data, this enabled bedform symmetry to be analysed.", "Third, the setting of each bedform field was analysed, i.e. are the bedforms confined by a definitive lateral margin or are they unconfined.", "Fourth, each bedform field was analysed for any notable additional features such as an identifiable headscar.", "Analysis of the seismic data from the bedform fields was carried out using automatic picking routines in Kingdom Suite.", "This was followed by manual picking of the seismic reflectors in order to ensure greater accuracy, and identify any artefacts present in the automated routines.", "Repetitive bedforms have developed over large parts of the southwest and northern flanks of Macauley Volcano (Figs. 2 and 3).", "These semi-continuous undulations extend outwards for over 20 km, from a major break-in-slope formed by the external rim of the submarine caldera.", "In planform these bedforms have a repetitive convex morphology and have poorly defined lateral margins.", "The wavelengths of bedforms on the caldera's southern flank decreases downslope from 1500 to 250 m, whilst their amplitude decreases from 140 to 10 m.", "Seismic data from the southwest bedform field shows multiple high amplitude reflectors making up a chaotic facies (Figs. 3b, c and 4).", "Separated from this facies by a stratigraphic unconformity, a series of thin, ordered lower amplitude reflectors are apparent.", "The lower amplitude reflectors make up the bedforms themselves (Figs. 3 and 4).", "An internal unconformity is also identifiable within the lower amplitude reflectors.", "The strength of the reflector that marks this unconformity is highly variable along the seismic line (Fig. 4).", "Our interpretation of this unconformity is marked by the green line in Figs. 3 and 4, and represents either a change in the seismic character of the reflectors (e.g. an abrupt change in orientation), a truncation of other reflectors, or a specific higher amplitude reflector (see Fig. 4).", "If this surface represents a significant unconformity, then there are >20 planar reflectors beneath this surface and >30 above.", "The planar reflectors below the interpreted unconformity are truncated at high angles on their lee sides (Fig. 4).", "Each reflector is truncated by the proposed unconformity (Fig. 4f).", "The upslope limb length of the bedforms in this lower set of reflectors is on average 1.84 times the length of the downslope limb length.", "The greater upslope limb length of bedforms in the lower set of reflectors defines this set of reflectors as asymmetric.", "In comparison, the upper set of reflectors are more clearly defined and well-developed.", "These reflectors conform to the underlying interpreted unconformity.", "The reflectors in the upper set have a dome-like apex, and their lee sides are thin but not truncated, instead becoming unresolvable in the seismic data (see Fig. 4b–d).", "As a result, the upper set of reflectors that make up the bedforms are more symmetrical.", "The upslope limb is on average only 1.55 times the length of the downslope limb.", "In some bedforms, some continuous and well-layered reflectors can be traced from one flank to the other, but it is difficult to correlate reflectors between one bedform and the next.", "Proximal to the caldera, where the wave amplitudes are greatest, the regular reflectors cut into the chaotic reflector patterns which are thought to represent the underlying substrate.", "Ponded facies have also developed closest to the break-in-slope indicated by the chaotic reflector patterns on the seismic data (Fig. 4).", "The total bulk volume of these bedforms around the whole island is at least ∼27.5 km3, with ∼17.5 km3 on the calderas southern flank, and ∼10 km3 on the northern flank.", "This volume estimate uses the basal scoped reflector, shown in orange on Fig. 3.", "A number of possible mechanisms exist which could have resulted in the emplacement of the observed bedforms.", "First, they could be emplaced by poorly confined sediment density flows resulting from the radial collapse of an eruption column.", "In this instance, each bedform is a consequence of flow interaction with the sedimentary bed.", "The bedform architecture results from sediment deposition occurring mainly on the upcurrent flank of each bedform, while erosion and lower rates of erosion dominate the downstream flank.", "The bedforms migrate upslope and prograde upcurrent as a result of these flows.", "Second, the observed bedforms could be a consequence of sediment density flows unrelated to an eruption.", "Third, they could be the surface expression of a single (or multiple) submarine landslide(s), each bedform resulting from the downslope movement of material.", "In this case the internal architecture of the bedforms is a consequence of the deformation of material contained within each slide block during its downslope transition.", "Of these possible emplacement mechanisms, we interpret the bedforms to the southwest of Macauley Volcano to have originated as a consequence of eruption-fed density derived from eruption column collapse; the bedforms originating as a consequence of instabilities between a flow and the sedimentary bed.", "We favour this interpretation for the following reasons.", "1) The bedform crests are radially extensive and sub-parallel to the caldera rim which fan out in a regular pattern, as might be expected for voluminous, poorly-confined sediment density flows formed by radial collapse of an eruption column.", "2) The bedforms do not originate from a distinct headscarp, nor are they strongly confined by submarine valleys, as might be expected for a landslide.", "3) Perhaps most importantly, our interpretation of the regular reflectors incised into the underlying substrate as troughs suggests that these bedforms are not related to landslide activity.", "Were these bedforms related to landslides, translation of material would result in deformation within each slide block and thus may produce the internal reflector patterns observed (Hampton et al., 1996).", "It would, however, be unlikely to periodically erode into the substrate below the failure surface.", "4) No compressional zone is identifiable on the bathymetry or seismic data as might be expected from a landslide.", "5) The bedform wavelength and amplitude is similar to that produced by fine-grained and mainly supercritical turbidity currents in non-volcanic settings (see Fig. 1).", "6) An origin for the bedforms being generated from sediment density flows that are unrelated to eruptions is discounted.", "It is not obvious how such energetic flows, which are needed to erode several tens of meters into the subsurface, could have originated from a narrow submerged caldera rim (Fig. 3a).", "An eruption source is also more likely given the nearby caldera.", "Having interpreted sediment density flows as responsible for the emplacement of the observed bedforms, the bedform morphology and internal architecture will now be analysed to suggest bedform type and therefore the possible flow dynamics which resulted in their emplacement (see Fig. 5).", "The bedforms symmetry and internal architecture suggests that stoss-side deposition and lee-side erosion occurred during their formation.", "This suggests that the bedforms migrated upstream and thus we can exclude any bedform type which is downstream migrating.", "This leaves two candidates for the bedforms; anti-dunes and cyclic steps.", "In comparison to anti-dunes, cyclic steps are considered to generally be longer wavelength and more asymmetrical bedforms (Cartigny et al., 2011).", "They are characterised by depositional subcritical flow over their stoss side and erosive supercritical flow over their lee side.", "The systematic transition between the two flow states is characterised by a hydraulic jump (Kostic and Parker, 2006).", "Meanwhile anti-dune bedforms form in phase (i.e. the flow elevation mimics the bed elevation) with surface waves in a supercritical flow (Cartigny et al., 2011).", "In the lower part of the stratigraphy (see Fig. 4), the internal reflectors of the longer wavelength bedforms are strongly asymmetric.", "In contrast, the internal reflectors in the upper part of the stratigraphy are shorter and more symmetrical.", "We therefore interpret this geometry to be representative of cyclic step deposits overlain by a series of anti-dunes.", "Superposition of more symmetrical bedforms on top of longer cyclic steps as has been previously observed in experiments (Cartigny et al., 2014) and modelling (Kostic, 2014).", "This superposition often reflects a drop in Froude number through time (Cartigny et al., 2014; Zhong et al., 2015).", "However, in both cases the bedform emplacing flow is dominated by supercritical flow conditions.", "Distally, the boundary between the two sets of waves is not resolvable, and the wavelength and wave height decrease away from source (Fig. 2b).", "This may indicate that sediment concentration, grain size, and/or velocity decreased with distance, reducing the flow's ability to modify the seafloor.", "Seafloor sampling reveals the uppermost sequence of the sediment wave fields offshore Macauley Island is covered by pumice clasts from multiple eruptions (Barker et al., 2012, 2013).", "Interestingly, Sandy Bay Tephra compositions were not identified, indicating that at least part of the uppermost stacked planar bedded seismic reflectors (Fig. 2) were not related to the 5.7 ka eruption.", "Current seismic data suggests a maximum burial depth of a few tens of meters below the seafloor.", "However, coring and further geochemical analyses will be required to resolve the exact post-climactic eruption sediment thickness and the timing of the eruption(s) that generated the conspicuous wave fields.", "The submarine flanks of Raoul Island are characterised by large areas of wavy seafloor morphology (Figs. 2 and 6).", "A series of bedforms occur to the west and south of the Island.", "Each bedform train contains at least 30 individual bedforms.", "These bedforms have concave or linear crests when analysed in planform (Fig. 6).", "Each bedform train originates at an arcuate headwall located near to the shelf break, extends downslope for ∼10 km, and occur on relatively steep slopes (5°).", "They are confined to a zone that is 0.5–2 km wide and have sharp lateral boundaries.", "Individual bedforms are strongly asymmetrical (the upslope limb being 1.65 times longer than the downslope limb) with wavelengths of ∼650 m and heights up to 80 m in proximal areas.", "Distally, the bedforms become more symmetrical (the upslope limb is 1.09 times longer than the downslope limb) and smaller in height (10 m).", "The seismic data from the bedforms offshore Raoul Island (Fig. 6b, c) are more chaotic than those seen from the Macauley Volcano bedforms (Figs. 3 and 4).", "This is partly a consequence of the Raoul seismic line not being perfectly perpendicular to the bedform crests, as was the case for Macauley.", "Although the noise within the Raoul seismic data makes it difficult to define internal layering, these bedforms do not clearly exhibit the internal reflectors visible in the Macauley Volcano seismic data.", "Instead, a seismic transect from the western slope of Raoul Island shows a chaotic seismic unit which is bounded at its base by an interpreted scoop-shaped basal surface which truncates well-bedded, seaward-dipping planar stratigraphy beneath.", "The chaotic seismic unit is interpreted as probable back-rotated stratigraphy, dissected by inferred normal faults.", "A second basal scoop-shaped surface is also visible suggesting that the one chaotic unit is made of two separate units.", "The end of the shear surface cannot, however, be defined.", "Some of the Raoul Island bedforms end in a toe-shaped area of positive bathymetric relief, which is ∼40 m higher than the surrounding seafloor.", "The greater symmetry of bedforms in the distal parts of these bedform fields may also be a consequence of the compression of sediment in these locations as a consequence of landslide processes.", "The geomorphology and seismic stratigraphy of these bedforms is consistent with that of previously described submarine landslides (Hampton et al., 1996; Shea et al., 2008; Watt et al., 2014).", "It could be proposed that these bedforms were generated by eruption-fed density flows, which were relatively thin and thus confined within gullies.", "Equally, it has previously been proposed that small mass-wasting events have led to the formation of crescent-shaped bedforms through the downslope evolution of these flows, such as offshore volcanoes in the Aeolian Archipelago (Romagnoli et al., 2013; Casalbore et al., 2014a, 2014b).", "However, it is likely that eruption-fed density flows would be less confined than the presented examples and would lead to a gradual transition to overbank bedforms not seen here.", "Some of these bedforms might also originate via turbidity currents initiated at the shelf edge by non-eruptive processes (cf. Hughes Clarke, 2016), although the limited hydrologic system of Raoul Island prevents large-scale fluvial input to the ocean.", "Nevertheless the abrupt initiation of the bedforms at a distinct head scarp favours a landslide origin, and the scale of these bedforms is larger than those formed by non-volcanic density flows in similarly confined settings, where bedforms typically have wavelengths of <200 m, and heights of <10 m (Symons et al., 2016).", "We interpret these bedform trains as most likely the result of slope failure for the following reasons.", "1) Bedforms trains originate abruptly from a distinct arcuate headwall, which is most consistent with landslides.", "2) The bedform trains are laterally confined in valleys, and absent on intervening interfluves.", "3) There is some evidence in the seismic data for backward-rotated blocks.", "4) A toe-shaped area of positive bathymetric relief exists at the base of the slope which we interpret to represent the compressional zone exhibited by other known submarine landslides (Hampton et al., 1996).", "The greater symmetry of bedforms in the distal parts of these bedform fields may also be a consequence of the compression of sediment in these locations as a consequence of landslide processes.", "Together this evidence suggests that the identified bedforms are the result of slope failure.", "However, it must be acknowledged that eruption-fed density flows cannot be ruled out as the formative process and therefore further higher resolution seismic data is needed to be certain (Table 1).", "Although spectacular, the bedform fields observed on the flanks of Macauley Volcano and Raoul Island are not unique.", "Bedform fields have been observed on the submarine flanks of volcanic islands worldwide in other subduction zone, collision zone and hot spot settings (see supplementary Table 1).", "These sites include Stromboli (Kidd et al., 1998), Canary Islands (Wynn et al., 2000), the Bismark Volcanic Arc (Hoffmann et al., 2008; Silver et al., 2009), the West Mariana Arc (Gardner, 2010), South Sandwich Islands (Leat et al., 2010), Reunion Island (Sisavath et al., 2011) and the Madeira Archipelago (Quartau et al., 2018).", "Nonetheless it has often been difficult to unambiguously infer the origin of these bedforms (Quartau et al., 2018).", "The following section will therefore compare the bedform fields observed offshore Macauley Volcano and Raoul Island with other identified bedform fields and outline a set of general criteria for determining bedform origin.", "Macauley Volcano and Raoul Island bedforms reported in this study have wave heights ranging from 10 m to 140 m and wavelengths of 250 m to 2000 m. Globally, reported bedform wave heights have been reported ranging from 2.5 m to 340 m (see Fig. 1; Babonneau et al., 2013; Leat et al., 2013).", "Globally, wavelengths vary between 10 m and 3.5 km (Moore and Chadwick, 1994; Ollier et al., 1998).", "Although larger than average (see Fig. 1), the wave heights and lengths reported in this study are therefore fairly typical of bedform sizes reported in other locations.", "The planview morphology of the Macauley and Raoul bedforms varied from convex to concave with some classified as wavy (see Fig. 2 and Table 1).", "Their setting was also variable; some bedform fields were confined between sidewalls (see Fig. 6) whilst others remained unconfined (Fig. 3).", "The confined bedforms were often associated with a distinct upper headscarp.", "The morphology of these features is similar to those seen on other volcanic islands.", "The northern flanks of La Palma exhibit a series of unconfined convex, sinuous and bifurcating bedforms which are parallel to the regional slope (Wynn et al., 2000).", "They also exhibit internal architectures similar to those described for the bedforms offshore Macauley Island (see Section 3).", "In contrast, the flanks of Montagu and Bristol Islands are characterised by laterally confined bedforms near to the shelf edge which are associated with arcuate headwalls (Leat et al., 2010, 2013).", "These bedforms are similar to those interpreted as landslide sourced offshore Raoul Island (see Section 3).", "The bedforms reported in this study are therefore fairly typical of bedforms seen on other volcanic islands and are thought to be the result of similar processes.", "Observations/interpretations from Macauley and Raoul should therefore be able to inform the interpretation of other volcanic island bedforms.", "One specific type of submarine bedform field type observed elsewhere is, however, absent from Macauley Volcano and Raoul Island.", "On a number of large volcanic islands (e.g. Reunion, Madeira etc.) bedforms associated with the mouths of fluvial systems have been identified (Babonneau et al., 2013; Lebas et al., 2011; Quartau et al., 2018).", "A consequence of sediment gravity flows related to fluvial discharge and deposition of sediment rather than volcanic activity, the wave height and wavelength of these bedforms is limited compared to their volcanically sourced counterparts.", "They are also found in association with deep-sea fan systems (Sisavath et al., 2011).", "The presented bathymetry and seismic data in this study suggests that widespread bedforms that fan out radially from a caldera suggest eruption-fed density flows (i.e. Type 1 bedforms in Table 1), whereas a distinct upper headscarp conversely favours a landslide origin.", "The strongest evidence for eruption-fed density flows comes from seismic data that can resolve internal layering within bedforms (Fig. 4b).", "Where such layering indicates up-slope migration, it indicates formation through supercritical density flows.", "Rotation of planar-bedded blocks within a landslide can also form up-slope dipping reflectors, thus continuous internal reflectors are needed to preclude a landslide origin (Lee et al., 2002).", "Detailed seismic data is thus critical for determining the origin of bedforms with confidence, as was also found for studies of bedforms in non-volcanic settings (Lee et al., 2002).", "Although difficult to obtain due to the coarseness of volcanic sediment (Jutzeler et al., 2014b), sediment cores may increase confidence in how bedforms originate, especially if dating shows the bedforms are coeval with a known subaerial eruption.", "From our observations of bedforms offshore Macauley Volcano and Raoul Island, we therefore propose general criteria for understanding the origin of such bedforms (Fig. 7 and Table 1).", "To assess the applicability of these criteria, we now consider bedforms offshore from the South Sandwich Islands and New Britain.", "Originally reported by Hoffmann et al. (2008, 2011), the flanks of Dakataua Caldera and Kimbe Bay in the New Britain Arc (Figs. 8a and 8b) are characterised by bedforms similar to those described on Macauley Volcano.", "Here, wavelengths range from 500 to 3700 m, and heights from 10 to 100 m.", "The bedforms are mostly asymmetrical (steeper and shorter lee sides) and decrease in wavelength downslope.", "Chirp sub-bottom data through the Kimbe Bay wavefield suggests thicker sediment packages on the upslope limb of waves and upslope migrating sediment packages (Hoffmann et al., 2008), therefore similar to those identified as Type 1 (eruption-fed in Table 1).", "The flanks of the Candlemas Islands and Zavodovski Island (South Sandwich Arc) are characterised by bedforms originating at arcuate headwalls, and are laterally confined on the upper slope (Figs. 8c and 8d).", "These bedforms have lengths from 830 to 2500 m, and heights from <10 to 150 m (Leat et al., 2010).", "The bedforms observed offshore the Candlemas Islands and nearby Adventure Volcano (Leat et al., 2013) are similar to those identified as landslide-generated offshore Raoul Island and can therefore be categorised as Type 2 (due to landslides) in Table 1.", "The origin of bedforms offshore Zavodovski Island is harder to ascertain.", "Bedforms to the east of Zavodovski originate from headwalls and are confined within gullies suggesting a landslide origin (Type 2 in Table 1).", "However, the bedform shape changes with distance offshore from concave to convex in some locations.", "Moreover, shallow seismic data also shows a veneer of downslope prograding sediments containing internal reflectors, suggesting downslope movement of sediment by density flows (Leat et al., 2010).", "Overbank sediment deposits have also been identified (Leat et al., 2010).", "The observed bedforms could therefore be interpreted as a consequence of initial mass-wasting (landslide) events followed by eruption-fed density flows thus exhibiting both Type 1 and Type 2 features (Table 1).", "These examples demonstrate that it is possible to use the general criteria set out in Table 1 as a means to make initial assessment of the processes resulting in different volcanic island flank morphologies.", "However, the complexity shown offshore Zavodovski Island indicates that caution is required in terms of prescribing specific formation processes in these locations without suitably detailed seismic data in addition to high resolution bathymetric data.", "There are strong differences in morphology of volcanic bedforms between subaerial and submarine domains.", "These morphological discrepancies between subaerial and submarine deposits reflect the different hydrodynamic properties of these flows, subject to the density of the interstitial fluid (gas against water).", "Another influencing factor may be the slope morphology, coarseness and composition of the substrate.", "Pyroclastic density currents produced by subaerial volcanism create deposits that range from massive, to non to poorly-stratified deposits (Sparks, 1976), and to deposits recording more dilute conditions, forming sets of dunes and anti-dunes (Sigurdsson et al., 1987; Brown and Branney, 2004).", "However, subaerial-deposited bedforms do not match the size and extent of the submarine bedforms presented in this study.", "Dilute pyroclastic density current deposits have bedform heights of <5–10 m and wavelengths reaching several hundred meters (Brown and Branney, 2004).", "In contrast, the submarine bedforms described here have heights of 10–150 m, and wavelengths can be up to 1700 m and are therefore at least one order of magnitude greater.", "Dunes formed by dilute pyroclastic density currents can extend across areas up to ∼150 km2 (Sigurdsson et al., 1987), but can be much more localised (Brown and Branney, 2004), and much less extensive than the studied submarine bedforms (>400 km2).", "Large-magnitude silicic eruptions (i.e. Plinian in the subaerial realm) produce high-flux, sustained pyroclastic density currents that can last hours to days (Wolfe and Hoblitt, 1996; Maeno and Imamura, 2011).", "Under such conditions, voluminous sediment input in the ocean may be continuous for long periods of time, favouring instalment of prolonged sedimentation conditions on the seafloor.", "This eruptive behaviour is in strong contrast with instantaneous, essentially mass-wasting events such as collapse of an active lava dome or sector collapse (Voight et al., 2002; Trofimovs et al., 2006, 2008), which are associated with gravitational instabilities or vulcanian explosions.", "Moreover, volumes of transported sediment through lava dome collapses are commonly less voluminous.", "It is therefore difficult to use literature examples of such collapses to infer hydrodynamic properties in sustained volcaniclastic density flows.", "Offshore Montserrat, the density flow deposits derived from dome and sector collapses lack bedforms, which we interpret as resulting from rapid accumulation of sediment from short-lived, non-sustained events (Trofimovs et al., 2006, 2008).", "Here, we propose that eruption-fed density flows produce larger and more extensive bedforms in submarine slope settings for the following reasons.", "First, submarine density flows have smaller density contrasts between their interstitial fluid (seawater) and the transported sediment than for subaerial pyroclastic density currents (air and magmatic gases).", "This low density contrast in submarine density flows favours supercritical flows (Kostic and Parker, 2006; Cartigny et al., 2014).", "Moreover, this implies that submarine density flows are slower than their subaerial equivalent, and respond more slowly to changes in slope, such as on the stoss and lee slopes of bedforms (Gray et al., 2006).", "Such hydrodynamic characteristics lead to formation of larger and more extensive bedforms on underwater slopes (Gray et al., 2006).", "The small density contrast between particles and seawater also induces slower settling rates of grains (Cashman and Fiske, 1991; Jutzeler et al., 2015), which favours suspension and longer runouts, and hence more extensive bedform fields.", "Second, bedform development is strongly affected by the concentration of particles within the lowermost part of the flow.", "For example, pyroclastic block-and-ash flows (formed by collapse of an active lava dome) often contain a dense granular layer at their base, which is typically confined within topography; it is overlain by dilute pyroclastic density currents (surges) that can travel much further (Calder et al., 1999).", "Sediment settles more rapidly in air than in water, and this favours the generation of dense near-bed flows on land (Breard et al., 2016) rather than underwater (Sohn, 1997).", "Bedforms produced by such stratified flows are significantly shorter (Cartigny et al., 2014; Postma and Cartigny, 2014; Symons et al., 2016).", "Future modelling is necessary to test how these various factors may combine to favour bedform generation in submarine settings.", "Sector collapse of a volcano triggers debris avalanches, which can form hummocks and Toreva blocks in their deposits (e.g. Crandell et al., 1984), but their morphology differ from results of this study.", "The Type 2 bedforms (Table 1) that we ascribe as most likely originating from landslides (Figs. 6 and 7) differ markedly from the hummocky terrain typically associated with subaerial debris avalanches.", "Submarine landslide hummocks are chaotic and tend to lack elongate crests; they are not so regularly distributed as the submarine bedforms described here (Watt et al., 2015).", "Debris avalanches can produce rotated Toreva blocks with linear crests (e.g. Clavero et al., 2002); however, the regular spacing and linear crests of the Type 2 submarine bedforms more closely resembles the morphology of non-volcanic submarine landslides, such as the Eureka Slump offshore Alaska or the Storegga Slide offshore Norway (e.g. Hampton et al., 1996; Micallef et al., 2008).", "Submarine linear crests are created in response to normal faulting by extension along a seafloor-parallel detachment surface, which produces rows of tilted fault blocks (e.g. Micallef et al., 2008).", "It records situations in which the failed material does not disintegrate fully, and where marine sediment provides extensive weak layers for regional detachment.", "The submarine landslide morphology discussed also differs from many previously described submarine landslide, such as deeper-seated and larger failures offshore from the Canary Islands (Masson et al., 2006) in which the landslide material has more fully disintegrated.", "Here, we present two detailed marine geophysical datasets from Macauley and Raoul Islands in the Kermadec Arc, which include detailed seismic sub-seafloor profiles through bedforms.", "Some bedform fields result from eruption-fed submarine density flows.", "These eruption-fed bedforms are radially extensive around a caldera structure, and seismic profiles show that bedforms migrated up-slope.", "However, other types of bedform represent rotated blocks in submarine landslides.", "We therefore propose a series of generally applicable criteria for distinguishing bedforms formed by eruption-fed density flows and landslides (Table 1).", "As in non-volcanic settings, it may be necessary to collect and analyse detailed seismic data to avoid ambiguity.", "Submarine density flows derived from sustained silicic explosive eruptions produce larger and more extensive bedforms than pyroclastic density currents, their subaerial equivalent.", "This is mainly due to the lower density difference between the transported sediment and the interstitial fluid.", "Relatively slow settling of sediment in water favours formation of bedforms, whereas fast settling of pyroclastic density currents in air favour formation of dense basal layers and flow stratification.", "Moreover, the reduced density difference causes a slower response of submarine flows to changes in seafloor gradients generating large and extensive bedforms.", "Bedform occurrence and geometry suggests that the dynamics of submarine and subaerial mass flows are markedly different." ]	[ "High resolution seismic data of volcanic island bedforms is presented.", "Bedforms related to eruption-fed flows migrated up-slope.", "Diagnostic criteria distinguishing eruption-fed and landslide bedforms are proposed.", "Subaerial and submarine bedform contrasts relate to eruption-fed flow dynamics." ]
Control of immune escaped human papilloma virus is regained after therapeutic vaccination	High-risk human papillomaviruses infect the basal cells of human epithelia. There it deploys several mechanisms to suppress pathogen receptor recognition signalling, impeding the immune system to control viral infection. Furthermore, infected cells become more resistant to type I and II interferon, tumour necrosis factor-α and CD40 activation, via interference with downstream programs halting viral replication or regulating the proliferation and cell death. Consequently, some infected individuals fail to raise early protein-specific T-cell responses that are strong enough to protect against virus-induced premalignant disease and ultimately cancer. Therapeutic vaccines triggering a strong T-cell response against the early proteins can successfully be used to treat patients at the premalignant stage but combinations of different treatment modalities are required for cancer therapy.	[ "IMMU" ]	[]	[ "2406" ]	[ "About 80% of sexually active individuals become infected with a high-risk HPV type (hrHPV).", "While most hrHPV infections (90%) are controlled within two years [1], viral persistence may lead to malignancies.", "The hrHPV are responsible for ∼5% of all human cancers.", "Of the 14 different hrHPV types detected in cervical carcinoma, HPV16 and 18 are the most prevalent.", "HPV16 is the dominant type in all other HPV-induced cancers [2,3].", "HPV exclusively infect keratinocytes (KCs) in the basal layer of the epidermis and mucosal epithelia, through micro-wounds and abrasions.", "In the large majority of exposed but healthy individuals strong type 1 (IFNγ, TNFα, IL-2 producing) T-cell responses to the structural protein L1 as well as the early proteins E2, E6 and E7 are detected [4–7].", "Stimulation of the L1-specific immune response most likely occurs via the uptake of virions, produced during the productive phase of the infection, by the Langerhans cells that reside in the epidermis.", "T-cell responses to L1 are detected in healthy individuals and in patients with premalignant lesions or cancer [7].", "While they reflect a productive infection, they do not contribute to the control of viral infection as L1 is not expressed in the first few layers of the proliferating infected basal cells.", "In these layers, however, the early proteins E2, E6 and E7 are produced and immunity may be induced if these proteins are taken up by professional antigen presenting cells.", "However, type 1 T-cell responses to the early proteins are weak at best in patients with persistent infections.", "The HPV infected skin expresses the cytosolic DNA sensors STING, AIM2 and IFI16.", "HPV DNA can trigger the latter two resulting in the secretion of IL-1β and IL-18 [8].", "These cytokines mediate local and systemic immune responses to infection [9] and might be critical for early immune control of virus replication [10–12].", "Hence, there is a period in which an HPV infection may trigger a protective T-cell response, dominated by CD4+ T-cells [5,6,12,13,14•], but if this response is too weak or too late HPV may deploy several mechanisms to suppress the pathogen recognition receptor pathways [15–19,20••,21–23].", "Importantly, as HPV infection does not cause viremia or cell lysis, either intact immune signalling or minor trauma to the lesion [24] is crucial to induce protective immunity.", "Basal KCs express several pattern-recognition receptors (PRR) that can recognize viral DNA or RNA (Figure 1).", "PRR ligation results in the production of type I interferon and pro-inflammatory cytokine production through signaling via interferon regulatory factor (IRF) and nuclear factor of kappa-light-chain-enhancer of activated B cells (NFκB) activating pathways.", "Several genome-wide transcription studies reported that hrHPV types have found means to suppress PRR- and type I IFN-induced signaling pathways [22].", "Recently it was found that the cells in hrHPV-positive low-grade lesions display higher levels of E2 than normal hrHPV-infected cells, and this coincided with downregulation of STING [20••].", "Furthermore, hrHPV upregulated UCHL1, a deubiquitinase which was shown to inactivate TRAF3 and mediates the degradation of NEMO [15] and it may inhibit TLR9 expression [25].", "Notably, prednisolone- and hydroxychloroquine-mediated downregulation of TLR7 and TLR9, respectively, is associated with HPV infections [26].", "As a consequence, persistently hrHPV-infected cells will be less equipped to attract and activate the adaptive immune response via the production of interferons and cytokines (Figure 1).", "Especially, the secretion of the potent immune activating cytokine IL-1β is suppressed by hrHPV by targeting pro-IL-1β for destruction [27].", "However, even when the immune system manages to mount a type 1 T-cell response it will be difficult for these T cells to control a persistent infection as hrHPV adapts the infected cells to become less sensitive to immune control mechanisms (Figure 1).", "The virus interferes with T-cell recognition via the reduction of MHC class I and II expression but also by affecting the downstream signalling pathways of CD40, and the TNFα and IFNγ receptors which normally will mitigate the infection by arresting cell proliferation and inducing cell death, but will also lead to amplification of the local immune response via the direct (CD40, TNFα) and indirect (IFNγ) activation of NFκB (Figure 1).", "Persistently hrHPV infected cells display lower levels of STAT1 but this does not completely impair signalling [28,29••].", "Therefore, hrHPV also downregulate the interferon-induced transmembrane protein 1 (IFITM1) thereby preventing the upregulation of the antiproliferative gene RARRES1 [29••].", "A similar suppression of RARRES upregulation is noted after CD40 ligation [30].", "In addition, hrHPV evades TNFα-induced cell death of infected cells by the downregulation of RIPK3, a crucial regulator of necroptosis [29••].", "Local amplification of immunity by the secretion of cytokines and the attraction of immune cells is dampened by hrHPV through an increased expression of interferon-related developmental regulator 1 (IFRD1), which attenuates the transcriptional activity of NFκB via deacetylation of RelA [31••] as well as by interfering with downstream signalling of CD40, probably via the interaction of UCHL1 and TRAF6 [15,30].", "Finally, there is evidence that hrHPV-infected cells create a local immune suppressive microenvironment by altering the phenotype and function of local antigen dendritic cells [32] and the attraction of mast cells [33].", "Therapeutic vaccines aim to stimulate strong type 1 helper T-cell and cytotoxic T-cell responses (Th1/CTL) to attack infected cells.", "They come in many flavours [34] and are also developed to treat HPV-induced diseases [35].", "Clinical success has been obtained in women either with infected cells or with hrHPV-induced high-grade lesions.", "GTL001 in combination with the TLR7 agonist imiquimod topically applied to the vaccine site as adjuvant, stimulated E7-reactivity and a post-hoc analysis suggested increased and sustained clearance of HPV, albeit that the group size was small [36].", "Four different types of vaccines were tested for their capacity to treat hrHPV-associated high-grade cervical lesions (CIN2-3).", "The DNA vaccine VGX-3100 was shown to induce strong E6/E7-specific Th1/CTL responses [37••] and was subsequently tested in a large randomized placebo-controlled trial [38••].", "The spontaneous clearance rate of CIN2-3 was 30% and this was increased to 50% by vaccination.", "Post-hoc analyses revealed a relation between a clinical response and the strength of the vaccine-induced immune response [38••].", "Also the DNA vaccine GX-188E induced E6/E7-specific Th1/CTL responses that resulted in viral control and lesion regression in 7 out of 9 patients [39] while another (pnGVL4a-CRT/E7 DNA) failed to induce strong Th1/CTL reactivity or clinical reactivity exceeding the spontaneous clearance rate [40].", "GLBL101c, an orally administered bacterial vector vaccine expressing HPV16 E7 protein [41] did not lead to overt systemic immunity but HPV-specific T-cells were detected in the cervix.", "A downgrade of disease stage was found in 5 of 13 patients [41], just above the spontaneous clearance rate.", "Similarly, PepCan, an HPV16 E6 peptide-based vaccine with Candida skin test reagents as adjuvants induced T-cell reactivity in <50% of the subjects and there was no relation between immunity and lesion regression or an increase in clearance rate [42,43].", "The spontaneous clearance of HPV16-induced high-grade lesions of the vulva is less than 1.5% and treatment with the synthetic long peptide vaccine ISA101 considerably increased this percentage to more than 50% as shown in two subsequent medium-sized trials [44,45••].", "Clinical reactivity was strongly related to the strength of the vaccine-induced Th1/CTL response as found during the post-hoc analyses of the first trial [44,46] and confirmed as pre-defined marker in the second trial [45••].", "The general observation from these trials is that if a strong Th1/CTL response is evoked one has the best chance for a clinical response.", "This fits with studies showing that hrHPV increases the resistance of infected cells to the effects of type 1 cytokine mediated signals but does not make them insensitive [15,23–25,29••,31••,42].", "In addition, it should be appreciated that the viral gene expression changes during the progression of disease and this may impact on the immune evasive strategies deployed [22].", "For example, STING expression is regained in progressive lesions, consistent with the loss of E2 protein expression [23].", "Currently 20 different ongoing trials focus on the treatment of premalignant or cancerous lesions (Table 1).", "Bearing in mind that local immune suppression hampers the efficacy of therapeutic vaccines [34] there are a couple of trials attracting the attention.", "Two trials try to circumvent general immune suppression by vaccinating patients during cancer surgery or after successful standard treatment, aiming to prevent recurrences (NCT00002916; NCT02405221).", "In three trials vaccination is combined with chemotherapeutics that may alleviate immune suppression mediated by regulatory T cells (NCT02865135) or myeloid cells (NCT 02526316; NCT02128126) [47•,48•].", "Last but not least, activated T cells may express PD-1, which after engagement with PD-L1 on tumor cells or myeloid cells, suppresses their effector function.", "In one trial this is prevented by combining vaccination with the PD-1 blocking antibody nivolumab (NCT02426892).", "The high incidence of HPV infections, the quick clearance of infections in spite of HPV’s stealthy behaviour, and the detection of early protein-specific T cells in most healthy subjects while seroconversion is low, indicates that in general pathogen recognition of hrHPV occurs after which a protective T-cell response is launched.", "The production of IL-1β may be crucial for the activation of a strong T-cell response during hrHPV infection.", "IL-1β is important for the acute phase response and it also enhances the expansion, differentiation and tissue localization of CD4+ and CD8+ T-cell responses [49,50].", "However, polymorphisms in the IL-1 gene [12,51] and active downregulation of a network of IL-1β interconnected genes by hrHPV [16] as well as inhibition of IL-1β secretion at higher stages of disease [27] may stifle the development of protective type 1 T-cell responses in a minority of cases, with weak T-cell reactivity as result.", "In addition, hrHPV lowers the sensitivity of infected cells to key type 1 cytokines which otherwise will help infected cells to control the virus and it creates a local suppressive environment.", "This raises the bar for the type 1 T-cell responses to gain control of infection but therapeutic vaccines can stimulate type 1 HPV-specific T cell responses with a magnitude that readily exceeds the weak responses in patients and this is associated with regained control of hrHPV infection.", "In time, however, additional layers of immune suppression develop within the hrHPV-induced lesion necessitating combinations of vaccines with other treatment modalities to alleviate these suppressive mechanisms.", "Papers of particular interest, published within the period of review, have been highlighted as:", "• of special interest", "•• of outstanding interest" ]	[ "Immune detection of HPV requires pathogen receptor recognition or local trauma.", "Type 1 T-cell reactivity to the HPV early but not structural proteins is protective.", "HPV-infected cells display lower sensitivity to the effects of type 1 cytokines.", "Therapeutic vaccine-induced T-cell reactivity may regain control of infection.", "Combinations of treatment modalities are key to successful treatment of HPV lesions." ]
A Landscape of Pharmacogenomic Interactions in Cancer	Systematic studies of cancer genomes have provided unprecedented insights into the molecular nature of cancer. Using this information to guide the development and application of therapies in the clinic is challenging. Here, we report how cancer-driven alterations identified in 11,289 tumors from 29 tissues (integrating somatic mutations, copy number alterations, DNA methylation, and gene expression) can be mapped onto 1,001 molecularly annotated human cancer cell lines and correlated with sensitivity to 265 drugs. We find that cell lines faithfully recapitulate oncogenic alterations identified in tumors, find that many of these associate with drug sensitivity/resistance, and highlight the importance of tissue lineage in mediating drug response. Logic-based modeling uncovers combinations of alterations that sensitize to drugs, while machine learning demonstrates the relative importance of different data types in predicting drug response. Our analysis and datasets are rich resources to link genotypes with cellular phenotypes and to identify therapeutic options for selected cancer sub-populations.	[ "BIOC" ]	[]	[ "1300" ]	[ "Cancers arise because of the acquisition of somatic alterations in their genomes that alter the function of key cancer genes (Stratton et al., 2009).", "A number of these alterations are implicated as determinants of treatment response in the clinic (Chapman et al., 2011; Mok et al., 2009; Shaw et al., 2013).", "Studies from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) have generated comprehensive catalogs of the cancer genes involved in tumorigenesis across a broad range of cancer types (Lawrence et al., 2014; Tamborero et al., 2013b; Zack et al., 2013).", "The emerging landscape of oncogenic alterations in cancer points to a hierarchy of likely functional processes and pathways that may guide the future treatment of patients (Ciriello et al., 2013; Hanahan and Weinberg, 2000; Stratton et al., 2009).", "Clinical trials are complex and expensive, and pre-clinical data that helps stratify patients can dramatically increase the likelihood of success during clinical development (Cook et al., 2014; Nelson et al., 2015).", "Thus, pre-clinical biological models that, as much as reasonably possible, capture both the molecular features of cancer and the diversity of therapeutic responses are a necessity.", "Human cancer cell lines are a facile experimental model and are widely used for drug development.", "Large-scale drug sensitivity screens in cancer cell lines have been used to identify clinically meaningful gene-drug interactions (Barretina et al., 2012; Basu et al., 2013; Garnett et al., 2012; Seashore-Ludlow et al., 2015).", "In the past, such screens have labored under the limitation of an imperfect understanding of the landscape of cancer driver genes, but it is now possible to view drug sensitivity in such models through the lens of clinically relevant oncogenic alterations.", "Here, we analyzed somatic mutations, copy number alterations, and hypermethylation across a total of 11,289 tumor samples from 29 tumor types to define a clinically relevant catalog of recurrent mutated cancer genes, focal amplifications/deletions, and methylated gene promoters (Figure 1A; Tables S1A–S1D).", "These oncogenic alterations were investigated as possible predictors of differential drug sensitivity across 1,001 cancer cell lines (Figures 1B and 1C; Table S1E) screened with 265 anti-cancer compounds (Figures 1D and S1; Table S1F).", "We have carried out an exploration of these data to determine (1) the extent to which cancer cell lines recapitulate oncogenic alterations in primary tumors, (2) which oncogenic alterations associate with drug sensitivity, (3) whether logic combinations of multiple alterations better explain drug sensitivity, and (4) the relative contribution of different molecular data types, either individually or in combination, in predicting drug response (Figure 1E).", "We built a comprehensive map of the oncogenic alterations in human tumors using data from TCGA, ICGC, and other studies (Figure 1A; Table S1C).", "The map consisted of (1) cancer genes (CGs) for which the mutation pattern in whole-exome sequencing (WES) data is consistent with positive selection, 2) focal recurrently aberrant copy number segments (RACSs) from SNP6 array profiles, and 3) hypermethylated informative 5′C-phosphate-G-3′ sites in gene promoters (iCpGs) from DNA methylation data, hereafter collectively referred to as “Cancer functional events” (CFEs).", "We identified CFEs by combining data across all tumors (pan-cancer), as well as for each cancer type (cancer specific) (Tables S2A, S2D, and S2H).", "The WES dataset consisted of somatic variant calls from 48 studies of matched tumor-normal samples, comprising 6,815 samples and spanning 28 cancer types (Tables S1A–S1D).", "CGs were detected per cancer type by combining the outputs of three algorithms: MutSigCV, OncodriveFM, and OncodriveCLUST (Lawrence et al., 2013; Rubio-Perez et al., 2015; Tamborero et al., 2013a).", "This identified 461 unique pan-cancer genes (Table S2A).", "We further added nine genes identified as putative tumor suppressors (Wong et al., 2014).", "We mined the COSMIC database to identify likely driver mutations in 358 of the 470 CGs (Table S2B; Supplemental Experimental Procedures).", "Most tumors harbored only a few driver mutations (median n = 2, range 0–64), consistent with previous reports (Kandoth et al., 2013; Vogelstein et al., 2013).", "RACSs were identified using ADMIRE for the analysis of 8,239 copy number arrays spanning 27 cancer types (van Dyk et al., 2013) (Table S1D; Supplemental Experimental Procedures).", "In total, 851 cancer-specific RACSs were gained (286 segments) or lost (565 segments), with a median of 19 RACSs per tumor type (Table S2D).", "The median number of genes within each RACS was 15 for amplified regions and one for deleted regions.", "The majority of known driver gene amplifications (e.g., EGFR, ERBB2, MET, and MYC) and homozygous deletions (e.g., CDKN2A, PTEN, and RB1) were captured, with 320 RACSs (38%) containing at least one known putative cancer driver gene, in addition to 531 RACSs (62%) without known driver genes.", "A smaller pan-cancer set (due to overlap in RACSs across cancer types) was constructed by pooling these results, comprising 425 RACSs (117 amplified and 308 deleted) (Tables S2D–S2F).", "iCpGs were identified using DNA methylation array data for 6,166 tumor samples spanning 21 cancer types (Table S1D).", "We defined 378 iCpGs based on a multimodal distribution of their methylation signal in at least one cancer type (Tables S2H and S2I).", "This also established a discretization threshold used to define such regions as hyper-methylated in the cell lines (Table S2J; Supplemental Experimental Procedures).", "In total, our multidimensional analysis of >11,000 patient tumor samples identified 1,699 cancer-specific CFEs, which were further merged into 1,273 unique pan-cancer CFEs (Figure S2A).", "Next, we assessed the extent to which the mutational landscape of cancer cell lines captures that seen in primary tumors.", "We utilized a panel of 1,001 human cancer cell lines analyzed through WES (n = 1,001), copy number (n = 996), gene expression (n = 968), and DNA methylation (n = 957) (http://cancer.sanger.ac.uk/cell_lines) (Figure 1B) and which we reclassified according to the TCGA tissue labels (Figure 2A; Tables S1A and S1E).", "Molecular alterations identified in cell lines were filtered using the CFEs identified in the primary tumor samples, providing a set of clinically relevant CFEs for the cell lines (Figure 1C).", "Of the 1,273 pan-cancer CFEs identified in patient tumors, 1,063 (84%) occurred in at least one cell line, and 1,002 (79%) occurred in at least three (Figure 2A).", "This concordance was greatest for the RACSs (100% of 425; Table S2G), followed by iCpGs (338 of 378, 89%; Table S2J) and CGs (300 of 470, 64%; Table S2C).", "When considering cancer-specific CFEs, concordance was highest for CFEs occurring in at least 5% of patients (median of 86% of CFEs covered across cancer types; Figure 2A; Data S1A).", "Coverage of CFEs varied by cancer type, and when we include infrequent CFEs (occurring in < 5% of patients), this concordance is markedly lower for the majority of cancer types (median coverage = 46%; Figure S2B).", "CFEs absent in cell lines are reported in Table S2K.", "The correlation between the frequency of CFEs in cell lines and patient tumors was high for the majority of the cancer types and for all three classes of CFEs (Figures 2B and S2C; Table S2L; Supplemental Experimental Procedures).", "Using a simple nearest-neighbor classifier based on the presence of CFEs in cell lines and tumors across cancer types, we could correctly match the tissue of origin of cell lines to primary tumors (and vice versa) for 71% of the cases (27 out of 38 alteration profiles [randomly expected 1%]) (Figures S2D and S2E; Table S2M; Supplemental Experimental Procedures).", "This percentage increased to 81% and 92% (randomly expected 2% and 5%), when considering the second and fifth nearest-neighbors, respectively (Figure S2E).", "The frequency of alterations in 13 canonical cancer-associated pathways was highly correlated between cell lines and tumors of the same cancer type (median R = 0.75 across all 13 pathways) (Figure 3A; Table S3A).", "A previous hierarchical classification of ∼3,000 tumors identified two major subclasses: M and C class (dominated by mutations and copy number alterations, respectively) (Ciriello et al., 2013).", "We expanded this analysis by including methylation data and by jointly analyzing cell lines and tumor samples.", "This integrated analysis of 3,673 samples (composed of 1,001 cell lines and 2,672 primary tumors for which all three data types were available and that were positive for at least one of the 1,250 CFEs [Tables S3B and S3C]) yielded four classes referred to as M, H, CD, and CA (Table S3D; Supplemental Experimental Procedures).", "Class M is enriched for CG mutations, class H for hypermethylation of iCpGs, and classes CD and CA for deleted and amplified RACSs, respectively (Figures 3B and S3; Tables S3E, S3F, and S3H; Data S1B).", "We observed a high concordance between the predominant class of CFEs in primary tumors and cell lines of the same tissue type (80% of cancer types, exceptions being GBM, KIRC, and PRAD) (Figure 3C; Table S3G; Data S1B).", "Taken together, these results show that a sufficiently large panel of cell lines is able to capture individual clinically relevant genomic alterations, in addition to pathway alterations and global signatures of driver events.", "To investigate how CFEs detected in primary tumors impact drug response, we first mapped these on our panel of cell lines (Figure 1C; Tables S2C, S2G, and S2J).", "Cell lines underwent extensive drug sensitivity profiling, screening 265 drugs across 990 cancer cell lines and generating 212,774 dose response curves (median number of screened cell lines per drug = 878, range = [366, 935]; Figure 1D).", "This is an expansion on previous pharmacogenomic datasets (Barretina et al., 2012; Basu et al., 2013; Garnett et al., 2012; Seashore-Ludlow et al., 2015).", "The effect of each drug on cell number was used to model sensitivity as IC50 (drug concentration that reduces viability by 50%) or AUC (area under the dose-response curve) values (Tables S4A and S4B).", "Screened compounds included cytotoxics (n = 19) and targeted agents (n = 242) selected against 20 key pathways and cellular processes in cancer biology (Figure 1D; Table S1F).", "These 265 compounds include clinical drugs (n = 48), drugs currently in clinical development (n = 76), and experimental compounds (n = 141).", "We screened seven compounds as biological replicates and observed good correlation between replicate IC50 values with a median Pearson correlation (R) = 0.65 (0.78 for the compounds with most of IC50 values falling within the range of tested concentrations) and consistent classification of cell lines as sensitive or resistant to a compound (median Fisher’s exact test [FET] log10 p value = −26) (Figure S1).", "Cluster analysis based on AUC values confirmed that compounds with overlapping nominal targets or targeting the same process/pathway had similar activity profiles (Table S1G; Supplemental Experimental Procedures).", "We used three distinct analytical frameworks to define the contribution of CFEs to the prediction of drug sensitivity (Figure 1E).", "ANOVA was used to identify single CFEs as markers of drug response.", "Logic models identified combinations of CFEs that improve the prediction of drug response.", "Lastly, we used machine-learning algorithms to assess the contribution of each molecular data type (CGs, RACS, iCpGs, and gene expression) in explaining variation in drug response.", "For consistency, all analyses used IC50 values.", "We carried out a pan-cancer, as well as a cancer-specific, analysis (for those 18 cancer types of sufficient sample size, n > 15 cell lines).", "For pan-cancer ANOVA, the set of CFEs included 267 CGs, 407 RACSs, and three gene fusions (BCR-ABL, EWSR1-FLI1, and EWSR1-X).", "Overall, for the 265 compounds, we identified 688 statistically significant interactions between unique CFE-drug pairs (p value < 10−3 at a false discovery rate [FDR] < 25%; Figure 4A), with 540 pan-cancer and 174 cancer-specific hits (Figure S4A; Table S4C).", "A subset of 262 CFE-drug pairs was additionally defined as large-effect interactions (Figure 4A).", "The effect size was quantified through Glass deltas (Δs) and Cohen’s D (CD) (Supplemental Experimental Procedures).", "The majority of CFE-drug interactions was exclusively identified in either the pan-cancer or cancer-specific analysis (n = 662 of 688 significant interactions, 96%, and n = 254 of 262 significant large-effect interactions, 97%), with few overlapping interactions (Figure 4A; Table S4C).", "The effect size was frequently greater for the cancer-specific associations than for pan-cancer associations (CD > 1 for 100% and 30% of hits, respectively) (Table S4D).", "A possible explanation for this observation could be that cancer-specific associations, with fewer cell lines, require a larger effect size to be statistically significant.", "However, downsampled pan-cancer analyses confirmed that the increased effect size of cancer-specific associations is greater than expected by downsampling alone (Figures S4B and S4C; Supplemental Experimental Procedures).", "This indicates that sensitivity to many drugs is modulated by genomic alterations in the context of a defined tissue lineage.", "Overall, 233 of 674 (34%) CFEs were significantly associated with the response to at least one compound, and more RACSs (62%) were associated with response than were CGs (38%).", "The importance of these two classes of CFEs varied by cancer type and was related to their prevalence (Figures 3C and S4G).", "We identified significant associations for the majority of compounds (85%; n = 225 of 265).", "When compounds were classified by their nominal target into 20 specific biological processes (Figure S4H; Table S1F), CFEs best explained sensitivity to compounds targeting EGFR and ABL signaling, mitosis, and DNA replication and least explained sensitivity to compounds targeting TOR, IGF1R, and WNT signaling.", "For the latter, alternative non-genomic events may be the primary modulators of drug sensitivity.", "The proportion of cytotoxic and targeted compounds (Table S1F) associated with at least one significant large-effect interaction was similar (63% and 60%, respectively).", "However, compared to targeted agents, the significant interactions between CFEs and cytotoxics tended to be of a smaller effect size (average CD 0.96 vs. 1.32) and less significant (average –log10 p value 3.68 vs. 4.56).", "We performed ANOVA on randomly downsampled subsets of cell lines (500, 300, 150, and 60 cell lines) and evaluated our ability to retain the set of statistically significant associations.", "The number of associations exponentially decreased as the number of cell lines was reduced, with a loss of ∼80% of pan-cancer associations when using 500 cell lines (Figures S4D–S4F; Supplemental Experimental Procedures).", "This highlights the utility of using a large cell line collection to increase statistical power and to preserve representation of diverse genotypes and histologies.", "Among the individual CFE-drug associations, we identified many well-described pharmacogenomics relationships (Figure 4B).", "These included clinically relevant associations between alterations in BRAF, ERBB2, EGFR, and the BCR-ABL fusion gene and sensitivity to clinically approved drugs in defined tumor types, as well as associations between KRAS, PDGFR, PIK3CA, PTEN, CDKN2A, NRAS, TP53, and FLT3 with drugs that target their respective protein products or pathways (Figure 4B; Table S4C).", "Moreover, we observed a secondary T790M EGFR mutation in lung adenocarcinoma (LUAD) and resistance to EGFR-targeted therapies (Gefitinib and Afatinib) (Godin-Heymann et al., 2008) (Figure 4D), as well as resistance of NRAS mutated melanoma patients to a BRAF inhibitor (Figure 4B; Table S4C) (Su et al., 2012).", "A pathway-centric view highlighted the number of interactions between CFEs in cancer pathways (EGFR, ERK-MAPK, PI3K-MTOR, and DNA repair and cell-cycle-related pathways) and drugs targeting those CFEs (Figure S4I).", "For example, compounds targeting EGFR signaling showed potent activity in cells with EGFR and ERBB2 alterations, but were ineffective in cells with downstream alterations in ERK-MAPK signaling, such as mutant RAS.", "To explore the most important CFE-drug interactions, we focused on 262 associations with a large effect on drug sensitivity (p < 10−3, FDR < 25%, and Δ > 1, for both the cell line populations included in the test) (Figure 4C; Table S4C).", "For example, at the pan-cancer level, U2AF1 mutations associate with sensitivity to multiple FLT3 inhibitors, such as AC220 (p = 8.3 × 10−8, CD = 2.5), Sorafenib (p = 3.04 × 10−6, CD = 2.8), Sunitinib (p = 5.6 × 10−5, CD = 2.5), and XL-184 (p = 1.3 × 10−4, CD = 1.9); PTEN mutations associate with sensitivity to an AKT inhibitor in COAD/READ (p = 3.5 × 10−6, CD = 2.4).", "The chemotherapeutic Mitomycin C is widely used to treat BLCA, and here, we detect, in the BLCA specific analysis, a sensitizing interaction with mutations in TP53 (p = 9.9 × 10−5, CD = 2.8) that are highly prevalent in this cancer type.", "In LUSC cells, loss-of-function mutations in the DNA methyltransferase MLL2 are associated with sensitivity to the clinical anti-androgen Bicalutamide (p = 6.02 ∼ 10−4, CD = 3); the BCL-2 inhibitor, ABT-263, shows activity in COAD/READ cells that harbor focal amplifications of MET (p = 1.02 × 10−4, CD = 2.8) or FOXA1/CRNKL1 (p = 1.31 × 10−4, CD = 2.2), events found in almost 60% of colorectal tumors; and truncating mutations in the co-repressor of BCL6, BCOR, statistically interact (p = 2.04 × 10−5, CD = 3.5) with sensitivity to a PKC beta inhibitor in STAD (Figure 4D), and deletions of a RACS (2q37.3) containing MTERFD2 and SNED1 is associated with resistance to the HDAC inhibitor Vorinostat (p = 5.4 × 10−7, CD = 4; Figure 4D) in OV cell lines.", "Interestingly, 24 of the 262 associations are driven by RACSs that do not contain known cancer genes (Tables S4C and S2D).", "For these regions, the patterns of drug sensitivity may give clues as to the likely contained driver cancer gene(s).", "Many genomic alterations occur together or in a mutually exclusive way that suggests a biological function (Babur et al., 2015).", "We hypothesized that combinations of CFEs could, in some contexts, improve our ability to explain variation in drug sensitivity.", "We employed a computational approach termed “logic optimization for binary input to continuous output” (LOBICO) to find the optimal logic model combining CFEs to explain the IC50 values for a drug, for example, “if RAS or RAF mutated, then sensitive to MEK inhibition” (Knijnenburg et al., 2016).", "LOBICO binarizes the IC50s, labeling cell lines as sensitive or resistant, and uses these together with the continuous IC50s to find optimal models (Table S5C) (Supplemental Experimental Procedures).", "We employed 5-fold cross-validation (CV) to select the appropriate model complexity from a set of eight possible models, ranging from single CFE predictor models to complex multi-input models with up to four CFEs.", "We required solutions to have specificity greater than 80%.", "The input features included the CGs, RACSs, gene fusions, and binarized pathway activity scores derived from the basal gene expression profiles of the cell lines (Figure S5A; Tables S5A, S5B, and S5D).", "The latter is based on 11 transcriptional signatures of pathway activation (Parikh et al., 2010) (Table S5B; Supplemental Experimental Procedures).", "LOBICO was executed for each drug separately utilizing pan-cancer and cancer-specific molecular datasets.", "This led to the inference of 1,112 logic models (Table S5E).", "In the pan-cancer dataset we found that for 69% (182 of 265) of the drugs, the IC50s were better explained than expected by chance (p value < 0.05 and FDR < 5%).", "Across the cancer-specific datasets, on average, 24% of the drugs were explained by the inferred logic models (Figure 5A).", "We termed these logic models (182 from the pan-cancer dataset and 208 from the 18 cancer-specific datasets) “predictive models”.", "When considered together, the pan-cancer and cancer-specific LOBICO analyses identified predictive models for 208 out of 265 (78%) drugs.", "Importantly, for 85% of the 390 predictive models, a multi-input model achieved better performance than did the best single-predictor model (Figure 5B).", "Although the pan-cancer dataset produced the largest number of predictive models, the CV error was consistently higher than for cancer-specific datasets (Figure S5B).", "This is in agreement with the ANOVA analysis, where larger effect sizes were observed for the cancer-specific datasets.", "The response to drugs that target the p53 or ERK-MAPK pathway were especially well-predicted by LOBICO (Figure S5C).", "We observed that CGs had the largest role in explaining drug response, followed by RACSs and the pathway activities derived from gene expression (Figure S5A; Supplemental Experimental Procedures).", "The small number of pathway signatures had a disproportionately large effect in the logic models, showing that basal pathway activation scores provide relevant information to predict drug response beyond the genomic CFEs (Costello et al., 2014) (Figure S5D).", "LOBICO uncovered many known, as well as novel, associations (Table S5F).", "Figure 5C depicts a selection of particularly strong and consistent “and/or” combinations found for clinically approved drugs.", "For example, in the pan-cancer dataset, the “or” combination of KRAS or BRAF improved the precision and recall compared to single predictor models to explain cell line sensitivity to a number of MEK and RAF inhibitors (e.g., Trametinib in Figures 5C and 5D).", "In general, the “or” combinations led to models with higher recall (Figure 5C, right quadrants) as compared with the single-predictor model.", "For example, HNSC cell lines that have an EGFR amplification or a SMAD4 mutation account for 45% (10 out of 22) of cell lines sensitive to the ERRB2/EGFR inhibitor Afatinib, whereas considering only the EGFR amplified cell lines accounts for only 32% (7 out of 22) of the sensitive cell lines (Figure 5E).", "Conversely, “and” combinations led to models with higher precision (Figure 5C, left quadrants).", "For example, BRCA cell lines that lack a deletion of the FAT1/IRF2 locus and are TP53 mutant show increased sensitivity to the ERRB2/EGFR inhibitor Lapatinib.", "This is achieved at higher precision (57% instead of 45% for the single predictor model), but at a lower recall (80% instead of 100%) (Figure 5F).", "Collectively, LOBICO analysis highlights the importance of considering combinations of oncogenic alterations as biomarkers for drug response.", "We sought to validate our pharmacogenomic models using independent drug sensitivity datasets from the Cancer Cell Line Encyclopedia (CCLE) (Barretina et al., 2012) and the Cancer Therapeutics Response Portal (CTRP; second version) (Seashore-Ludlow et al., 2015).", "This analysis was for necessity restricted to only those compounds and cell lines shared with our own study (hereafter referred to as GDSC).", "The shared set consisted of 466 cell lines and 76 compounds from the CTRP study (Tables S4I–S4K) and 389 cell lines and 15 compounds from the CCLE study (Tables S4E–S4G; Supplemental Experimental Procedures).", "Validation was performed using IC50 values from the GDSC and CCLE studies and AUC values from the CTRP study (where IC50 values were not reported).", "We performed ANOVA on the overlapping set of cell lines/compounds.", "We validated 53% (19 of 36 on CTRP) and 86% (6 of 7 on CCLE) of the testable sensitivity associations identified in the GDSC, and 21% (6 of 29 on CTRP) and 0% (0 of 7 on CCLE) of testable resistance associations (p < 0.05, Fisher’s exact test CTRP: p = 8.1 × 10−9; CCLE: p = 0.01; Figures S4J and S4K; Tables S4H and S4L; Supplemental Experimental Procedures).", "A significant Pearson correlation of the CFE-drug interaction significance was observed between the GDSC dataset and the other two datasets (R = 0.86 for CTRP and R = 0.86 for CCLE; Figures S4J and S4K).", "Similarly, using LOBICO, we validated 44% (17 of 39) of testable models using the CTRP, including both single and multi-input models, and observed a significant Pearson correlation of the interaction significance between the two datasets (R = 0.96; Figures S5E and S5F; Data S1C).", "Thus, even within the relatively limited set of overlapping drugs and cell lines, resulting in reduced statistical power, we observed reasonable-to-good rates of validation for the set of pharmacogenomic interactions identified in our study, including a number of novel associations.", "Complete summaries of these comparisons are provided in Tables S4E–S4L and S5G, Data S1C, and Supplemental Experimental Procedures.", "To investigate the power of different combinations of molecular data to predict drug response, we built linear and non-linear models of drug sensitivity (elastic net [EN] regression and Random Forests [Costello et al., 2014]).", "As input features, we used CGs, RACSs, iCpGs, and gene expression data.", "In a pan-cancer analysis, the most predictive data type was gene expression, closely followed by the tissue of origin of the cell lines (Figure 6A).", "By comparison, genomic features (CG mutations and RACSs alterations) performed poorly.", "The predictive power of gene expression and tissue type was strongly correlated, while RACSs and CGs are less correlated with the tissue type (Figure S6A).", "This is consistent with the tissue specificity of gene expression (Ross et al., 2000).", "Next, we compared the most predictive data types in pan-cancer versus cancer-specific analyses (Figures 6B and 6C).", "For each drug, we identified the best-performing combination of data types and the corresponding model, referred to as the “lead model”.", "Notably, paired molecular data types contributed to the most lead models in both the pan-cancer (∼42% of all models) and the cancer-specific analyses (∼45% for all cancer types) (Figures 4B and 4C).", "In the pan-cancer analysis, all of the lead models use gene expression data (Figures 6D and 6E), but for 211 drugs (∼86%), the models are improved by including methylation, RACSs, CGs, or any combination of those additional data types.", "In addition, we identified 379 predictive (non-lead) models (∼17%) independent of gene expression (Figures S6B–S6E).", "In a cancer-specific analysis, the majority of lead models are based solely on genomics features (Figures 6D and 6E).", "For 120 cases (∼38%) the lead model is based on genomics alone (CGs and RACS).", "We found that genomics in combination with methylation provided an additional 117 lead models (∼37%), whereas genomics in combination with gene expression contributed 19 (∼6%).", "The remaining lead models use methylation alone (∼7%), gene expression alone (∼3%), or a combination of genomic, epigenetic, and transcriptomic features (12%).", "Therefore, in the context of a cancer-specific analysis, ∼74% (237 of 319) of lead models were explained by genomics, either alone or when combined with methylation (Figures 6D and 6E).", "Cancer cell lines are important tools for drug development.", "Here, we have extended previous efforts with the systematic expansion of the pharmacological, genomic, transcriptomic, and epigenetic characterization of 1,001 human cancer cell lines.", "These datasets can be investigated through the COSMIC and Genomics of Drug Sensitivity in Cancer Web portal (http://www.cancerrxgene.org).", "To the best of our knowledge, this is the largest and most extensively characterized panel of cancer cell lines and should enable a broad range of studies linking genotypes with cellular phenotypes.", "Our analysis of >11,000 patient tumor samples and the subsequent superimposing of salient cancer features on cell lines exemplifies how large-scale cancer sequencing can be used to empower biological research and maximizes the potential clinical relevance of the pharmacological models reported.", "The majority of CFEs identified from a broad range of tumor types is captured within a large cell line panel and often at a frequency similar to that observed in patient cohorts.", "However, the picture is far from complete; many CFEs occurring at low to moderate frequency (2%–5%) are represented by a single cell line or not at all, and coverage by cancer type is variable.", "As we enter an era of precision cancer medicine, where many drugs are active in small molecularly defined subgroups of patients (e.g., only 3%–7% of lung cancer patients harbor the drug sensitizing EML4-ALK gene fusion [Soda et al., 2007]), the scarcity of models for many cancer genotypes and tissues is a limitation.", "New cell culturing technologies enable derivation of patient cell lines with high efficiency and thus make derivation of a larger set of cell lines encompassing the molecular diversity of cancer a realistic possibility (Liu et al., 2012; Sato et al., 2011).", "Pharmacogenomic screens in cancer cell lines are an unbiased discovery approach for putative markers of drug sensitivity.", "We identified a wealth of molecular markers of drug sensitivity, including completely novel associations not easily explained with our current knowledge.", "With appropriate validation and follow-up studies, these putative biomarkers may aid patient stratification and help to explain the heterogeneity of clinical responses.", "Going beyond single gene-drug interactions, “logic” combinations of CFEs consistently perform better than single events in sensitivity prediction.", "Clinical support for this comes from the observation that BRAF mutant melanoma patients treated with BRAF inhibitors show heterogeneity of response that may be explained by the presence of additional molecular alterations (Chapman et al., 2011).", "Our analyses suggest that clinical studies in cancer patients should be designed to enable combinations of genomic alterations to be detected, which has implications for both trial size and the statistical approaches employed.", "We validated our pharmacogenomic models using independent datasets from the CCLE and CTRP.", "Consistent with previous reports, this demonstrated good consistency in the set of markers identifiable across these studies (Cancer Cell Line Encyclopedia Consortium, 2015) and lends additional support to the results presented here.", "However, our ability to validate some pharmacogenomic associations was restricted by the limited number of overlapping cell lines and compounds between these studies.", "Furthermore, the consistency between datasets is not perfect, and efforts toward standardization to reduce methodological and biological differences across the different studies are likely to improve future correlation between datasets.", "For many of our pharmacological models, the defining CFE is present in clinical populations at a frequency that would make testing in a clinical trial setting feasible (Figure 7).", "For example, the alkylating agent Temozolamide (used to treat glioblastoma multiforme) shows activity in MYC amplified colorectal cancer lines (present in 33% of primary tumors) (Figure 7A).", "Overall, we found that a median of 50% of primary tumor samples harbor at least one CFE, or logic combination of CFEs, associated with increased drug response; ranging from 0.63% (OV) to 83.61% (COAD/READ) (Figure 7; Tables S7A–S7C; Supplemental Experimental Procedures).", "This suggests that there are likely to be a number of molecular subtypes within many cancers that, following appropriate validation, could be tested in the clinical trial setting using these stratifications for treatment selection.", "Using machine learning, we determined that within each specific cancer type, genomic features (either driver mutations or copy number alterations) generated the most predictive models, with the addition of methylation data further improving our models.", "While informative in the pan-cancer setting, baseline gene expression data was less informative in the more clinically relevant tissue-specific setting.", "Prioritizing the design of diagnostics that deliver driver mutations, copy number alterations, and DNA methylation profiles might be the most cost effective means in the short-term to stratify patients for cancer treatment.", "The clinical development of molecularly targeted cancer therapies remains a formidable challenge.", "Our current analysis is restricted by the availability of patient genomic datasets, the cell lines and compounds screened, and methodological and biological variables, as well as the inherent limitation associated with the use of in vitro cancer cell lines.", "Nonetheless, our results represent a comprehensive attempt to describe the landscape of clinically relevant pharmacogenomics interactions in cellular models of cancer, complementing previous efforts (Barretina et al., 2012; Basu et al., 2013; Garnett et al., 2012; Seashore-Ludlow et al., 2015).", "The data resource and analyses described here should enable the matching of drug response with oncogenic alterations to provide insights into cancer biology and to accelerate the development of patient stratification strategies for clinical trial design.", "Genomic data for a panel of 1,025 genetically unique human cell lines were assembled from the COSMIC database.", "1,001 cell lines were included in this study (Table S1E).", "Variants and copy number alterations were identified as described in the Supplemental Experimental Procedures.", "Microsatellite instability data were assembled as detailed in the Supplemental Experimental Procedures.", "Gene fusions from a subset cell lines (∼700) were identified by targeted PCR sequencing or split probe fluorescence in situ hybridization (FISH) analysis (Table S2C).", "Variant data from sequencing of 6,815 tumor normal sample pairs derived from 48 different sequencing studies were compiled (Rubio-Perez et al., 2015).", "To aid in the analysis, the tumor data were reannotated using a pipeline consistent with the COSMIC database (Vagrent: https://zenodo.org/record/16732#.VbeVY2RViko).", "For primary tumors, raw data for 6,035 methylation samples, covering 18 tumor types, were downloaded from the TCGA data portal.", "For the cell lines, data were generated in-house as described in the Supplemental Experimental Procedures.", "In both cases, Infinium HumanMethylation450 BeadChip arrays were preprocessed using the R Bioconductor package Minfi.", "Only CpG site probes falling on the promoter region of the known genes were considered, i.e., TSS1500, TSS200, 5′ UTR, and 1st exon.", "Probes containing SNPs and non-specific probes, falling on sex chromosomes, and not associated with a gene were discarded.", "Methylation beta values of CpG islands were averaged across CpG sites.", "The selection of cancer-driver genes (together with the variant recurrence filter) of the recurrently copy-number-altered chromosomal regions and the informative CpG islands is detailed in the Supplemental Experimental Procedures.", "Cell line pellets collected during exponential growth in RPMI or DMEM/F12 were lysed with TRIzol (Life Technologies) and stored at −70°C.", "Following chloroform extraction, total RNA was isolated using the RNeasy Mini Kit (QIAGEN).", "DNase digestion was followed by the RNAClean Kit (Agencourt Bioscience).", "RNA integrity was confirmed on a Bioanalyzer 2100 (Agilent Technologies) prior to labeling using 3′ IVT Express (Affymetrix).", "Microarray analysis was performed as described in the Supplemental Experimental Procedures.", "All analyses evaluating the extent to which cell lines resemble primary tumors are detailed in the Supplemental Experimental Procedures.", "Experimental protocols used for compound screening are detailed in the Supplemental Experimental Procedures.", "Effects on cell viability were measured, and a curve-fitting algorithm was applied to this raw dataset to derive a multiparameter description of the drug response (half maximal inhibitory concentration (IC50),and area under the curve [AUC]) through a multilevel mixed model (Vis et al., 2016) (Supplemental Experimental Procedures).", "For each drug an ANOVA model was fitted to correlate drug response with the status of Cancer Functional Events (CFEs), as described in Garnett et al. (2012), implemented in GDSCtools (http//gdsctools.readthedocs.io) and detailed in the the Supplemental Experimental Procedures.", "The downsampling ANOVA simulation studies are detailed in the Supplemental Experimental Procedures.", "We applied the LOBICO (Knijnenburg et al., 2016) framework as detailed in the Supplemental Experimental Procedures.", "Machine learning models were computed as detailed in the Supplemental Experimental Procedures.", "Conceptualization, F.I., T.A.K., D.J.V., G.R.B., M.P.M., M.Sc., L.F.A.W., J.S.-R., U.M., and M.J.G.; Methodology, F.I., T.A.K., D.J.V., G.R.B., M.P.M., M.Sc., S.B., U.M., and M.J.G.; Software, F.I., T.A.K., D.J.V., M.P.M., M.Sc., T.C., H.L., and E.v.D.; Validation, F.I., T.A.K., D.J.V., M.P.M., N.A., S.B., H.L., P.G., and M.J.G.; Formal Analysis, F.I., T.A.K., D.J.V., M.P.M., and M.Sc.; Investigation, G.R.B., S.B., P.G., T.M., and L.R.; Resources, D.J.V., G.R.B., M.Sc., E.G., S.B., H.L., P.G., E.v.D., H.C., H.d.S., H.H., T.M., S.M., L.R., X.D., R.K.E., Q.L., X.M., J.W., T.Z., N.S.G., S.S., D.T., N.L.-B., P.R.-M., M.E., D.A.H., C.H.B., U.M., and M.J.G.; Data Curation, F.I., D.J.V., G.R.B., M.Sc., E.G., H.L., P.G., H.C., H.d.S., H.H., S.M., S.S., M.So., D.T., N.L.B., P.R.-M., L.F.A.W., J.S.-R., U.M., and M.J.G.; Writing – Original Draft, F.I., T.A.K., D.J.V., G.R.B., M.P.M., U.M., and M.J.G.; Writing – Review & Editing, F.I., T.A.K., D.J.V., G.R.B., M.P.M., M.Sc., N.A., L.F.A.W., J.S.-R., U.M., and M.J.G.; Visualization, F.I., T.A.K., M.P.M., M.Sc., and E.G.; Supervision, D.A.H., M.R.S., C.H.B., L.F.A.W., J.S.-R., U.M., and M.J.G.; Project Administration, F.I., U.M., and M.J.G.; Funding Acquisition, D.A.H., C.H.B., M.R.S., L.F.A.W., J.S.-R., U.M., and M.J.G." ]	[ "We integrate heterogeneous molecular data of 11,289 tumors and 1,001 cell lines", "We measure the response of 1,001 cancer cell lines to 265 anti-cancer drugs", "We uncover numerous oncogenic aberrations that sensitize to an anti-cancer drug", "Our study forms a resource to identify therapeutic options for cancer sub-populations" ]
Classifying Sustainable Development Goal trajectories: A country-level methodology for identifying which issues and people are getting left behind	How useful are the Sustainable Development Goals for conducting empirical analysis at the country level? We develop a methodological framework for answering this question, with special emphasis on the SDGs’ normative ambition of “no one left behind.” We first classify all 169 SDG targets and find that 78 incorporate an outcome-focus that is quantitatively assessable at the country level, including 43 through a systematic approach to establishing “proxy targets.” We then present a framework for diagnosing the embedded diversity of absolute and relative indicator trajectories in a harmonized manner, based on a country's share of its starting gap on course to be closed by the relevant deadline. In turn, we present a method for estimating the human consequences of falling short on targets, measured by the number of lives at stake and people's basic needs at stake. As a case study, we apply the framework to Canada, an economy not commonly examined in the context of global goals. We are able to assess a total of 61 targets through the use of 70 indicators, including 28 indicators drawn from the United Nations’ official database. Overall, we find Canada is on course to succeed on 18 indicators; to cover at least half but less than the full objective on 7 indicators; to cover less than half the required distance on 33 indicators; and to remain stagnant or move backwards on 12 indicators. Among indicators assessed, the country is only fully on track to achieve one SDG. Shortfalls suggest approximately 54,000 Canadian lives at stake and millions of people left behind on issues like poverty, education, intimate partner violence, and access to water and sanitation. Our diagnostic framework enables considerable, if only partial, quantification of a country's SDG challenges, recognizing the wide range of contexts for underlying data availability and societal problems.	[ "ECON", "SOCI" ]	[ "Canada", "Methodology", "Mortality", "Poverty", "Sustainable Development Goals", "United Nations" ]	[ "2002", "3303", "3305", "3312" ]	[ "Since their adoption at the United Nations (U.N.) in 2015, the Sustainable Development Goals (SDGs) have gained increasingly widespread traction as a normative policy framework.", "But what is the empirical relevance of the goals?", "A headline in the elite Economist newspaper once described the goals as “worse than useless,” criticizing the substantive breadth and rhetoric embedded across 169 targets spanning 17 diverse policy realms (“The 169 Commandments,” 2015).", "At the country level, it is not ex ante clear how useful the global political framework is for conducting empirical analysis relevant to the lives of real people.", "Our paper considers this question, with a special emphasis on the SDGs’ stated ambition of “no one left behind.”", "There are many analytical challenges embedded in translating the SDGs from diplomatic text to quantitative assessment.", "The goals and targets touch on a wide array of topics and disciplines, each of which is anchored in its own norms of measurement and reporting, making it difficult to distill trends and gaps in a standardized manner across issues.", "Moreover, there is no overarching empirical logic guiding all the goals.", "Target ambitions range from the absolute universal elimination of one problem to a proportionate domestic reduction of another.", "Meanwhile, many targets are quantitatively ambiguous or focused on process ambitions rather than policy outcomes.", "Uncertainty regarding a target’s intrinsic empirical aspirations risks hindering that target’s efficacy in helping to stimulate improvements in policy action.", "Our methodology addresses these challenges by producing a multi-step analytical framework to translate the SDGs from a U.N. framework to a country-level diagnostic tool.", "Specifically, we consider three questions from the country-level perspective where sovereign policy decisions are made.", "First, which of the SDG targets lend themselves to quantitative assessment?", "Second, how can the information embodied in a vast range of SDG indicators be coherently synthesized to identify which issues are lagging?", "Third, how can such a diagnosis be interpreted in terms of absolute human consequences, measured by the number of people who will be left behind on each issue if the relevant target is not achieved?", "We then apply this framework to a case study of Canada, an economy not commonly examined in the context of global goals.", "Our methodology uses a multi-step logic that aims to be applicable to any country and, subject to data availability, at the subnational level too.", "We attempt a “by-the-book” approach that follows the U.N.’s formal SDG architecture of goals, targets, and indicators as much as practical.", "In cases where the official U.N. language is quantitatively vague, we develop a logic of “proxy targets” to enable empirical assessment where viable.", "One value of this framework is that it allows a straightforward and transparent methodology for translating the normative aspirations of the SDGs into specific estimates of the consequences of falling short on the targets.", "It also helps draw attention to population-specific data gaps and policy gaps.", "Whereas some studies have interpreted the SDGs as a scale for comparing progress across countries (OECD, 2017; Sachs, Schmidt-Traub, Kroll, Lafortune, & Fuller, 2018), our approach considers the extent to which the SDGs can be implemented as a tool for tracking progress within countries, relative to each society’s own needs on each issue.", "The remainder of the paper is presented in four sections.", "Following this introduction, section two describes previous studies relevant for domestic SDG assessment.", "Section three presents the framework’s key methodological elements.", "Section four presents the framework results when applied to Canada.", "Section five concludes.", "A number of studies have conducted initial attempts at translating the intergovernmental SDG targets into empirical assessments.", "Much of the early SDG benchmarking research takes stock of starting baselines across countries and issues (U.N., 2018; WHO, 2017; World Bank, 2018).", "Other studies have considered questions of SDG target sorting and empirical benchmarking.", "For example, the Organization for Economic Cooperation and Development (OECD, 2017) identifies targets in which the outcome level is specified in the target language and then categorizes targets based on whether this outcome is defined in the same absolute terms for all countries or defined relative to each country’s starting baseline.", "It uses this target classification to assess domestic challenges across advanced economies.", "For SDG targets that are not quantified as written, the authors either substitute targets from other international agreements or set the relevant standard as the 90th percentile among OECD countries as of 2010.", "Other studies have identified ways to systematize measures of progress across the goals.", "For example, Nicolai, Hoy, Berliner, and Aedy (2015) looks at aggregate global trajectories out to 2030 for a sample of 17 indicators, one for each goal.", "It classifies each indicator based on the share of distance the world travels toward the target if recent trends continue.", "The European Union (EU, 2017) uses short- and long-term trends to assess the EU’s aggregate performance on a selection of indicators.", "For indicators with quantified outcomes defined either in SDG target language or EU strategy, indicators are classified into four categories, using the ratio of recent rate of progress to rate required to reach the target.", "For indicators without quantified outcomes, indicators are categorized by recent rates of progress.", "The Sustainable Development Solutions Network and Bertelsmann Stiftung (SDSN, Sachs et al., 2018) presents an important country-level trend analysis for a subset of indicators across goals and classifies each based on the share of distance traveled toward absolute global thresholds.", "To set values for target achievement, SDSN identifies a range of values bounded by the “technical optimum” (e.g. 100 percent access to basic sanitation) and an absolute threshold at which a country is deemed to have achieved the SDG, which is set at a different value (e.g. 95 percent access to basic sanitation).", "In situations where a country has surpassed the absolute threshold prior to the start of the SDG period, SDSN classifies them as having already met the target, including when the target language defines an outcome in relative terms.", "As an example of the latter, target 3.4 calls for a one-third reduction in pre-mature mortality from non-communicable diseases (NCD).", "SDSN classifies a country as having achieved the relevant standard if it is at or below an absolute threshold of 15 percent of 30-year-olds dying from cardiovascular diseases, cancer, diabetes or chronic respiratory diseases before their 70th birthday.", "Some studies focus on specific sectoral issues.", "On extreme income poverty, for example, Cuaresma et al. (2018) presents country-level trajectories out to 2030.", "The Global Burden of Disease, 2017 Collaborators (2018) estimates country-level progress towards 2030 on specific health-related indicators.", "UNICEF (2018) looks at projections for a sample of child-focused indicators.", "McArthur, Rasmussen, and Yamey (2018) examines maternal mortality and child mortality trends and identifies the approximate number of “lives at stake” if each country continues on its recent trajectory.", "Some national governments have included gap and trend analysis in their Voluntary National Reviews (VNR) presented at the U.N.’s annual SDG-focused High-Level Political Forum.", "Kindornay (2019) finds that 32 of 46 countries that presented in 2018 included some form of baseline or gap analysis.", "Some countries consider indicator trajectories.", "For example, Egypt’s VNR classifies multiple indicator trends per goal under three categories: positive change, negative change, and no change (Ministry of Planning, Monitoring, and Administrative Reform, 2018).", "Latvia similarly classifies recent trajectories but does so based on progress toward targets drawn from the SDGs and its own national sustainable development strategy (Cross Sectoral Coordination Centre of Latvia, 2018).", "The current study builds on previous research in multiple ways.", "Our approach presents a standardized filtering logic for assessing several dozen targets across all countries and at subnational levels.", "Adhering to the formal SDG target language and framing as closely as possible, we offer a method for quantifying and classifying SDG target trajectories relative to each country’s own situation, where appropriate, rather than to global aggregates.", "Consistent with the SDG aim of “no one left behind,” this also includes a literal interpretation of universal coverage targets where relevant.", "Our analytical framework is comprised of five key steps.", "First, we identify which SDG targets are quantitatively assessable at the country level.", "Second, in cases where the official U.N. language is quantitatively vague, we present an approach for establishing “proxy targets.”", "Third, we present a decision tree logic for identifying relevant data sources.", "Fourth, we classify forward-looking trajectories of several dozen corresponding indicators into a harmonized analytical framework.", "Finally, for targets focused on human outcomes, we demonstrate how shortfalls in trajectories to 2030, or corresponding SDG target year, can be translated into approximate numbers of lives and people’s needs at stake.1", "These steps are described further below and the Appendix includes target-specific details for readers interested in more detailed examination or replication of the results.", "To identify which targets can be used to assess progress, we use a filtering logic as outlined in Fig. 1.", "Of the 169 total SDG targets, we first identify those that are outcome-focused at the country level.", "An initial cut at this is provided by the official SDG framework, which distinguishes between outcome targets (numbered 1.1, 1.2, and so forth) and “means of implementation” (MOI) targets (lettered 1.", "a, 1.", "b, and so forth).", "Consistent with that structure, we filter out all “lettered” targets that focus on MOI.", "We also filter out all targets under Goal 17, a process-focused goal that seeks to “strengthen the means of implementation and revitalize the Global Partnership for Sustainable Development.”", "We then examine the remaining numbered targets to confirm whether they are outcome-focused at the country level.", "We find that 13 numbered targets are not outcome-focused or assessable at the country-level and filter them out of the core sample, reducing the number of examined targets down to 94.", "Among those filtered out, target 15.9, for example, aims to, “integrate ecosystem and biodiversity values into national and local planning.”", "This focuses on policy planning, so we classify it as a MOI target rather than an outcome target.", "Similarly, target 10.5 aims to, “improve the regulation and monitoring of global financial markets and institutions,” which we deem not to be outcome-focused at the country level.", "Some category determinations inevitably entail a degree of subjectivity, so the Appendix reports relevant details on our classifications for all numbered targets.", "The second branch of the decision tree of Fig. 1 shows how we next divide the 94 targets into two groups: those that are both quantified and measurable versus those that are not.", "Targets are considered quantified if their language includes either an explicit numerical target or an absolute verbal target, such as “conserve at least 10 percent of coastal and marine areas” (target 14.5) or “end hunger” (2.1), respectively.", "Targets are considered measurable if they have a clearly identifiable outcome and an objective direction for progress.", "For example, target 11.2 aims to “provide access to safe, affordable, accessible and sustainable transport systems for all.”", "We deem this to be conceptually quantified (i.e., access for all) but not measurable, because the target language is unclear as to how accessible and sustainable transport systems would be measured.", "Conversely, target 16.1 to “significantly reduce all forms of violence and related death rates everywhere” is conceptually measurable (i.e., the rate of violence should decline) but not quantified, since the amount of reduction to be achieved is unclear.", "These distinctions guide the identification of 35 targets that are outcome-focused, quantified, and conceptually measurable for any country as written.", "We deem 27 of these to be absolute targets, applying the same outcome standard to all countries, such as ending extreme poverty or reducing child mortality to no more than 25 deaths per 1000 live births.", "We classify the other eight as relative targets, such as cutting domestic poverty by half or reducing non-communicable disease mortality by one-third, whereby each country’s outcome objective is set in relation to its 2015 starting point.2", "Separately, we identify 59 targets where the official U.N. language is either not quantified or not measurable.", "For the special case of the least developed countries (LDCs), the corresponding breakdown at the second branch of the Fig. 1 decision tree adjusts to 37 and 57 targets, respectively, (rather than 35 and 59) since two targets are specifically quantified and measurable for the LDC context.3", "The third branch in the Fig. 1 decision tree applies to the 59 targets for which the U.N. framework language is not adequately quantified and measurable to be numerically assessable.", "For these cases, we adopt an expansive “proxy target” approach, aiming to apply a consistent logic that can allow country-level progress to be assessed wherever data permit.", "A first step in this direction is to identify any existing, equivalent national targets within the country of interest that are quantified and measurable.", "For example, target 7.2 is to “increase substantially the share of renewable energy” by 2030.", "The phrase “increase substantially” is quantitatively ambiguous, but in the Canadian context there is a relevant target in the country’s Federal Sustainable Development Strategy, “By 2030, 90% and in the long term, 100% of Canada’s electricity is generated from renewable and non-emitting sources” (ECCC, 2016).4", "In cases where we are not able to identify a corresponding national target, we propose a logic for establishing proxy targets, illustrated in Fig. 2.", "The horizontal axis segments targets by whether the official U.N. language defines a desired outcome level.", "The vertical axis segments by whether the official language includes a measurable outcome and objective direction of progress.", "As shown in the top-left quadrant, when targets are both measurable and quantified they are relatively straightforward to assess.", "In other cases, we attempt to assign a proxy benchmark, proxy indicator, or both, while recognizing the degree of subjectivity inherent in determining whether to interpret the wording of some targets as measurable or quantified.", "The Appendix shows our classification for each of the 59 relevant targets considered.", "The top-right quadrant returns to the example of target 16.1 on violence, which is measureable but not quantified as written, and for which we can assign a proxy benchmark for assessing outcomes.", "As a general approach, we define benchmarks as cutting a relevant problem by half by 2030 – in this instance the intentional homicide rate.", "However, for targets that aim to increase a metric but do not have a natural data ceiling, such as target 9.5’s aim of, “substantially increasing the number of research and development workers per 1 million people,” we assign a 50 percent increase as the proxy benchmark.", "For targets under Goal 5 on gender equality, we use gender parity as a quantified benchmark for equality.", "A total of 17 targets fall under this top-right quadrant.", "The bottom-left quadrant of Fig. 2 represents the cases where targets are quantified but not measurable, such as target 11.7’s aim to, “provide universal access to safe, inclusive and accessible, green and public spaces.”", "For seven such targets, we assign a proxy indicator, using official SDG indicators where practical.", "For example, target 11.7’s ambition of “universal access” to green and public spaces can be measured through a proxy indicator of the share of people living less than 10 min from a park or green space.", "When a target is neither quantified nor measurable, as reflected in the bottom-right quadrant, the establishment of a proxy target is particularly subjective.", "In 19 instances, we are able to set a proxy target using a combination of the above approaches.", "Target 16.6, for example, is to “develop effective, accountable and transparent institutions.”", "In this instance we use a Statistics Canada indicator on public confidence in the justice system and courts and assign a proxy benchmark of halving the share of the population without confidence by 2030.5", "For 16 targets, we deem it impractical to set a proxy because too much subjectivity is required.", "For example, target 12.2 is to, “achieve the sustainable management and efficient use of natural resources.”", "It is unclear what defines “efficient use” and the direction likely varies depending on which natural resource is considered, and in which context.", "Meanwhile, target 8.5 aims to, “achieve full and productive employment and decent work for all women and men.”", "In this case, the empirical standard for “full and productive employment” is unclear, especially in the context of debates about underemployment and “decent” wages.", "Fig. 3 shows the spread of our target classifications across the 17 SDGs.", "On some goals, such as Goal 3 for health and wellbeing, we identify several directly quantified and measurable targets.", "For others goals, such as Goal 9 for industry, innovation, and infrastructure and Goal 11 for sustainable cities and communities, we are only able to make assessments through the use of proxy targets.", "Overall, the additional use of proxy targets results in a total of 78 quantitatively assessable outcome targets.6", "The categorization of 35 quantified and measurable targets, 43 proxy targets, and 91 other forms of targets (here not assessed) applies generally across economies and levels of development.", "For least developed countries, the corresponding numbers are 37 quantified and measurable targets, 43 proxy targets, and 89 other forms of targets.", "For the assessable targets, we identify relevant data sources using another decision tree, as outlined in Fig. 4.", "Here we first examine whether a target has any relevant data for the country of interest in the U.N. SDG Indicator Global Database, which we use as the default data source to allow for comparable analysis across countries.", "Because this database and others are updated regularly, Fig. 4 does not include a specific breakdown of how many targets fall under each branch of the tree.", "Next, we identify whether there are adequate observations to conduct recent trend analysis for the country, defined as either (i) having at least two observations since 2000, ideally spaced 10 years apart, or (ii) the most recent observation hitting an indicator ceiling that achieves the relevant target outcome (e.g., 100 percent access to basic drinking water).", "If the answer is no at either of the first two branches of the tree in Fig. 4, then the next step is to consider alternate sources, including the relevant national statistics agency.", "In doing so, we prioritize using indicators that match those in the SDG framework.", "We aim to identify at least one indicator with data for each assessable target, although for nine targets we use two indicators to assess distinct outcomes that are embedded in the target language.", "For example, target 3.4 has two official indicators assessing two distinct issues: mortality from non-communicable diseases and suicide mortality rates.", "For targets with multiple official indicators, we prioritize using an indicator that assesses change in outcomes at the country-level and, where applicable, quantifies people being left behind.", "For example, on target 12.4 on sound management of chemicals and waste, we prioritize indicator 12.4.2 on hazardous waste generated per capita rather than 12.4.1 on parties to international agreements that meet their commitments and obligations in transmitting information.", "Here x represents the indicator value, t represents a recent index year, and n indicates the number of years prior to t, ideally 10 years.", "We then compare trajectories to each target’s desired outcomes, with particular attention to the SDG philosophy of “no one left behind.”", "As previously mentioned, for targets that commit to a desired outcome for “all” people or “universal” coverage, we interpret this literally as 100 percent of the population.", "Among other reasons, this is because each percentage point of population can represent a large number of lives.", "For example, if a country’s 2030 population is likely to be 50 million people, then every percentage point gap implies 500,000 people left behind.", "Even 98 percent population coverage on an indicator, which might generally be considered “success,” would still imply one million people left behind.", "We classify each indicator trajectory under a common standard, using four categories to inform consideration of which issues are making progress and which ones are being left behind.", "Each category is based on the share of starting distance to the target that will be covered on current trajectory: (i) On track, meaning already achieved or on track for target achievement; (ii) Acceleration needed, meaning the country is currently on course to cover more than 50 percent but less than 100 percent of its starting distance to the target; (iii) Breakthrough needed, meaning the country is on course to cover between 0 and 50 percent of its starting distance to the target; and (iv) Moving backwards, meaning the most recent available trend is negative.8", "For indicators focused on human outcomes, the final step in the methodology is to translate trajectories into estimates of the absolute numbers of people left behind.9", "We do so by calculating the approximate difference between the number of people affected on current trajectory and those affected under a trajectory that reaches the SDG target.10", "For each indicator, calculations are based on the relevant demographic group, ranging from total population to narrower reference points like children aged 2 to 4 for children underweight, or females aged 15 and older for intimate partner violence.", "For this portion of the analysis, we further segment targets into two conceptual categories: life-and-death targets like maternal mortality, traffic deaths, and homicides; and basic needs targets like food security, literacy, and access to water.", "For life and death targets, we estimate the cumulative number of “lives at stake” from a present year, here 2019, through to the target deadline, usually 2030.", "For basic needs targets, we estimate the number of people’s needs at stake in only the final target year, in order to avoid double counting.", "As with any analytical methodology, our approach has some inherent tradeoffs embedded.", "First, because we implement a literal interpretation of the SDG normative ambition to leave no one behind, our approach draws attention to shortfalls, however small, in reaching targets, rather than celebrating relative proximity to achieving an objective.", "For example, if access to some basic service is on course to climb from 99.4 percent in 2015 to 99.5 percent by 2030, then it is classified as a source of concern with a “breakthrough needed,” rather than an achievement, since less than half the remaining distance to the finish line of 100 percent would be covered.", "Similarly, if access to the same basic service had declined from 99.6 to 99.5 percent coverage in recent years, the target falls under the most problematic category of “moving backwards,” instead of something like “still close.”", "Second, in instances where there are multiple options to choose from in selecting an indicator to assess a target, the choice of one indicator over another might provide different impressions of how a country is doing.", "For example, for target 3.3 on infectious diseases, we use the official indicator of tuberculosis incidence, due to its ongoing relevance across all countries.", "Using malaria incidence, another official indicator, might produce a different result, depending on its relevance to a particular country’s disease burden.", "Third, targets anchored in relative domestic benchmarks risk conveying a negative narrative on indicators making considerable absolute gains but modest relative gains.", "To illustrate figuratively, if one indicator starts the SDG period 100 km from its target and only covers 40 km in 15 years, then this covers less than half the distance required and would be categorized under Breakthrough needed.", "Meanwhile, another indicator that starts the period 10 km away from its target and is on course to cover only 6 km, for a 60 percent gain, is categorized more positively as Acceleration needed.", "Fourth, because we use a linear extrapolation for a number of trajectories (those not related to mortality or economic growth), recent fast-moving trends might overlook forthcoming “last mile” challenges en route to universal coverage and thereby risk overestimating current trajectories for 2030.", "Using a logistic function or similar adjustment would require inserting ad hoc assumptions regarding inflection points in basic needs trend lines, so we instead adhere to the straightforward calculations as described above.", "To demonstrate the types of insights generated by our methodology, the following section applies the analytical framework to Canada.", "Although some readers might consider Canada to be a surprising case study for the issues, due to its higher values on many socioeconomic indicators than most low- and middle-income countries, it still grapples with many challenges of poverty and exclusion, most prominently among its indigenous peoples.", "The country has long fallen short, for example, in achieving universal access to basic drinking water.", "In line with the intentionally universal nature of the intergovernmental policy agenda, many SDG targets are also set relative to the domestic nature of each country’s challenge, such as its own national poverty line, so a methodology needs to be able to accommodate cross-country variations in this regard.", "Moreover, there are some issues, like greenhouse gas emissions per capita and protection of coastal areas, on which Canada faces much bigger absolute challenges relative to many countries.", "Analytically, the country’s relatively good data availability also permits us to consider which types of insights can be generated through our methodology, which would be untestable in, for instance, an extremely resource-constrained country with no official statistics.", "Implementing the methods described above, we identify relevant data for 61 outcome targets, using 70 indicators available as of March 2019.", "Twenty-eight of these indicators are drawn from the official U.N. SDG Indicator Global Database.", "Of these 28 indicators, Canada is missing trend data for two indicators but hits a relevant data ceiling.", "The other 42 indicators are drawn from complementary sources, including official Canadian government sources, as all described in the Appendix.", "In considering potential data constraints to applying our framework to other countries, we note that Canada is not unique in at least some aspects of its data availability.", "It is beyond the scope of this study to look at all potential domestic data sources for all countries, but if one looks at the Group of 20 countries as a relevant cross-section, then of the 26 indicators for which Canada has trend data available in the U.N. SDG Indicator Global Database as of March 2019, all G-20 countries have relevant trend data for at least 20 indicators, and four countries – Argentina, Italy, Mexico, and Turkey – have trend data for the same 26 indicators.", "Some developing G-20 countries also have more indicators available in the U.N. database than Canada does.", "As our first main empirical result, Table 1 presents a goal-by-goal summary classification of all 70 indicators examined for Canada.", "Solid circles represent indicators for targets that are quantified and directly measurable as written.", "Hollow circles represent indicators for targets that are assessed by a proxy measure.", "The Appendix provides each indicator’s underlying numerical values and corresponding classification.", "Overall, the table suggests that Canada is so far only fully on track for one of the first 16 SDGs, Goal 1 on ending poverty.", "For Goals 2 through 16, the country requires faster progress on at least one indicator, even if for many indicators the absolute distance to the SDG benchmark is small.", "In total, the country is on track for 18 indicators; requires acceleration on 7; needs a clear breakthrough on progress on 33; and requires a reversal of trends on 12.", "While Canadian society has undoubtedly achieved success on many fronts, efforts are still needed to cover a “last mile” of success on many issues and to achieve faster overall progress on others.", "The “On track” column on the right of Table 1 shows the positive aspects of the results.", "Before the establishment of the SDGs in 2015, Canada had already surpassed absolute global standards for targets including extreme income poverty (under Goal 1), neonatal mortality (Goal 3), and maternal mortality (also Goal 3), and had achieved universal access to services like social protection (under Goal 1), modern energy (Goal 7), and legal identity (Goal 16).", "Canada’s current trajectory also places it on track to meet multiple targets including halving the share of domestic poverty (under Goal 1), and achieving universal access to early childhood education and universal upper secondary graduation rates (under Goal 4) by 2030.", "Issues falling under this column suggest related policy approaches have generally been working well.", "The “Acceleration needed” column captures indicators that are making good but not quite enough progress to achieve the targets.", "For example, Canada requires acceleration to meet targets on preventing marine pollution (under Goal 14) and increasing renewable energy generation (under Goal 7).", "These are issues where targeted efforts might be needed to “nudge” efforts toward greater success (Sunstein & Thaler, 2008; Biggs & McArthur, 2018).", "At the other end of the spectrum, the far-left column of Table 1 draws attention to national challenges where indicators have been moving backwards.", "Under Goal 2, for example, indicators of food insecurity and children overweight have been worsening.", "Remarkably, reported access to drinking water (under Goal 6) has recently declined, the problem being particularly concentrated among indigenous people.", "Under Goal 4 on education, the proportion of lower secondary students who lack basic numeracy skills has been increasing.", "The “Breakthrough needed” column reflects indicators that are either stagnant or making slow progress toward the targets.", "National breakthroughs are needed for Canada to achieve gender equality, Goal 5, as measured by the wage gap, gender disparity in unpaid work, violence against women, early marriage, and women in managerial positions.", "For indicators falling under either the Breakthrough needed column or the Moving backwards column, recent policy approaches appear not to have been working well enough, so new strategies are likely required.", "In addition to reviewing results by column category, our methodology enables mapping of diverse issue-specific dynamics within each goal domain.", "For example, the range of issues encompassed in Goal 3 draws attention to Canada’s mixed trajectories for health and well-being.", "Mortality from non-communicable diseases is declining, although needs acceleration in order to achieve a one-third reduction by 2030.", "Faster progress is also needed to reduce substance abuse and to cut, by 2020, traffic deaths by half.", "The country is nearly but not quite on track to achieve universal coverage of nine key health interventions by 2030.", "A breakthrough is required on suicide mortality, infectious diseases like TB, and universal access to reproductive health services.", "Meanwhile, Canada’s outlook on environmental issues is mixed.", "The federal government has recently established, and made concrete steps toward meeting, SDG-consistent targets for protecting its uncommonly large land and marine areas by 2020, although faster progress is still needed to achieve desired outcomes on both fronts.", "Meanwhile, the country seems to be on course to end overfishing and keep forest harvests within sustainable levels, but a breakthrough is required to halt the loss of biodiversity.", "On climate change, Canada has some of the world’s highest per capita emissions and requires breakthrough rates of progress to meet its own 2030 emissions targets, again despite recent policy advances.", "Relevant indicators also show the need to increase energy efficiency and the share of renewables in energy consumption.", "In light of Canada’s international reputation for good governance, the results under Goal 16 for peace, justice, and strong institutions offer potential surprise.", "Many aspects of the country’s public institutions are strong, but only 57 percent of the population has clear confidence in the justice system and courts.", "Indicators are moving in the wrong direction for reported sexual violations against children and unsentenced detainees as a share of the prison population.", "Future research focused on Canada’s domestic Goal 16 challenges would clearly be valuable.", "Our methodology also permits a deeper dive on such issues at the subnational level.", "As an illustration, Table 2 maps status on one component of SDG target 3.4, major cardiovascular disease mortality, for each of Canada’s ten provinces and three territories.", "Whereas the country as a whole needs acceleration to achieve the overall NCD mortality target, it is on track to achieve a one-third reduction in the cardiovascular disease mortality component.", "Unpacking the national trend by geography, fully seven provinces and two territories are currently off track.", "The results suggest that the Northwest Territories and Nunavut merit particular attention due to slow and even backwards rates of progress.", "These two territories each have populations of less than 50,000 people, but more than half of each population is comprised of indigenous people, drawing attention to Canada’s unique historical challenge in supporting relevant communities.", "Further national disaggregation by location, gender, indigenous status, income group, age, immigrant status, and disability status can reveal similar insights wherever data permit.", "As an empirical reference point, our national results for Canada can be compared to those produced by SDSN (Sachs et al., 2018).", "Some findings are a naturally direct match.", "For example, on targets 3.1 for maternal mortality and 3.2 for neonatal mortality, both we and SDSN identify Canada as already meeting the absolute thresholds identified in the targets.", "On target 6.2 for access to safely managed sanitation, SDSN classifies Canada as “Stagnating” or “increasing at a rate below 50% of the growth rate needed to achieve the SDGs,” which is similar to our classification of Breakthrough needed.", "Meanwhile, one important source of difference between our results and those of SDSN is our literal interpretation of “no one left behind” for absolute targets.", "As mentioned earlier, SDSN uses a lower threshold for targets aiming at universal coverage.", "For example, under target 6.1 for access to drinking water, SDSN classifies high-income and OECD countries as achieving the SDG if they have at least 95 percent of the population using safely managed water services and classifies all other countries as achieving if they have 98 percent or more using at least basic water services.", "While SDSN does not classify a drinking water trend for Canada, World Bank (2019) data reports Canada’s 2015 value for basic water services as above the SDSN threshold but short of universal access and moving backwards.", "We interpret the SDG target to require fully 100 percent coverage and our approach draws attention to both the gap and the trajectory.11", "Recognizing the importance of real-time measurement for this issue in Canada and other contexts, in light of the large amount of Canadian public attention focused on shortfalls in drinking water access for many of the country’s indigenous communities, we believe our methodology aligns well with the “no one left behind” intention that underpins the SDGs.", "A second key source of difference in results is anchored in our by-the-book treatment of relative targets as proportional objectives for each country, rather than common absolute objectives across countries.", "To illustrate again with target 3.4 on NCDs, as mentioned earlier, SDSN defines SDG achievement as meeting an absolute global mortality threshold, which Canada has already surpassed, whereas our method sets Canada’s benchmark relative to its own initial baseline.", "Our calculations indicate that Canada is not yet on course to reduce its NCD mortality rate by one-third by 2030, and we therefore classify the relevant indicator as Acceleration needed.12", "The findings produced by our methodology do not amount to predictions, nor a suggestion that an assessed country can or cannot meet the SDGs.", "Instead, the results draw attention to a country’s gaps: the issues and people that are currently being left behind amid a society’s pursuit of economic, social, and environmental progress.", "We next translate the gaps on a subset of outcome trajectories into their absolute human consequences.", "We stress that these findings are only approximate estimates, meant to demonstrate a transparent quantitative methodology by which analysts and policymakers could consider the people-focused implications of intergovernmental commitments.", "To that end, Table 3 presents the estimated number of people left behind for a cross-section of targets that are measurable and for which Canada is not currently on track.", "The right-side column indicates the relevant reference population.", "The 2017 U.N. population projection suggests that, by 2030, Canada will have a total population of 40.6 million people, up from 37.6 million in 2020, the deadline year for the traffic death target.", "Across life and death targets, we estimate that current shortfalls translate to more than 54,000 Canadian lives at stake between 2019 and 2030.", "This includes 44,000 lives lost to gaps in reducing premature mortality from non-communicable diseases.", "On suicide, the shortfall translates to 8000 lives at stake.", "On traffic deaths, Canada has been making progress toward the 2020 target deadline, but shortfalls in cutting mortality by half will amount to 900 additional lives lost in 2019 and 2020.", "Meanwhile the homicide rate has seen little progress, and the shortfall toward achieving a proxy benchmark of 50 percent reduction by 2030 translates to an additional 2000 deaths.", "For measures of basic needs in Table 4, we estimate the number of people’s needs at stake in the final target year, if trajectories fall short of the desired outcome, again separating out results for proxy targets.13", "Unlike for lives at stake, the numbers in Table 4 are not strictly comparable from one row to the next because indicators are measured relative to different reference populations, as again listed in the final column.", "Access to water, for example, is measured relative to the entire population of Canada, whereas children overweight is reported only for children aged 2–4.", "The results in Table 4 show that, although Canada is often close in percentage point terms to achieving many of the SDG targets, the shortfalls frequently translate to millions of people left behind.", "The extrapolation of recent trends implies more than one million people without access to basic drinking water by 2030 and almost eight million without safely managed sanitation services.", "On education, approximately 3.2 million Canadians aged 15–79 might lack core literacy skills by the same year, while more than 6.4 million might lack core numeracy skills.", "As an indicator for local quality of life, more than 3.7 million people would not have access to a park or green space within ten minutes of their home.", "If hunger trends continue, more than 4.4 million people in Canada will suffer from moderate or severe food insecurity in 2030.", "Concurrently, more than 29 percent of children aged 2–4 are on trend to be overweight by the same year.", "This is equivalent to 356,000 children and implies much greater overall numbers of Canadians of all ages struggling with overweight or obese status over the coming decade.", "The results also draw attention to the number of Canadian women and girls who will be left behind if recent trends continue.", "As a general proxy for gender discrimination, we interpret the share of women in managerial positions as representative of overall barriers to women’s equal access to leadership positions in society.", "On current trajectory, only 36 percent of managers in Canada will be women in 2030, far short of half.", "Extrapolating this gap across society implies 5.5 million Canadian women and girls being excluded from equal opportunities.", "On the issue of intimate partner violence, the shortfall to elimination translates to more than 900,000 women in 2030.", "This paper began by asking how useful the SDGs are in informing country-level empirical analysis across economic, social, and environmental indicators.", "We pursue this question with an aim of identifying a quantitative methodology for assessing which people and issues are being left behind.", "We provide a tractable analytical framework for generating answers to these questions, adhering as much as practical to the formal U.N. targets, indicators, and database.", "Our results can inform the ongoing evolution of methodological debates regarding best approaches to SDG measurement.", "Of the 169 SDG targets, we find that many, although not a majority, are useful for empirical assessment.", "Specifically, we classify 35 targets as directly outcome-focused, quantified, and measurable at the national level for all countries.", "Another two targets meet the same criteria for LDCs.", "We identify another 43 targets that are plausibly assessable across all countries through the use of “proxy” benchmarks and indicators.", "This yields a total of 78 assessable SDG outcome targets at the country level.", "That slightly less than half the SDG targets are assessable at the country level forms something of a Rorschach test: some readers might see this as a cup half full of assessable targets; others might see it as a cup half empty of missed opportunities.", "As a case study, we apply our methodology to Canada.", "In attempting to identify data sources to inform trajectory analysis, we are only able to identify relevant data for 61 targets, using 70 indicators, of which 28 indicators can be directly sourced from the U.N. SDG Indicator Global Database.", "Although it might be reasonable to presume that Canada is close to a global upper bound in terms of SDG data availability, its indicators drawn from the U.N. database have similar availability across G-20 countries.", "But it is again a matter of interpretation as to whether 61 targets and 70 indicators amount to large or small numbers in the SDG context.", "They represent less than a third of all SDG targets and indicators but still add up to several dozen measures of progress.", "We apply a classification scheme to harmonize and distill trajectory diagnoses across all indicators, including those aiming at absolute global targets and those aimed at domestically-referenced relative targets.", "We classify each indicator under one of four categories – On track, Acceleration needed, Breakthrough needed, or Moving backwards – based on the share of the initial target gap that the country is on course to address by the relevant SDG deadline.", "The categorization of indicators can help inform policy making.", "Issues that are on track likely merit the continuation of recent approaches.", "Those with acceleration needed might need a policy tweak or targeted effort to close the remaining gap.", "Indicators that are relatively stagnant and register under breakthrough needed likely merit a change in strategy.", "Indicators that are moving backwards require a turnaround strategy, potentially requiring a wholesale new approach from all relevant stakeholders.", "In the Canadian context, we find 18 indicators to be on track for success, 7 requiring acceleration, 33 requiring a breakthrough, and 12 moving in the wrong direction.", "Overall, looking across our assessed indicators, Canada is only wholly on track for one of the first 16 SDGs: Goal 1 on poverty.", "Despite the country’s many notable successes, it exhibits a pattern of issues and population segments being consistently left behind.", "This type of trajectory analysis could help researchers and policy leaders identify priority areas for focusing attention on the need for change.", "The final step of the framework is to estimate the human consequences of SDG shortfalls if recent trends continue.", "In the Canadian case study, our results suggest that more than 54,000 lives are at stake if trajectories continue to fall short of SDG targets.", "For measures of basic needs at stake relative to SDG achievement, shortfalls might imply more than 4.4 million people subject to food insecurity, more than 6.4 million adults lacking core numeracy skills, and more than 900,000 women subject to intimate partner violence.", "These estimates should not be interpreted with false precision, since they are based on a number of methodological assumptions in extrapolating recent trends.", "But the results do offer a practical approach to translating abstract statistical percentages in to specific numbers of people.", "We show how corresponding analysis can be conducted for indicators at the subnational level.", "In Canada, there are tremendous variations across geographies and demographic groups.", "Among the considerable populations currently being left behind, the SDGs draw particular attention to the profound and longstanding challenges still faced by indigenous people in Canada.", "Our methodology is not able to assess status on all SDG-relevant issues, often because the relevant target is not defined in a clear or relevant manner.", "For example, the targets on industry and infrastructure are not ideal for assessing performance in an advanced economy, nor are many of the targets on decent work and economic growth.", "Some countries might want to set their own SDG targets in these realms.", "Future research might also consider more refined approaches to establishing proxy targets.", "Overall, this study shows that, despite data gaps and limitations, a subset of assessable and outcome-focused SDG targets offer a useful framework for conducting an empirical diagnosis of country-level economic, social, and environmental trends.", "The relevance for empirical diagnosis in a high-income country like Canada suggests a more universal applicability across countries grappling with lower general levels of development and greater intensities of human deprivation.", "Ideally, a quantitative distillation of diplomatically-defined challenges can then inform necessary policy debates on how best to solve problems if, in fact, people and issues are not to be left behind." ]	[ "A harmonized methodology can translate SDG targets into an empirical framework pertinent to each country’s challenges.", "Of 169 SDG targets, 35 include quantified and measurable country-level outcomes; 43 are assessable through proxy targets.", "A clear SDG diagnostic framework can distinguish between policies that are succeeding and those meriting a new approach.", "A methodological focus on “no one left behind” suggests even Canada is only fully on track for one of the 16 SDGs assessed.", "The human scale of SDG shortfalls can be estimated; Canada has approximately 54,000 lives at stake." ]
Identification of the presence of ischaemic stroke lesions by means of texture analysis on brain magnetic resonance images	Background: The differential quantification of brain atrophy, white matter hyperintensities (WMH) and stroke lesions is important in studies of stroke and dementia. However, the presence of stroke lesions is usually overlooked by automatic neuroimage processing methods and the-state-of-the-art deep learning schemes, which lack sufficient annotated data. We explore the use of radiomics in identifying whether a brain magnetic resonance imaging (MRI) scan belongs to an individual that had a stroke or not. Materials and methods: We used 1800 3D sets of MRI data from three prospective studies: one of stroke mechanisms and two of cognitive ageing, evaluated 114 textural features in WMH, cerebrospinal fluid, deep grey and normal-appearing white matter, and attempted to classify the scans using a random forest and support vector machine classifiers with and without feature selection. We evaluated the discriminatory power of each feature independently in each population and corrected the result against Type 1 errors. We also evaluated the influence of clinical parameters in the classification results. Results: Subtypes of ischaemic strokes (i.e. lacunar vs. cortical) cannot be discerned using radiomics, but the presence of a stroke-type lesion can be ascertained with accuracies ranging from 0.7 < AUC < 0.83. Feature selection, tissue type, stroke subtype and MRI sequence did not seem to determine the classification results. From all clinical variables evaluated, age correlated with the proportion of images classified correctly using either different or the same descriptors (Pearson r = 0.31 and 0.39 respectively, p < 0.001). Conclusions: Texture features in conventionally automatically segmented tissues may help in the identification of the presence of previous stroke lesions on an MRI scan, and should be taken into account in transfer learning strategies of the-state-of-the-art deep learning schemes.	[ "COMP", "HEAL", "MEDI" ]	[ "Radiomics", "Small vessel disease", "Stroke", "Texture analysis", "White matter hyperintensities" ]	[ "1704", "1707", "2718", "2741", "3614" ]	[ "The differential quantification of brain atrophy, white matter hyperintensities (WMH) and stroke lesions (i.e. either acute or old, symptomatic or asymptomatic) is important in studies of stroke and dementia.", "However, stroke lesions, in magnetic resonance images (MRI), can have similar signal intensities as WMH and cerebrospinal fluid (CSF) (i.e. a proxy for brain atrophy), and may be accidentally considered WMH or CSF by image processing methods.", "Disentangling the effect that each of them has in cognitive and health indicators is crucial for individual prognosis of stroke outcomes and for understanding the pathophysiology of stroke and ageing.", "For example, although the most serious consequence of a stroke is neuronal death, indicators of neurofibrillary degeneration have been associated with 1-year post-stroke brain atrophy (Ihle-Hansen et al., 2017), assessed from MRI.", "Brain tissue atrophy and stroke lesion volume, but not WMH volume have been cited as neuroimaging determinants of post-stroke cognitive performance (Puy et al., 2018).", "On the other hand, there is evidence that WMH and not old stroke lesion volume is associated with brain atrophy and cognitive decline in normal ageing (Habes et al., 2016; Valdés-Hernández et al., 2013).", "The differential assessment of these imaging markers (i.e. WMH, stroke lesion and brain atrophy) is also necessary in the design of clinical trials that use these parameters as outcome measurements, for estimating the sample size.", "The latter is driven by a measure of “uncertainty” in the estimation of the outcome measurements, as well as the absolute difference between these measurements in the groups of individuals involved in the trials.", "A study showed that failure to exclude stroke lesion volume in the volume reported as WMH added an uncertainty of 20%, which could cause an increase in the sample size calculation and consequently impact in the trial duration and cost (Wang et al., 2012).", "Adjudicating treatment response to an increase/decrease in WMH instead of stroke lesion due to miscalculating their true volumes potentially could miss effective treatments or make ineffective treatments look as if they were beneficial.", "The same study found that for individual patients, failing to consider the tissue loss due to stroke when measuring brain atrophy could increase the apparent brain volume loss by up to 21.25 ml more, representing up to 4 times more atrophy than the true value.", "Neuroimaging studies of stroke benefit from the use of diffusion weighted images, which are part of routine clinical stroke neuroimaging protocols as they facilitate the identification of stroke lesions.", "However, it has been reported that these images do not identify the presence of stroke on approximately a third of patients seen in clinics with a non-disabling stroke (Makin et al., 2015).", "Also, they are not always part of the neuroimaging protocol for studies of ageing and dementia.", "In the last few decades, the increase in computational power of affordable computer platforms has given a boost in the application of machine-learning methods to medical image analysis.", "A recent review on machine learning methods applied only to the study of dementia using MRI (Pellegrini et al., 2018), found 5747 studies, excluding reviews and animal studies, published up to January 2016.", "From 2016 onwards, deep learning methods, principally convolutional neural networks (CNN), have dominated advances in this field (Litjens et al., 2017; Shen et al., 2017).", "However, the requirements of still expensive hardware and a large amount of annotated data have limited their applicability, favouring the application of the more conventional ones (Giger, 2018).", "Moreover, CNN, the deep learning method by excellence in Computer Vision, suffer a considerable loss in performance in the following scenarios: 1) in the presence of pathologies that differ from those in the training set, 2) with datasets acquired with different imaging protocols, or using different sequences (i.e. the task domain changes), 3) when performing tasks that are related to but not the same as that on which they were trained (e.g. lesion segmentation vs. lesion assessment) (Rachmadi et al., 2018).", "To overcome these limitations, there are several ways to enhance the performance of CNN architectures without modifying the architecture itself.", "These are known as “enhanced learning techniques” and comprise: 1) modifying the input data by adding information derived from internal and external sources (i.e. data augmentation), and/or 2) re-purposing a model trained for one task to perform a second related task (i.e. transfer learning).", "For these techniques to be successful, it is important to understand which imaging features are relevant for the machine and integrate this “domain” knowledge in (re)training the CNN (Liu et al., 2018).", "Radiomics is a promising field that aims to improve precision in diagnosis, assess prognosis or predict treatment response by extracting a large number of quantitative descriptors from medical imaging data.", "Radiomics practice is based on the hypothesis that medical images contain valuable information at the tissue/organ level (macroscopic level) that reflects the underlying pathophysiology of the tissue (microscopic level) and that these relationships can be revealed by means of quantitative imaging features (Gillies et al., 2016; Lambin et al., 2012, 2017).", "Texture features are the imaging features most widely used in radiomics since they have proved to be efficient in describing the voxel interrelationships and the grey level distributions within images, allowing quantification of the intrinsic heterogeneity (vs. homogeneity) that may not be visually perceived, thus facilitating the characterisation and classification of different tissues (Castellano et al., 2004).", "These parameters can be correlated or combined with other medical information such as demographic, clinical, histologic or genomic data in order to improve decision-making tasks (Avanzo et al., 2017; Kumar et al., 2012; Larue et al., 2017).", "Texture analysis can be applied to different/multiple imaging modalities, and the selection of the appropriate technique to investigate each disease or lesion depends on several factors.", "Recently, MRI has increased in popularity in radiomics studies due to its growing incorporation into routine clinical practice and its ability to produce high-quality images (Larroza et al., 2016).", "Specifically for the study of SVD and stroke lesions, several studies that used MRI have successfully applied texture analysis to different tasks (González-Castro et al., 2017; Kassner et al., 2009; Leite et al., 2015; Valdés-Hernández et al., 2017; Viksne et al., 2015).", "One of them found that texture in normal-appearing tissues showed promise for stratifying patients according to their SVD and WMH burden (Valdés-Hernández et al., 2017).", "As normal-appearing tissue can be assessed using well-established automatic image processing tools (e.g. SPM (https://www.fil.ion.ucl.ac.uk/spm/), FSL (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/), FreeSurfer (https://surfer.nmr.mgh.harvard.edu/), BrainVisa (http://brainvisa.info/web/index.html) to mention just a few) with a high level of accuracy, and given the effect that a stroke is known to have not only in the affected region, but also in unaffected tissue, we investigated the feasibility of using texture analysis in the tissues conventionally identified in structural MR images to identify the presence/absence of a previous stroke.", "Moreover, as WMH (i.e., a confound for the automatic identification of ischaemic stroke lesions) have been defined as having a mainly vascular origin (Wardlaw et al., 2013), we also analysed whether texture in WMH can help increase the likelihood of accurately identifying the presence of a major ischaemic stroke lesion.", "We therefore hypothesised that a conventional classifier that uses textural features in WMH and tissues commonly segmented by current automatic image processing techniques, can discriminate the brain MRI of individuals that had a stroke from those who had not, so as a post-processing step of removing the stroke effect could be done, although the type of stroke (i.e. cortical vs. lacunar) might be difficult to be ascertained.", "Specifically, our research questions are: 1) Can a texture-based automatic classifier discriminate a routine clinical structural brain MRI scan of a patient with a recent lacunar stroke from a brain MRI scan from a patient with a recent mild cortical stroke?", "2) Can a texture-based automatic classifier discriminate between the MRI data of an individual who had a previous stroke from an individual of similar age who never had a stroke?", "We also investigated the effect of age in the classification.", "To answer our two research questions we used MRI data from individuals enrolled in two different prospective studies: one study of stroke mechanisms (Wardlaw et al., 2017) and one study of cognitive ageing (Taylor et al., 2018).", "The sample from the first study included MRI data from 100 patients (54 women and 46 men, mean age 65.3 years old, SD 11 years) which had a lacunar (n = 50) or mild (i.e. mRS <3) cortical (n = 50) ischaemic stroke less than 2 weeks prior to the MRI scan (i.e. post-acute stage).", "The sample from the second study included MRI data from 100 individuals from a year-of-birth cohort (53 women and 47 men, mean age 73.2 years old, SD 0.6 years) who were either stroke free (n = 50) or had a prior ischaemic stroke in the non-acute (i.e. chronic) phase identifiable on imaging (n = 50).", "The data selection was performed randomly and fully automatically, only taking account that the four subgroups (i.e. 1) recent lacunar stroke, 2) recent cortical stroke, 3) no stroke, 4) old stroke) were equal-sized.", "To evaluate the influence of age in the classification of scans having a stroke or not, we used brain MRI data from 36 individuals from another year-of-birth cohort.", "They were also enrolled in a study of cognitive ageing (Taylor et al., 2018) (20 women and 16 men, mean age 91 SD 0.5 years).", "From this sample, 22/36 individuals never had a stroke (i.e. at least identifiable in imaging).", "All studies that provided data and involved human participants were conducted in accordance with the 1964 Helsinki declaration and its later amendments, with protocols and ethical standards approved by the following Scottish Research Ethics Committees: Lothian Research Ethics Committee (09/S1101/54, LREC/2003/2/29, REC 09/81101/54), the NHS Lothian R + D Office (2009/W/NEU/14), and the Multi-Centre Research Ethics Committee for Scotland (MREC/01/0/56) (Valdés-Hernández et al., 2015a,b; Wardlaw et al., 2017).", "All brain MRI data were acquired on a 1.5 T GE Signa LX clinical scanner (General Electric, Milwaukee, WI), equipped with a self-shielding gradient set and manufacturer supplied eight-channel-phased array heal coil.", "The MRI acquisition protocols of the studies that provided data for these analyses differed.", "The MRI sequences considered in this work were 3D T1-weighted (T1W) inversion recovery-prepared spoiled gradient echo (SPGR), axial 2D T2-weighted (T2W) and axial 2D fluid-attenuated inversion recovery (FLAIR) brain images.", "For the stroke study, the T1W had TR/TE/TI = 7.3/2.9/500 ms, 8° flip angle, 33 × 21.5 cm2 field of view (FoV), 256 × 146 acquisition matrix, 100 × 1.8 mm slices, the T2W sequence had a propeller acquisition with TR/TE = 6000/90 ms, 24 × 24 cm FoV, 384 × 384 acquisition matrix, 28 × 5 mm slices, 1 mm slice gap, and the FLAIR had TR/TE/TI = 9000/153/2200 ms, 24 × 24 cm FoV, 384 × 224 acquisition matrix, 28 × 5 mm slices, 1 mm slice gap.", "Both year-of-birth cohort (normal ageing) studies had the same MRI acquisition protocol: T1W had TR/TE/TI = 9.7/3.984/500 ms, 25.6 × 25.6 cm2 FoV, 192 × 192 acquisition matrix, voxel size 1 × 1 × 1.3 mm3, T2W had TR/TE = 11320/102 ms, 25.6 × 25.6 FoV, 256 × 256 acquisition matrix, 80 × 2 mm slices, no inter-slice gap; and FLAIR had TR/TE/TI = 9000/140/2200 ms, 256 × 192 acquisition matrix, and 40 × 4 mm slices.", "All acquisitions were zero-filled and resampled to either 256 × 256 or 512 × 512 in-plane resolution matrices.", "The segmentation of the brain tissues and structures was performed following the protocol described in (Valdés-Hernández et al., 2015a,b).", "Briefly, binary masks of normal appearing white matter (NAWM) and WMH were obtained using a multispectral segmentation method (Valdés-Hernández et al., 2010) (www.sourceforge.com/projects/bric1936) followed by manual editing to correct for possible errors.", "The structures of the basal ganglia and thalami were fully automatically extracted using a combination of three tools from the FMRIB software library (FSL) (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki) (Jenkinson et al., 2012): Smallest Univalue Segment Assimilating Nucleus (SUSAN), FMRIB's Linear Image Registration Tool (FLIRT) and a model-based segmentation/registration tool (FIRST), combined on an automatic pipeline developed in-house, and also manually corrected if necessary.", "Binary masks of NAWM, WMH and subcortical structures were mapped into the T1W, T2W and FLAIR sequences as illustrated in Fig. 1", "Textural features were extracted from a total of 1800 3D sets of images (2 prospective studies × 100 individual data × 3 MRI sequences × 3 brain tissues/structures).", "A simple approach to capture the volumetric information of each 3D image was implemented: we first extracted the 2D texture features from each slice of each 3D image, and then the 3D texture features of the image were obtained by computing the median of the values of all the slices.", "This process is illustrated in Fig. 2.", "Using this approach, the grey-level distributions in the third dimension are not considered; however, it has been shown that features computed with this 2D averaging method are more discriminative than features extracted from a single slice (Larroza et al., 2016).", "Additionally, all features were standardized to zero mean and unit variance to improve numerical stability in the model training process.", "Also, zero-variance and near-zero-variance predictors were removed for the same reason (Kuhn and Johnson, 2013b).", "Finally, some features failed to give a valid numeric value for some patients (e.g. while attempting to be calculated on small WMH clusters), so these features were also removed to avoid computational problems in the training process.", "The feature extraction process was performed in MATLAB (R2015b; The MathWorks Inc., Natick, MA, USA) taking as a reference the code implemented in (Alegre et al., 2012).", "A total of 114 features were extracted from each of the 1800 3D images and grouped into five different sets of textural features according to the texture analysis method employed: Grey-level Co-occurrence Matrix features (GLCM: 13 parameters), Grey-Level Run-Length Matrix features (GLRLM: 11 parameters), Local Binary Patterns features (LBP: 40 parameters), Wavelet Statistical features (WSF: 26 parameters) and Wavelet Co-occurrence features (WCF: 24 parameters).", "Table 1 shows all textural features extracted from each method.", "The Grey-level Co-occurrence Matrix (GLCM) is a second-order statistical matrix-based texture analysis method that was first proposed by (Haralick et al., 1973) to describe local heterogeneity information in images.", "This method quantifies the relationship between grey levels in an image by counting the pairs of pixels separated by a predefined distance (d) and direction (θ) that have the same distribution of grey-level values.", "Each pixel of the resulting matrix represents the number of times that the grey level of a reference pixel and the grey level of the neighbour pixel in the predefined distance d and direction θ are seen in the image.", "Consequently, the size of the GLCM will be Ng × Ng, being Ng the number of grey levels of the image.", "Several statistics representing the homogeneity or heterogeneity of the image can be mathematically computed from the GLCM.", "In our study, images were uniformly quantised to Ng = 32 grey levels to reduce the computational cost of the feature extraction process and to improve the signal-to-noise ratio (Gibbs and Turnbull, 2003).", "A distance of d = 1 pixel was chosen to enhance mainly the local properties when computing the GLCM.", "To achieve rotation invariance, the features extracted from the GLCMs in the four directions of the 2D space (θ = 0°, 45°, 90° and 135°) were averaged.", "Rotation invariance is important in the context of our work because some texture methods like GLCM are dependent on the direction and different texture values could be obtained if the image is rotated, thus affecting the results when images from different patients have different orientations (Larroza et al., 2016).", "At the end, 13 texture features were extracted from the GLCM of each image, as shown in Table 1.", "The equations to compute these features and more details can be found in (Haralick et al., 1973).", "The Grey-Level Run-Length Matrix (GLRLM) is also a statistical matrix-based texture analysis method of a higher-order than the GLCM that describes regional heterogeneity information.", "This method, first proposed by (Galloway, 1975) and extended by (Chu et al., 1990) and (Dasarathy and Holder, 1991), examines the times that each grey level value is seen consecutively in an image in a predefined direction.", "The GLRLM is constructed by detecting and counting the runs (sequences of consecutive pixels with the same grey level) of different grey levels and lengths in the image.", "Each row of the GLRLM represents a grey level and each column represents a specific length, so each pixel of the matrix indicates the number of runs of a specific grey level and length in the image.", "The features extracted from the GLRLM can be used to define fine textures (dominated by short runs) or coarse textures (dominated by longer runs) (Nailon, 2010).", "To compute the GLRLMs, images were previously quantised to Ng = 32 grey levels as in the case of GLCMs.", "The GLRLM features are also affected by the orientation of the image, so features extracted from the GLCMs in the four directions of the 2D space (θ = 0°, 45°, 90° and 135°) were averaged to achieve rotation invariance.", "A total of 11 GLRLM features were computed, as shown in Table 1, and further details can be found in (Chu et al., 1990; Dasarathy and Holder, 1991; Galloway, 1975).", "Once the Eq. (1) is applied to all the pixels in the image, a LBP image is obtained and all the bins of the histogram of this image are used as texture features.", "Other statistics can be extracted from the LBP image and used as texture features like the mean or the variance.", "In this work, the original LBP operator (patches of 3 × 3 pixels: P = 8, R = 1) was employed to preserve the texture analysis as local as possible because regions like WMH are not very large.", "Rotation invariance was achieved by performing a circular bit-wise right shift operation (rotating the neighbour pixel set clockwise) and assigning the smallest LBP binary number (Ojala et al., 2002).", "Using this approach, 36 unique rotation invariant histogram-based LBP features were obtained, as only 36 LBP binary numbers can occur for P = 8.", "Additionally, 4 statistics derived directly from the LBP image (median, variance, skewness and kurtosis) were added to the LBP features set.", "The MR images were not quantised to compute the LBP texture feature since the rotation invariant LBP approach is robust to intensity variations (Unay et al., 2007).", "The discrete Wavelet transform (DWT) is a technique that examines the spatial frequency patterns of an image within different scales and frequency directions, considering that frequency is directly proportional to grey level variations in an image.", "The DWT applied to an image produces four matrices of coefficients (sub-images) that represent the approximations or low frequencies (LL: low-low) and the details or high frequencies in the vertical (LH: low-high), horizontal (HL: high-low) and diagonal (HH: high-high) directions.", "An example of this matrices is shown in Fig. 3.", "The DWT can be repeated consecutively to achieve a major image decomposition: the first level of decomposition (LL1, LH1, HL1 and HH1) is applied to the original image as mentioned before and the subsequent levels are applied to the matrix of approximations of the previous level (LLi, LHi, HLi and HHi, where i is the level of decomposition).", "The DWT can be used as a transform texture analysis method by processing these sub-images to obtain parameters that describe the spatial frequency information of the image.", "These parameters have been previously used in some studies with successful results (Alegre et al., 2012; Arivazhagan and Ganesan, 2003; González-Castro et al., 2017).", "In this work we examined two groups of texture features derived from the DWT.", "The first group was the Wavelet statistical features (WSF), consisting of 26 descriptors that are the mean and SD of the histograms of the original image and the sub-images yielded after three levels of decomposition.", "The second group was the Wavelet co-occurrence features (WCF), consisting of 24 descriptors that are obtained by extracting six of the GLCM features (energy, contrast, correlation, homogeneity, entropy and variance) from the sub-images yielded after the first DWT decomposition.", "The Haar family of wavelets was used to perform the DWT decomposition.", "A preliminary statistical analysis was conducted to evaluate the discriminative power of each feature independently between populations.", "Its final purpose was to assess the feasibility of these features individually as biomarkers of stroke.", "To compare the distributions of each textural feature for each of the classes, the Mann-Whitney U test was applied.", "This non-parametric test is equivalent to the independent samples t-test but without the requirement of the normality assumption and is recommended for relatively small sample sizes (McDonald, 2014).", "As the number of statistical tests performed increases, the contrast test becomes more permissive, thus rejecting the null hypothesis more easily and increasing the number of false positives (McDonald, 2014).", "To counter this effect, usually known as the multiple comparisons problem, we decided to apply the Holm-Bonferroni correction before assuming the statistical significance of the features.", "This relatively strong (conservative) method controls the family-wise error rate, thus compensating the Type I error (incorrectly rejecting the null hypothesis) and attempting to limit the probability of even one false discovery.", "After the statistical analysis, we applied machine learning algorithms to analyse the groups of texture features without removing any feature initially, since features that may be completely useless by themselves can provide a significant performance improvement when combined with others within a machine learning approach (Guyon and Elisseeff, 2003).", "We evaluated the performance of two well-known conventional classifiers: Support Vector Machine (SVM) with linear kernel and Random Forest (RF).", "The SVM classifier (James et al., 2013; S. Wang and Summers, 2012; Wu et al., 2008), in a binary classification task like ours, tries to maximize the margin distance between the classification boundary (i.e. hyperplane) and the closest samples of both classes by adjusting internal parameters in the training process.", "One of these parameters is the cost C, which controls the trade-off between misclassification of the training data and the size of the margins.", "Values of C = 2−3, 2-2, 2-1, 1, 2, 22 and 23 were tuned to obtain the optimal classification results.", "We used a linear kernel after an initial evaluation where non-linear kernels did not produce notably better results even after a lengthy training process.", "The RF classifier (Fernández-Delgado et al., 2014), combines the results of a multitude of independent and decorrelated decision trees in the training process, thus improving generalization of the model and robustness against overfitting in small samples (i.e. our sample is small for machine-learning algorithms).", "One of the advantages of the RF model is the little parameter tuning required.", "The parameter mtry, which identifies the number of random variables used in each tree, controls the strength (how accurate the individual trees are) and the correlation (the dependence between trees) of the RF model.", "Another tuning parameter is the number of trees to be built.", "In this work, values of mtry = 2, 4, 6, 8, 10 and 12 were evaluated and the number of trees was set to 250, as higher values of this parameter did no produce notably better results on a preliminary evaluation.", "For evaluating the efficiency of the classification models, we employed a 5-fold cross-validation (CV) approach.", "This resampling method randomly partitions each texture dataset into five equally sized subsets of samples or folds, maintaining a balanced amount of both classes in each fold.", "Then, five models were trained and tested so that each of the five folds was used once as the test set, while the four remaining folds were used to train the model.", "This process was repeated ten times to reduce the variance of the cross validation results and to avoid possible bias in the random separation of the folds (Kuhn and Johnson, 2013c), so at the end 50 models (5 test folds × 10 repetitions) were built using different sets of patients for training and testing each time.", "The classification performance was evaluated using the averaged area under the curve (AUC) of the receiver operating characteristic (ROC) that resulted from averaging the AUC values obtained from the 50 iterations (mean ± SD).", "Good estimates of the model performance can be obtained using the validation data when the sample size is not large (Kuhn and Johnson, 2013c).", "Other metrics like sensitivity, specificity and accuracy were also obtained to validate the results.", "The 9000 texture combinations (18 × 100 sets of images × 5 texture analysis methods) were firstly examined with the classifiers without excluding any texture feature, as previously mentioned.", "However, the texture combinations that provided the highest AUC values were analysed again using the same cross validation structure with a feature selection step included within the model-building process to avoid overfitting (Ambroise and McLachlan, 2002).", "This way, we could test if reducing the number of features improved the classification results.", "Two filter feature selection methods were applied to obtain rankings of features based on the discriminative power of each feature independently without analysing the relation between features and without involving any predictive model (Kuhn and Johnson, 2013a).", "The first method used the p-value provided by the Mann-Whitney U test for independent groups of samples.", "The second method used the Maximal Information Coefficient (MIC), which measures the strength of the linear or non-linear association between two variables.", "The model evaluation process was implemented with the Caret package (Kuhn, 2008) in R language, version 3.2.5 (R Development Core Team, Vienna, Austria), and illustrated in Fig. 4.", "The patients for which the best models performed well 80% or more of the times were identified.", "From these best models, the patients for which both classifiers (i.e. SVM and RF) performed well 80% or more of the times, were also identified.", "For each patient, we extracted the following data: 1) proportion of times the images were correctly (and wrongly) classified, 2) proportion of times in which both classifiers correctly (and incorrectly) classified the images using the same descriptors, 3) clinical stroke classification into no stroke, large cortical, small cortical or lacunar, 4) age at the time of scanning, 5) general WMH factor considering visual scores and computational measurements, as per (Aribisala et al., 2014), 6) percentage of lesion and normal tissue volumes in intracranial volume, 7) percentage of brain tissue volume in intracranial volume, 9) perivascular spaces visual scores in the basal ganglia and in the centrum semiovale.", "We calculated the bootstrapped bivariate Pearson’s correlations between the first two variables (i.e. results from the classification processes) and the rest (i.e. clinically derived variables) to evaluate possible influence of the clinical biomarkers and age in the outcome of the classification schemes.", "Sixty-one texture features of a total of 1026 features (114 features × 3 MRI sequences × 3 brain tissues/structures) were statistically significantly different (p < 0.05) between post-acute cortical and lacunar stroke patients, but only two features derived from the GLCM (FIMC and SIMC (see Table 1), with p = 0.0218 and p = 0.0096 respectively) were significant after applying a Holm-Bonferroni correction for multiple comparisons.", "Table 2 shows the distribution of significant features according to the MRI sequence and the brain tissue/structure.", "T1W sequences seem to be more suitable for using texture information to discriminate between cortical and lacunar stroke patients, especially when analysing the brain subcortical structures.", "Nevertheless, the texture data extracted from these images and these brain tissues/structures did not seem to have enough discriminative power to differentiate cortical and lacunar stroke patients in general.", "Table 3 shows the averaged AUC (mean ± SD) computed from the 50 iterations when examining all the texture datasets with the two models under analysis (SVM with linear kernel and RF), and for all the MRI sequences and brain tissues/structures.", "A relevant AUC value could not be obtained (AUC < 0.7) in any case.", "The best result was obtained using the GLCM parameters in the T2W images of the NAWM region using SVM with linear kernel (AUC = 0.667 ± 0.117), but this value was not accurate enough to determine that a good classification can be achieved using these data.", "Many texture features showed capability for discriminating between “no stroke” and “old stroke” individuals.", "In this case, 349/1026 texture features (114 features × 3 MRI sequences × 3 brain tissues/structures) were statistically significant (p < 0.05) when applying a Mann-Whitney U test for independent groups of samples, and 235 features remained significant after applying a Holm-Bonferoni correction for multiple comparisons.", "Table 4 shows the distribution of significant features according to the MRI sequence and the brain tissue/structure.", "The information collected in this table indicates that the texture features extracted from the brain subcortical structures are more effective in discriminating between “no stroke” and “old stroke” individuals, regardless of the MRI sequence is used.", "As per the AUC values obtained, certain groups of textures allowed “no stroke” and “old stroke” individuals to be classified with a good degree of precision.", "Table 5 shows the averaged AUC (mean ± SD) obtained from the 50 iterations when examining all the texture datasets with the two models (SVM with linear kernel and RF), and for all the MRI sequences and brain tissues/structures.", "Good results (i.e. AUC > 0.7) were not achieved with all groups of textures.", "Local binary patterns features extracted from T2W and FLAIR images of the subcortical structures delivered good results, as expected from the previous statistical analysis (see Table 4).", "However, other feature datasets like GLRLM features extracted from FLAIR images of the WMH or WCF features extracted from FLAIR images of the NAWM provided satisfactory results although the previous statistical analysis was not very optimistic with features extracted from these groups of images (Table 4).", "It should be noted that parameters extracted from the T1W images as well as parameters derived from the GLCM did not provide AUC values higher than 0.7 in any case.", "The predictive model employed for classifying the patients influenced the results, but there was not a firm conclusion on which model was better as SVM worked better with some texture datasets and RF with others.", "We applied two filter feature selection methods to the five texture datasets that yielded good results (i.e. AUC > 0.7) to see if classification results improve when reducing the number of features.", "Rankings of features based on the Maximal Information Coefficient (MIC) and the p-value provided by the Mann-Whitney U test were computed from the training folds in each of the 50 iterations of the CV procedure.", "Table 6 shows the new AUC values obtained when reducing the number of features according to the computed rankings in the best texture datasets.", "In general, better AUC values were obtained when reducing the number of features.", "In particular, it is remarkable the substantial improvement achieved for LBP parameters extracted from T2W images of the subcortical structures when using the SVM model: a final value of AUC = 0.828 ± 0.075 was obtained when only using the 7 more relevant LBP characteristics according to the MIC coefficient.", "Fig. 5 shows the AUC values obtained for all possible subsets of features according to the MIC ranking and the ROC curves provided by the model when using all the features and when using the optimal number of features.", "Table 7 shows the results obtained with and without including the image data from the older individuals (i.e. age ˜91 years old) in the texture datasets that performed better (i.e. AUC > 0.7) in the previous analysis.", "The results show that the classification performance got worse when introducing older patients in general, suggesting that age influences the classification results by increasing the misclassification rate.", "None of the clinical biomarkers analysed correlated with the proportion of times the images were correctly (and wrongly) classified, and neither with the proportion of times in which both classifiers correctly (and incorrectly) classified the images using the same descriptors.", "Only age was significantly correlated with these measurements (p ≤ 0.001).", "However, the strength of the significance was only r = 0.31 and r = 0.39 respectively.", "The pattern of the classification performance of the different types of images (i.e. no stroke, large cortical stroke, small cortical stroke, lacunar stroke) was similar irrespective of the classifier used (i.e. SVM and RF) and when the analysis accounted for whether the images were correctly (or incorrectly) classified by both classifiers: “no stroke” images achieved the greatest proportion of well classified, followed by “lacunar”, “small cortical” and “large cortical” (Fig. 6).", "In this paper, the performance of several texture features extracted from different brain tissues and structures (i.e. NAWM, WMH and subcortical structures) in different MRI structural sequences (i.e. FLAIR, T2W and T1W) was analysed, in two conventional machine learning approaches, to identify the presence of stroke in the images of post-acute stroke patients and normal ageing individuals.", "Differentiation of post-acute cortical vs. lacunar stroke subtypes using texture analysis was examined as well as classification of images with vs. without chronic stroke lesions.", "Only two textural features from the subcortical structures in T1W images resulted with high discriminatory power between images from post-acute lacunar vs. cortical stroke.", "These features were the first and second information measures of correlation (FIMC and SIMC) derived from the GLCM, which quantify the linear dependency or correlation between intensities, thus representing homogeneity but adding some desirable properties that are not represented by the original correlation descriptor extracted from the GLCM (Haralick et al., 1973).", "It can be questioned the reason behind using all Haralick features in our models, given that some of them maybe correlated, carrying repetitive information.", "A review on feature selection methods (Guyon and Elisseeff, 2003) agrees that perfectly correlated variables are truly redundant in the sense that no additional information is gained by adding them, so the removal of perfect correlated variables will not negatively impact the learning performance.", "However, the authors also state that very high variable correlation (or anti-correlation) does not mean absence of variable complementarity: features that do not carry any discriminatory or added value by themselves can provide a significant improvement in performance when combined with others within a machine learning approach.", "A previous study that evaluated the use of texture analysis as an alternative for characterising SVD and assessing possible blood brain barrier leakage (Valdés-Hernández et al., 2017) reported differences in the texture of the FLAIR deep grey matter between post-acute lacunar and cortical stroke patients, but only with borderline significance.", "That study reported the texture in the deep grey matter was more ‘homogeneous’ in patients with recent lacunar stroke compared to those who had a cortical type.", "Statistically significant differences between the FLAIR images from both groups of patients were only found in the textural features measured in the post-acute stroke lesions.", "Our motivation was to explore whether the texture in the conventionally segmented tissues (i.e. normal and abnormal) could have enough discriminatory power to be used in machine learning schemes to identify the stroke subtype and if there was a stroke.", "Usually ischaemic stroke lesions and artefacts that mimic WMH are included within the burden of WMH by automatic WMH segmentation methods.", "A real-world scenario would have been to evaluate the output of the-state-of-the-art CNN WMH segmentation methods.", "However, corruption of the abnormal WMH signal with uneven burden of signal changes from other causes would have introduced a bias in the results.", "Therefore, we carefully excluded the stroke lesions from the burden of hyperintense T2W-based signal and did not analyse the texture in them.", "Our analysis found features with borderline statistical significance to discriminate between cortical and lacunar stroke patients in T1W MRI data but failed to find a conventional machine learning model to classify these patients accurately.", "The reason behind these results may lie in the fact that both types of stroke can be seen simultaneously in many cases, as reported by (Xu, 2014).", "The images from “no stroke” patients were, in general, better identified by the classifiers as opposed to the images that had “large cortical” chronic stroke lesions, which resulted in them being less well classified, and, instead, were classed as not having any stroke lesion at all.", "It might seem contradictorily, given that the images of individuals with chronic lacunar lesions (i.e. small lesions mainly in the region crossed by the corticospinal tracts (Valdés-Hernández et al., 2015a,b), which can be confounded by WMH, were the second best classified.", "However, lacunar, and not large cortical, strokes have been associated with blood brain barrier impairment, manifested in abnormal extracellular leakage (Wardlaw et al., 2017).", "Also, textural features in normal and abnormal tissues have been reported as being useful in detecting the subtle differences that this mechanism causes.", "One limitation of this work is the impossibility of combining the stroke and ageing datasets to analyse if images showing recent cortical or lacunar strokes could be distinguished from images of patients without stroke and patients with an old stroke lesion.", "This is because variations in acquisition parameters may result in differences in the texture outcome that are not due to the underlying biological characteristics of the tissues expressed by the texture (Mayerhoefer et al., 2009; Schad, 2004).", "Image normalization techniques help to reduce differences in imaging acquisition settings, but some residual effects may not be totally suppressed, thus obscuring the true texture differences due to the tissue properties only.", "Therefore, combining texture features extracted from both databases in the machine learning pipeline evaluated may lead to overoptimistic results caused by the differences in imaging acquisition protocols.", "Other limitation of the present work consists on the 3D texture analysis approach based on the median of 2D texture features.", "Although pure 3D texture analysis is usually preferred because it allows capturing more heterogeneity information of the tissue under analysis, this approach is not always feasible, especially when the slice thickness of the images is very large compared to the in-plane resolution (Depeursinge et al., 2014), as in our case.", "In these situations, 2D texture approaches or approaches like the one carried out are recommended.", "In this work we conducted a very detailed texture analysis study for identifying and characterizing ischaemic stroke lesions in structural brain MRI data by considering several regions or tissues and by testing a large amount of quantitative texture descriptors.", "The number of patients per group was sufficiently large to draw reliable conclusions and the machine learning pipeline was designed to avoid overoptimistic and overfitted results.", "We achieved promising results that suggested that texture features may help in the detection of previous stroke lesions, and identified the textural features that look promising on this task, so as they can be evaluated in transfer learning schemes with the-state-of-the-art deep CNNs.", "Additionally, the correlation of our results with clinical parameters was explored to find clinical patterns or characteristics that could be reflected in the textural features that resulted promising to the classification tasks evaluated.", "All authors declare no Conflict of Interests to report.", "This work was funded by the Row Fogo Charitable Trust (MVH, VGC) grant no. BRO-D.", "FID3668413, and the Wellcome Trust (patient recruitment, scanning, primary study Ref No. 088134/Z/09).", "The study was conducted independently of the funders who do not hold the data and did not participate in the study design or analyses.", "The Lothian Birth Cohort 1936 is funded by Age UK (Disconnected Mind grant) and the Medical Research Council (MRC; MR/M01311/1, G1001245, 82800), and the latter supported BSA.", "IJD was supported by the Centre for Cognitive Ageing and Cognitive Epidemiology, which is funded by the MRC and the Biotechnology and Biological Sciences Research Council (MR/K026992/1).", "David Moratal acknowledges financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) and FEDER funds under Grant BFU2015-64380-C2-2-R, and from the Conselleria d'Educació, Investigació, Cultura i Esport, Generalitat Valenciana (grants AEST/2017/013 and AEST/2018/021).", "Rafael Ortiz-Ramón was supported by grant ACIF/2015/078 and grant BEFPI/2017/004 from the Conselleria d’Educació, Investigació, Cultura i Esport of the Valencian Community (Spain)." ]	[ "Radiomics in conventionally segmented tissues can identify MRI scans that had a stroke.", "Patient’s advanced age can negatively influence classification results.", "Feature selection and stroke subtype influence but do not determine accuracy.", "Stroke subtype cannot be ascertained from texture analysis in brain tissues." ]
Non-linear thermogravimetric mass spectrometry of carbon materials providing direct speciation separation of oxygen functional groups	Thermogravimetric mass spectrometry (TG-MS) is an established way to analyze oxygen containing surface functional groups of carbon materials. Thermal stabilities and structures of functional groups influence their decomposition temperatures and products (CO, CO2). In this work, a non-linear procedure with isothermal steps is presented enabling a separation of functional groups by different decomposition temperatures. Nitrosulfuric acid functionalized carbon materials like multi-walled carbon nanotubes (MWCNT) and graphite were used to design the temperature program. Comparative studies of linear and non-linear heating experiments in argon and hydrogen containing atmosphere were performed to state the benefits and limits of both methods. The distinct advantage of non-linear thermal analysis is demonstrated by an application-oriented experiment where only selected functional groups are consumed.	[ "CHEM", "MATE" ]	[ "Atomic layer deposition", "Multi-walled carbon nanotubes", "Oxygen functional groups", "Selective reactivity", "Thermal decomposition" ]	[ "1600", "2500" ]	[ "Multi-walled carbon nanotubes (MWCNT) are highly desired materials for numerous applications.", "Due to their unique structure they combine graphitic properties like high conductivity, thermal and mechanical stability with high surface area (250 m2/g) [1–4].", "Properties of the MWCNT are strongly linked to the amount and kind of functional groups on the surface [5–7].", "With an increasing amount of functional groups the conductivity and stability of MWCNT will unavoidably change.", "In catalysis, MWCNT are often used as support materials for small active particles [1,8].", "The functional groups on the surface of the supporting MWCNT can influence the stability of the compound material.", "Hence, particle attachment and solubility will be influenced by the surface properties.", "In some cases functional groups show catalytic activities by themselves [9,10].", "To describe the interaction between metal catalysts and functional groups, or to determine the catalytic activity of the carbon material itself, it is necessary to qualitatively and quantitatively identify functional groups and describe the changes to such groups before and after their use in a catalytic reaction [11,12].", "Post-analysis therefore allows one to monitor the chemical changes, and to identify functional groups that are consumed or produced during a reaction.", "Several approaches are used to overcome this challenge.", "For instance titration methods use the different pka values of functional groups for the quantification.", "The conversion of functional groups, diffusion and acid residuals of MWCNT pre-treatments often impede a former characterization of functional groups [13].", "Catalytic tests like the oxidation of isopropanol are used to generate sum parameter where acidic and alkaline functional groups are separated.", "However, the reaction temperature applied during this catalytic test already changes the composition of functional groups and influences the quantification [14].", "Thermogravimetric mass spectrometry is a common way to analyze surface functional groups on carbon materials [15,16].", "During the thermal treatment, oxygen-containing functional groups, depending on their structure, decompose into CO2 or CO.", "Traditionally, TPD approaches use solely heating rates in argon atmosphere with a linear temperature program.", "Results gained out of these experiments do not enable the exact determination of the decomposition temperature of different functional groups.", "Overlapping signals due to the decomposition of multiple functional groups with similar decomposition temperatures hinders an exact determination of decomposition temperature [17–20].", "Recently, thermal stabilities of different surface functional groups were determined by a combination of temperature programmed desorption (TPD), X-ray Photoelectron Spectroscopy (XPS), titration, and catalytic test reaction experiments [21].", "Due to the combination of these methods an exact determination and assignment of the decomposition temperatures of functional groups, where maxima of the decomposition products are analyzed, was possible (see Table 1).", "Even if the decomposition temperatures of the functional groups have already been discussed the problem of overlapping signals during TPD measurements still exists, and prohibits differentiation of functional groups.", "For a quantitative analysis of functional groups via TPD, a separation during the decomposition process is required.", "The present work focuses on separating the decomposition products of functional groups during TPD measurements based on the observations summarized in Table 1.", "To accomplish this, we used a non-linear temperature program procedure.", "This method is now referred to as an isothermal temperature program.", "Due to the insertion of isothermal steps during the heating process, the overlay between different desorption process from chemically different functional groups can be avoided by completing each desorption process prior to increasing the decomposition temperature. .", "The temperatures of these isothermal steps were determined by analyzing the peak shape of the individual ion currents.", "Moreover, literature values were used to confirm the temperatures determined for the separation of functional groups [19–26].", "Applying an isothermal temperature program during the TPD analysis of carbon materials affords the following benefits:", "Isolation of different functional groups due to thermal separation", "Identification of decomposition temperature ranges for different functional groups", "Particular detection of decomposing products of functional groups with different decomposition temperatures", "Detection of interconversion of functional groups before and after reaction conditions (e.g. consumption, formation, conversion etc.)", "In addition to the influence of isothermal steps on the detected signals of decomposition products, the influence of different heating rates on the decomposition was investigated.", "Thereby, the influence of kinetic hindrances on the CO2+ and CO+ ion currents was investigated.", "The shapes of the ion currents were not affected by the heating rate and shoulders appearing during the analysis are related to different functional groups.", "Graphite (Merck Millipore) as well as MWCNT produced from Shangdong Dazhang Nano Materials Co. were treated with nitrosulfuric acid (vol% ratio: 1:1; nitric acid 65%: sulfuric acid 98%) to create oxygen containing functional groups on the surface of the MWCNT.", "The procedure of functionalization is described in detail elsewhere [27].", "Briefly 10 g of carbon material was mixed with 500 ml of acid.", "The mixture was heated to 105 °C and stirred for 4 h. Afterward, the modified carbon materials were filtered and residuals of acid were washed out over several hours in a washing cell.", "The oxidized carbon was dried in a vacuum-oven at 80 °C for 12 h. To test the applicability of the developed temperature program two commercially available graphitic reference materials, active char and Vulcan XC 75R, were also analyzed.", "These materials were not pre-treated before analysis.", "Thermal analysis was performed using a STA 449 F3 Jupiter® QMS4 setup from Netzsch with an argon gas flow of 70 ml/min.", "Isothermal steps were inserted during thermogravimetric mass spectrometry (TG-MS) analysis to separate functional groups with different decomposition temperatures.", "The optimum temperature of the implemented isothermal step was judged by the peak shapes of both CO+ and CO2+ signals.", "Heating rates were varied between 10 K/min and 50 K/min.", "Based on the investigated results a heating rate of 20 K/min was applied in-between the isothermal steps to avoid overheating while maintaining satisfactory signal intensities, whereas a heating rate of 50 K/min was chosen after the last isothermal step, due to higher signal intensities.", "Reproducibility was confirmed by three iterations.", "A commercial atomic layer deposition (ALD) setup (Savannah S200, Ultratech) was used to deposit vanadium oxide on the functionalized MWCNT.", "Vanadium(V) triisopropoxide (CAS: 5588-84-1, Sigma-aldrich) precursor was heated to 70 °C and exposed for 0.5 s to the acid pre-treated MWCNT.", "This procedure was repeated for 250 cycles, respectively.", "Organic residuals of the precursor were purged after each cycle using water pulses of 0.5 s.", "The temperature of the reaction chamber during the process was held constant at 150 °C.", "Thermal decomposition of oxidized carbon materials combined with simultaneous detection of released gaseous products enables the determination of different thermal stabilities of functional groups.", "To ensure a reliable analysis, the decomposition of surface functional groups at different temperatures needs to be independent of gas transport effects.", "Therefore, comparison of MWCNT, as representative of high surface materials with a surface area of 239 m2/g and a mesoporous character was compared with graphite, as nonporous material with a surface area of 20 m2/g.", "Non-porous MWCNT forms bundles due to agglomeration, corresponding empty spaces between MWCNT provides a mesoporous character of these samples [28].", "Specific surface areas were determined by N2 sorption experiments and analyzed by Brunauer-Emmet-Teller (BET) theory showing type II hysteresis for graphite and type IV for MWCNT (supporting information S1).", "Hence, both materials show similar behavior of relatively weak adsorbent-adsorbate interactions with molecular clustering at higher p/p0, and subsequent pore filling in the case of MWCNT.", "Different heating rates during the thermal analysis were applied to investigate how heating width affects peak width, position and intensity of the detected CO2+ and CO+ signals of the two representative carbon materials (Fig. 1.)", "as well as the influence on the detected ratio of these molecular fragments, as shown in supporting information Fig. 2.", "The comparison of MWCNT and graphite CO2+ and CO+ MS ion currents (Fig. 1) reveals clear differences in intensity and peak width, whereas the peak positions of MWCNT and graphite are similar.", "In general, the peak maxima are slightly shifted to higher temperatures with increasing heating rates caused by the accruing thermal gradient within the samples [29].", "This effect is most apparent in the case of MWCNT.", "Differences in ion currents are only minor, indicating that the decomposition temperatures of the functional groups for both materials are similar.", "The porous character of MWCNT affects only the diffusion of the released gases and results in a peak broadening.", "Additionally, Fig. 1 reveals that the peak shape of the ion currents becomes more pronounced with increasing heating rates.", "The exoergic reaction is thermodynamically hindered at higher heating rates, resulting in ostensibly lower CO at lower heating rates.", "Therefore, the secondary reaction complicates a quantitative analysis of functional groups based on thermal decomposition products.", "By gas flow variations of the carrier gas the influence of reaction 1on the TPD analysis can be quantitative investigated.", "In case of significant influences of reaction 1, the applied method of separation allows only a semi-quantitative determination of functional group amount.", "A heating rate of 20 K/min was chosen as an optimal compromise in comparison to higher heating rates to suppress secondary reactions.", "Furthermore, this heating rate generates well defined ion current profiles, and ensures less overheating at each isothermal step.", "In accordance to the presence of six types of oxygen functional groups with different decomposition temperatures in the carbon structure (Table 1), six isothermal steps had to be implemented and optimized.", "All determined decomposition temperatures correspond to the complete decomposition process of each functional group rather than the onset of the decomposition.", "The separation of different functional groups with isothermal steps during the TPD analysis is based on the theory that functional groups of certain kinds decompose at defined temperatures, whereas functional groups with higher thermal stability are not affected.", "The general procedure for the separation will be explained in detail for the carboxylic groups, exhibiting the lowest thermal stability.", "TG-MS profiles for group separation of additional functionalities are available in the supporting information (S3-6).", "Fig. 2 shows the ion current profiles of CO2+ (left row) and CO (right row) with three different isothermal steps.", "The middle panel (Fig. 2c/d) shows the ion current profiles of the accurately determined decomposition temperature of the carboxylic group at 252 °C, which is also in agreement with literature values [19,21].", "Exemplary, MS profiles with inserted steps at 210 °C (Fig. 2 a/b) and 290 °C (Fig. 2 e/f) of too-low and too-high decomposition temperatures are shown, respectively.", "The heating rates up to the first isothermal step were 20 K/min, as described earlier.", "After the isothermal steps, the heating rates were increased to 50 K/min to obtain a more pronounced peak shape.", "The determination of the isothermal steps was validated by the peak shapes of the CO2 and CO MS signals.", "The main decomposition product of carboxylic groups, is CO2.", "Nevertheless, fragmentation in the mass spectrometer results in a corresponding CO signal that additionally assists for the interpretation and analysis.", "In case of an isothermal step at 252 °C (Fig. 2 c/d) the first peaks have symmetric shapes and are marked with a green box.", "If the isothermal step is placed below 252 °C the decomposition is incomplete, which results in an interception and ongoing decomposition when the temperature is further increased.", "The consequence is the emergence of asymmetric peak shapes of the first and second arising peaks as seen in Fig. 2 a/b (red boxes), where the isothermal step was intentionally set below 252 °C.", "When the temperature of the isothermal step is above 252 °C, shoulders arise in the ion currents (red bar Fig. 2 e/f) due to the decomposition of the functional group with next-higher thermal stability.", "Comparison of peak shapes with different isothermal steps confirms that the thermal decomposition of carboxylic groups is concluded at 252 °C.", "The labeled arrows in the CO+ ion currents of Fig. 2 refer to the signals caused by the decomposition of different functional groups according to the numbers in Fig. 2.", "They illustrate, that an isothermal step at too-low temperature leads to a splitting of the decomposition of one functional group and an isothermal step at too-high temperatures impedes thermal separation.", "CO2 is generated from carboxylic, lactone (arm-chair, zig-zag), and anhydrate groups.", "CO is released by anhydride, phenol, ether and carbonyl groups (Fig. 3c).", "The optimization of each isothermal step on the basis of the CO2+ and CO+ ion currents is shown in the supporting information.", "The decomposition temperature of lactone groups under the release of CO2 depends on their structural connectivity.", "When they are located on zig-zag edges they exhibit lower stabilities in comparison to lactones located on arm-chair sites due to different strain of the rings as known from literature [23].", "This difference could be confirmed by the thermal separation method, and additionally the corresponding unknown decomposition temperatures, could be determined.", "Lactones at zig-zag edges decompose between 252 °C and 315 °C whereas lactones at arm-chaired structures show a specific decomposition temperature range between 510 °C and 620 °C.", "A special case among the functional groups is the anhydrides, due to the fact that they decompose in two subsequent steps under the release of equimolar amounts of first CO2 followed by CO.", "The release of CO2 between 315 °C and 510 °C is accompanied by the formation of a thermodynamically more stable intermediate that decomposes under the release of CO until 620 °C (Fig. 3a/b).", "A total decomposition of anhydrates is, therefore, only achieved at 620 °C.", "Separation of the phenol and ether functional groups is impossible as their decomposition temperatures overlap.", "With an isothermal step at 800 °C they could be separated from the decomposition of carbonyls.", "The step at 1000 °C is the highest technically feasible temperature, a complete decomposition of the carbonyl group is therefore not guaranteed additionally high temperature stable phenol groups as described by Pan and Yang can also not be detected [31].", "The ion current profiles of the optimized thermal separation procedure and the schematically visualized decomposition products and windows are shown in Fig. 3 a/b. Fig. 3c visualizes the decomposition temperatures up to the isothermal steps corresponding to the different functional groups of the optimized isothermal TPD analysis.", "The normed CO+ and CO2+ ion current profiles of nitrosulfuric acid functionalized MWCNT (orange) and graphite (blue) as well as untreated activated char (green) and Vulcan XC75C (red) with the six optimized isothermal steps are displayed.", "In principle, functional groups that decompose under the release of CO2 exhibit lower thermal stabilities than functional groups that decompose under the release of CO.", "As a result of the fifteen times higher surface area of MWCNT compared to graphite, more functional groups could be formed during the functionalization process.", "This results in a decreasing signal to noise ratio in case of the graphite CO+ ion current, since the same total masses were used for both samples.", "However, the CO2+ and CO+ ion current shapes of both materials are comparable after the implementation of the isothermal steps.", "The analysis of as received active char shows a different quantitative functional group distribution compared to the MWCNT and graphite samples.", "Active char exhibits a major fraction of higher stable functional groups of phenol/ether and carbonyl groups whereas carboxylic and zig-zag lactone groups are present in a minor scale.", "Anhydrides and arm-chaired lactones are also visible, but only in a minor extent as seen by the TG data (supporting information S7).", "Condensation reactions of adjacent carboxylic groups on the active char surface and the formation of anhydrites cannot be confirmed due to only minor water release during the measurement (supporting information S8).", "The thermal analysis of Vulcan XC75R exhibits that there are only a few functional groups present on the surface.", "TG data show a mass loss of only 0.2% apart from the loss of physisorbed water (supporting information S7).", "The CO+ ion current shows no peaks, small peaks can be observed for the CO2+ ion currents.", "The analysis of different graphitic carbon materials confirm, that the decomposition temperatures depend solely on the thermal stability of the functional groups and are not dependent on the carbon backbone, whereby the developed isothermal TPD analysis is applicable for different carbon materials with graphitic character.", "Nevertheless, it is noticeable that there is no baseline peak separation of the CO2+ and CO+ ion current of the MWCNT samples at temperatures over 620 °C.", "It is suggested that gas diffusion as well as adsorption and desorption processes affect the peak width of the desorbed gases in MWCNT samples.", "To determine the benefits and limitations of the linear and isothermal TPD methods, measurements from both heating procedures were compared.", "The analysis was performed in pure Argon as well in hydrogen-containing atmosphere (5% H2 in Ar).", "The resulting ion current profiles for CO2+, CO+ and H2O+ (m/z 18) are displayed in Fig. 4.", "The CO+ ion current profiles in Argon and hydrogen-containing atmosphere, recorded with a linear heating rate (Fig. 4b), exhibit significant differences.", "The most pronounced difference is marked with a yellow box where lower signal intensities are measured in the case of hydrogen containing atmosphere.", "The peak positions seem to be shifted to lower temperatures as well.", "The same trend can be observed in the CO2+ ion currents profiles (Fig. 4a).", "The lower thermal decomposition temperatures might due to the a dependence of decomposition mechanism on the atmosphere present, as is known from the literature [32].", "Furthermore an additional peak is observed between 600 °C and 750 °C.", "At the same temperature range, a water signal can be recorded in hydrogen containing atmosphere, which is absent during the measurement in inert gas atmosphere (Fig. 4c).", "The additional water peak at higher temperatures (blue box) is assumed to be caused by phenol groups, which release water during the decomposition [26].", "It cannot be differentiated between in-situ formed phenols and phenol groups that were generated during the acid functionalization procedure.", "The corresponding ion current profiles, determined with the isothermal heating program, are shown in the bottom graphs of Fig. 4.", "The CO signal in hydrogen atmosphere shows a pronounced difference in intensity for the phenol/ether group in comparison to the measurement in pure Argon (Fig. 4e).", "In inert gas atmosphere, OH species decompose in CO.", "In the presence of hydrogen, a concurrent reaction of the phenol group decomposition takes place with the formation of water.", "Therefore, less CO is generated during the thermal analysis in hydrogen atmosphere in step 5.", "In the case of isothermal heating procedure, the diminished CO intensities (yellow box, step 5) can be directly related to the absence of decomposing CO of the phenol groups, as other signal intensities are not influenced except of a slightly lower peak intensity of step 4.", "This might arise from the different decomposition temperature and the resulting lower thermal stability of phenol groups in hydrogen atmosphere, as discussed already for the linear heating range.", "Regarding the baseline of the CO+ ion current in both atmospheres, differences can be explained as well by changed decomposition temperatures in dependence of the atmosphere.", "Functional groups that decompose above 1000 °C in argon atmosphere, fall apart earlier in hydrogen atmosphere and cause the raised baseline [21].", "The isothermal steps were optimized for the case of argon atmosphere.", "This becomes even more pronounced regarding the CO2+ signals recorded in argon and hydrogen containing atmosphere (Fig. 4d).", "The ion current profiles in inert gas atmosphere show symmetric peak shapes, whereas the corresponding signals of the hydrogen atmosphere measurements reveal minor asymmetries.", "Analogous to the linear heating TPD, increased signal intensities can be obtained in hydrogen containing atmosphere.", "Furthermore, water can be detected in the same temperature range, where OH decomposes in the case of hydrogen-containing atmosphere.", "The m/z 18 signals cannot be detected in pure argon atmosphere (Fig. 4, blue box).", "The water signal at lower temperature is again an effect of a condensation reaction and removal of adsorbed water.", "In general the isothermal TPD procedure allows a much more detailed analysis of the ion currents due to the direct correlation to specific functional groups.", "Thermal separation will be validated by the application of a selective gas phase deposition technique, namely atomic layer deposition (ALD).", "Hereby, the metal-containing precursor reacts with specific functional groups of the support material resulting in the formation of a covalent bond.", "Further detailed information about the ALD process can be found in literature [33].", "In the present work vanadium(V) isopropoxide was used as a precursor in combination with water as a co-reactant to deposit vanadium oxides on functionalized MWCNT.", "The detailed synthesis and characterization of these catalyst materials are reported elsewhere [33,34].", "We show here that the precursor reacts selectively with specific functional groups (Fig. 5), and the reaction can be determined by the consumption of the reactive functional groups using the developed isothermal separation process.", "Therefore linear and isothermal TPD analysis was performed with a V-deposited MWCNT sample (V@MWCNT) and compared to the functionalized MWCNT base material without vanadium oxides.", "The detected CO+ and CO2+ MS signals of the linear and isothermal temperature program are shown in Fig. 6.", "The difference spectra illustrate the consumption of functional groups (negative peaks) and indicate an interaction between vanadium oxide and MWCNT (positive peaks) after the ALD process.", "In the case of the linear heating program (Fig. 6 a/b) a consumption of low thermally stable functional groups can be observed (Fig. 6a).", "The following temperature range exhibits a higher CO2 and a lower CO (Fig. 6b) release of the V@MWCNT sample than the functionalized support MWCNT.", "A distinct correlation to the consumption or formation of new functional groups after the ALD process cannot be assigned.", "The difference spectra in Fig. 6 a/b of the isothermal heating program reveal the selective consumption of functional groups after the ALD.", "The first negative CO2+ peak (Fig. 6a) is caused by consumption of carboxylic groups.", "An uncertain isothermal step would be seen by a consumption of the second peak, but here an increase of the CO2+ MS signal is detected confirming the thermal separation of carboxylic groups.", "Additionally derivatization and therefore consumption of the carboxylic groups was fulfilled with benzoyl chloride (supporting information S9) confirming the thermal separation due to the fact that only the CO2+ intensity of the first peak is influenced.", "The increased CO2+ signal of the difference spectra at step two can be assigned to newly formed, ester-like structures during the ALD process, as marked with an asterisk in Fig. 5.", "The increased CO2+ release is carried out over the whole range of step 2–5 and can be caused by ester-like groups with similar thermal stabilities as lactones.", "Direct interactions between vanadium oxide and MWCNT are also possible.", "The increased CO2 release has to be further investigated in detail.", "A crucial factor here is that during the isothermal step 2, up to 315 °C no CO release can be detected.", "This observation confirms that subsequent decomposition of the anhydrate group did not take place due to thermal separation during the isothermal step.", "The third isothermal step of CO2+ and CO+ MS signal and the fourth isothermal steps of the CO+ MS signal are attributed to anhydrates.", "After the deposition of vanadium oxide, a reduced CO+ signal is detected.", "This appearance can be explained as well by the formed ester-like structures of the anhydrate group with the precursor.", "Whereas one part of the precursor forms a O=C-O-V(O(OH)2) ester-like structure with the anhydrate group, the organic isopropyl part reacts with the appearing intermediate of the anhydrate group, forming again an ester-like structure O=C-O-V (Fig. 5).", "Both formed structures decompose with release of CO2, in contrast to the pure anhydrate group, where first CO2 is released and the formed intermediate decomposes to release CO.", "The thermal separation of step 5 from step 6 was already confirmed by the experiments performed in hydrogen containing atmosphere (Fig. 5).", "The arising water signal, observed at step 5 due to decomposition of phenol groups, cannot be detected at step 6.", "The drastic increase of the CO+ signal at step 6 is explained by the reduction of the vanadium species on the MWCNT.", "The reduction of vanadium was confirmed by XPS measurements, showing a reduced V oxidation state of the ex-situ annealed samples at 1000 °C in comparison to the annealed sample at 800 °C (Fig. S10).", "Quantifying the reduction of carboxyl and anhydride groups after the ALD process exhibits a total consumption of 0.3 mmol/g.", "The amount of deposited vanadium oxide detected via AAS shows a loading of 0.4 mmol/g on the MWCNT surface.", "Considering that only the consumption of carboxyl and anhydride groups can be taken into account, since the reduction of ALD active phenol groups cannot be analyzed, due to overlaying interactions between carbon and vanadium oxide in the temperature range of phenol group decomposition, the correlation between total consumption of functional groups and the amount of deposited vanadium oxide is clearly visible.", "The isothermal temperature program was also applied during the TPD analysis of differently pre-treated MWCNT (Fig. 7).", "The influence of different acid treatment methods on the formation of oxygen-containing functional groups was investigated.", "MWCNT treated with nitrosulfuric acid exhibit the highest amount of (and every kind of) oxygen functional group on their surfaces.", "Nitric acid treated MWCNT also exhibit every kinds of functional group, but in a lower quantity.", "Other treatment methods applied to the MWCNT exhibit only minor effects on the formation of functional groups compared to the untreated MWCNT.", "This finding is in very good agreement with previous studies, where the defect formation of differently treated MWCNT was investigated by Raman spectroscopy [27].", "It is notable that after the nitric acid treatment, phenol/ether and arm-chaired lactone groups are more dominant compared to the other functional groups, whereas, after nitrosulfuric acid treatment, the carboxylic, anhydride groups and phenol are very dominant (Fig. 7).", "The quantitative analysis of anhydride groups via CO2+ and CO+ illustrates again the high quality of peak separation.", "Anhydrides decompose during the thermal treatment into equal amounts of CO2 and CO, which could be detected by applying the investigated isothermal step TPD and quantitative analysis of both products.", "Furthermore, the comparison between pure MWCNT and hydrochloric and sulfuric acid-treated samples exhibit decreasing mass loss caused by the removed amorphous carbon from the surface (TG data supporting information S12).", "Hydrogen peroxide-treated samples exhibit a slightly increasing CO+ ion current between 800 °C and 1000 °C, which results from an increase of carbonyl (Fig. 7).", "In this work, an isothermal step TPD analysis was investigated and compared with linear TPD analysis.", "The isothermal procedure was used to separate functional groups with different decomposition temperatures.", "The separation was achieved by the implementation of isothermal steps at the end of each decomposition temperature of a given functional group.", "The temperature of these isothermal steps was optimized until the isolated CO2+ and CO+ ion currents showed symmetrical peak shapes.", "The separation by the isothermal steps allowed us to attribute a decomposition event to specific functional groups by interpreting of the detected CO+ and CO2+ signals.", "In general, CO2-releasing functional groups like carboxyls, lactones and anhydrides decompose at lower temperatures than CO releasing functional groups like phenols, ether and carbonyls.", "The behavior in hydrogen-containing atmosphere revealed the decomposition of phenol groups and the formation of H2O.", "Additionally, different acid-treated MWCNT were analyzed.", "It could be shown that only nitric and nitrosulfuric acid treatments have a significant influence on the generation of oxygen-containing functional groups.", "The ratios between the formed functional groups strongly depend on the treatment method and could be revealed by the isothermal TPD method.", "The isothermal analysis was also applied to vanadium containing MWCNT samples, synthesized with a selective gas phase atomic layer deposition (ALD) procedure.", "Hereby the reaction of specific functional groups (carboxyls, anhydrides, phenols) with the vanadium precursor could be identified.", "The consumption and generation of specific functional groups was discussed in detail.", "The direct correlation was not possible when the linear thermal analysis was applied.", "To conclude, due to the isothermal temperature program, changes of the CO2+ and CO+ ion currents could assigned.", "It allows a semi-quantitative analysis of the functional groups present on carbon surfaces.", "A quantification of the different separated functional groups is impossible by thermal analysis due to the dynamics during the annealing process.", "At high temperatures, reactions between released gases and functional groups or created defect sites influence the amount of detected gases, as well as conversions of functional groups during the heat treatment.", "The approach of isothermal TPD analysis is not limited to the analysis of surface functional groups on carbon materials.", "It may also be used to analyze serval overlapping events during TPD analysis of materials which were traditionally done with linear thermal TPD analysis.", "However, for these new applications the isothermal steps have to be optimized accordingly." ]	[]
Energy security and human security in a Dutch gasquake context: A case of localized performative politics	From the late 1980s, the natural gas extraction in the Netherlands has experienced an increasing number of ever stronger gasquakes (induced earthquakes due to gas extraction). This paper offers a security analysis of the accompanying debate on the material consequences and organization of the gas extraction between the threatened local population, the knowledge institutes analyzing the gasquakes, and the government and extraction industry. This paper studies how these parties make sense of the gasquakes through a combination of securitization theory and the flat relationality offered by new materialism, which forces the two conflicting securitization claims to be analyzed in their local sociotechnical and material context. The resulting analysis shows how the gas debate is structured by a shared security of supply understanding. An understanding which for a long time has been questioned by the local population on its safety and cost implications. However, it took 25 years until their claims were accepted and the security of supply understanding shifted to a focus on minimal extraction volumes. An acceptance that can only be explained through a self-reinforcing combination of security claims, actual material events, increasing measurements (following security calls), shifting value judgements and increasing audience acceptance (creating additional speech actors).	[ "ENER", "SOCI" ]	[ "Energy security", "Induced earthquakes", "Knowledge politics", "Security logics" ]	[ "2102", "2103", "2104", "2105", "3301" ]	[ "The Netherlands has been developing natural gas fields since the discovery of a large field in the region of Groningen in the 1950s.", "By 2015, this has resulted in 275 billion euro of state revenue as well as an infrastructure that connects almost all of the Dutch households to these gas fields for heating and cooking purposes.", "Simultaneously, the Groningen gas field is drained to roughly one-third of its original low calorific reserves (680 of its original 2800 billion m3).", "Unfortunately, from the mid-1980s onwards the areas above the fields have experienced light earthquakes, which have been increasing in magnitude and frequency (Fig. 1).", "For local residents, the everyday experience and (potential deadly) consequences of these earthquakes are conflicting with the long-standing national economic and security of supply concerns of the Dutch government and European energy markets.", "For a long time, the concerns for earthquakes remained limited to a small number of Groningen inhabitants.", "This changed with the 2012 Huizinge earthquake, which, as the strongest and most heavily experienced earthquake in the Groningen area to date, led to a large public debate and a string of reports on all aspects of the Dutch natural gas extraction and ultimately to a cap on extraction as of 2014.", "Consequently, the question is, why, after years of neglect, the security concerns of an initially small number of local residents suddenly superseded the energy security concerns of policy makers and energy scholars working on the Dutch gas and energy supplies.", "In other words, this paper studies the security politics behind an understanding of energy security.", "Within the literature, energy security has been described as a ‘slippery’, ‘fuzzy’ and ‘multidimensional’ concept [1,2].", "Definitions vary widely, but simultaneously often share common points of interest building around notions of security of supply, vital systems, and environmental and economic energy (in)security concerns [3,4].", "As such ‘the energy security concept nicely weaves together disparate policy issues into one basket ([5] p. 152).’", "Consequentially, the discussion on how energy security is used and defined seems ultimately a context bound one [1,6].", "As Pasqualetti ([7] p. 278) remarks in his reflection on a two-day meeting of 40 energy security experts: ‘Any discussion of energy security must recognize that it varies from one place and one culture to another, especially at the household level’.", "This is resolved in multiple ways.", "More traditional historical and geopolitical policy analyses draw conclusions from the debates they describe [8,9].", "Another prevalent approach is to map and develop the range of indicators and metrics used to analyze energy security [3,10,11].", "In a similar line, Cherp and Jewell ([6] p. 334), two strong proponents of a contextual approach, confirm the importance of ‘social reality in shaping perceptions of truth’ and identify a range of story lines about energy security, which they then categorize into a framework that can be used for further analysis.", "Elsewhere, they argue that ‘energy security is an instance of security in general’ ([84] p. 415) and thus needs to inquire about the often taken for granted values and assumptions behind energy security.", "This paper follows the notion that energy security, whether defined by scholars or as a shared understanding of participants, is part of a wider political spectrum.", "However, instead of defining what energy security is, it approaches the context bound nature of energy security by studying how it works.", "How one of its elements, in this case security of supply, becomes what those involved understand it to be, how it changes over time and how it shapes debates about energy production, transport and consumption in the meantime.", "‘The need for empirical investigation into the ramifications of using (…) energy security [claims], for what purpose and by whom ([5] p. 153)’ is not a new question, but due to its relatively small sample size remains an imperative one.", "Within the energy security literature it is studied mainly from a constructivist perspective on language and discourse [12,13] or from Securitization Theory [5,14–18].", "The discussion below builds on these studies in two ways.", "First, the gasquake debate offers an analysis of a central energy security concept, security of supply, in its broader societal context [15].", "As such, it does not study competing understandings of energy security [5] or the linguistic construction of a specific energy security understanding [12,17].", "Instead it shows how the Dutch security of supply understanding is influencing the debate and in turn is shaped by the resistance it faces coming from the safety concerns of the local population.", "This repetitive interaction between these two security concerns builds on a broader relational understanding that is at the heart of this paper.", "It is a relationality that, second, extends the discursive focus above by incorporating ideas of securitization into the flat relationality offered by New Materialist studies [19–21], in particular Actor-Network Theory [22,23].", "A flat relationality puts the shared understandings of security of supply and safety on an equal footing to the materiality of the earthquakes and the models used by the knowledge institutes.", "In other words, it reduces the analytical importance of the security claims by forcing the observer to study the security claims as part of their wider constantly changing context.", "The analysis itself builds on media coverage, news briefs, (court) statements and a number of reports, among them the 2015 report by the Dutch Safety Board (DSB) on the decision-making process behind the Dutch natural gas extraction from 1959 until 2014 [24].", "Although the debate is still ongoing, this paper focusses specifically on the period following the Huizinge earthquake in 2012 up to the autumn of 2015, as by then most of the major policy changes had taken place, including the decision to cap the extraction volume [25].", "This paper continues in Section 2 with an explication of the theory and subsequent contribution of this paper.", "Section 3 introduces the Huizinge earthquake and its consequences.", "Besides a discussion of the gasquake itself, this section touches in particular on a report from the main regulatory body, the State Supervision of Mines (SSM), which studied the Huizinge earthquake and shows how it is this report that actually shifted the value judgement behind the assessments of the decision-makers.", "Section 4 discusses the internationally encapsulated position of the gas-industrial complex, while Section 5 looks more closely at the safety and security claims of the locals.", "Section 6 moves on to discuss the knowledge politics behind the earthquakes in order to highlight the struggle over the uncertainty behind the scientific models and how security considerations play a role in this process.", "The reflection brings these lines together.", "This paper and the gasquake debate offers four main additions to the literature mentioned above.", "First, it completely conflates the distinction between security and politics.", "Second, it offers a long-term illustration that enforces a dynamic securitization analysis.", "Third, by placing security of supply in opposition to safety arguments, this debate not only studies two competing securitization processes but also places the resistance against current energy extraction practices in a Western context (contrary to insights from development studies on mining in Latin-America or Africa [26]) with an immediacy and geographical focus that is not always present when energy security is for example studied in relation to climate security.", "Lastly, the debate studies this immediacy itself and how it increased overtime by analyzing the security claims in relation to the actual gasquakes and the scientific knowledge about them.", "The reinforcing interaction between these elements and the actual material presence of the qasquakes (Sections 3 and 6) supports the theoretical argument that security should be placed in a flat relationality.2", "First, during the review of this article, the question was raised whether the below offered a discussion of energy security or instead was about energy politics.3", "The relation between security and politics has in fact long been a core issue within critical security studies, and one that is gaining traction once again [27,28].", "It stands at the center of Securitization Theory, developed in the 1990s, which sees security as a category separate from regular political and non-political issues ([14] pp. 23–24).", "In this theory, security is reserved for those extraordinary issues, like war, where an existential threat is believed to exist and extraordinary action are taken without concern for other social parties (e.g. outside normal political and economic routines) ([14] p. 26).", "Energy security does not often fall in that category and if it does it is subject to economic, climate, or military security ([14], [15] pp. 509–510; [17] p. 74).", "In this sense a discussion on energy security (contributing to its concepts and theories) as removed from a discussion on energy politics makes little sense as the former is always part of the latter and − as per the question above − the interaction itself deserves closer study.", "This is reflected in other security theories, where the distinction is more conflated.", "For instance, in Foucault’s biopolitics [29] or the security practices literature [30] security is not taken as a separate exceptionality but as a thoroughly routinized political process closely linked to knowledge practices (the ways people try to gain knowledge of the world) and the exercise of power.", "While these three theories approach the relation between security and politics differently, all share the view that security acts as a call for urgency based on distinctions between friend and enemy, safe and unsafe, risk and no risk, insecurity and security [31].", "They also share an understanding that threat images are performative.", "First, because the wording of these threats help shape the world we live in by defining an event (in doing so not only defining the event or enemy, but also oneself and the (referent) object in need of security).", "And second, because people act in the present to prevent these imagined future realities and through these acts materialize an alternative future.4", "On a political level, this implies that policy makers, scholars and other actors are not only responsible for a failure to counter threats, but also for the threats that they propose themselves: for the distinctions between friend/enemy, the choice of what to protect, and for the resources drawn from other areas in terms of actual resources and agenda setting ([32]p. xiii).", "Second, this paper also moves beyond the static nature of Securitization Theory with its implicit focus on a security claim made by a speech actor towards a single audience [33,34].", "With its focus on the speech act, the moment when a (securitizing) actor identifies and presents a future event as a threat and subsequently asks the audience for support to counter it, the theory is predicated to single acts from the perspective of the speech actor.", "While it would be fairly simple to write the Groningen gasquake debate solely in terms of the local population securitizing their safety concerns, it would offer a description of one actor (a representative of the local population) who calls for the threat (future consequences of more gasquakes) in the hope to convince the respective audience (decision maker).", "However, the illustration below shows how both the speech actor and the audience were not single entities and that they constantly evolved, as the growing audience acceptance of the gasquake threat subsequently meant a growing mass of speech actors, each calling for the gasquake threats in their own way and wording (see Section 5).", "Third, even though a more dynamic long term reading of Securitization Theory would pick up on this, it would still only focus on one event and its accompanying security claim (the threat of future gasquakes).", "While there is some work done on conflicting energy security claims and interpretations [16] and competing security claims are one of the main reasons for issues to lose their urgency [35], most of the work done on how energy security relates to other concerns sees energy security as subject to military and economic concerns (above) or involves negotiations in line with the energy trilemma [36].", "In contrast, the gasquake debate offers a case with two active security processes.", "A case where the securitization of the safety concerns interacts with the (institutionalized) security of supply concerns that dominate the debate.5", "For even though security of supply considerations often remained implicit, as the debate primarily focused on safety concerns, compensation and extraction volumes, it was central to it.", "For the governing bodies and gas industry, for whom security of supply was one of the core reasons to continue with business as usual.", "And subsequently, it was also central to the local population, for whom security of supply presented the boundary of what they could achieve.", "Irrespective popular calls for a complete halt of extraction, on a political and legal level the argument revolved around safe extraction and a minimalization of volumes (see Section 5).", "In doing so, the negotiations and subsequent actions of the actors involved reproduced security of supply as a central motif, but with a redefined understanding of what it meant.", "In contrast to an earlier shift in the meaning of security of supply from its initial unlimited extraction to the strategic ‘small gas field policy’, which resulted in equal measure from economic and security of supply reasoning (Section 4), this redefined understanding stems primarily from a security debate.", "A debate that resulted in an adaptation of the Dutch security of supply understanding away from economic gain towards a focus on the minimal extraction necessary for a continuation of energy demand within the existing sociotechnical infrastructure.", "Lastly, the debate nuances the discursive nature of security, something that comes across most strongly in the idea in Securitization Theory that security is ‘a self-referential practice’ ([14] p. 24) and that threats are always imaginary (as they have not happened yet) and hence have no material standing.", "As Van Wijk and Fischhendler ([37] p. 22 quoting Adam and Van Loon, 2000) argue in relation to risk: ‘the question of whether risk is genuine or not is irrelevant.", "What matters instead is the actualisation of risk in policy processes in general ….’", "Yet even when agreeing that threats are imaginary, the gasquake debate also offers two ways in which material elements played an active role.", "On the one hand, the support for the safety claims of the local population grew with the tangible experience of ever more and stronger gasquakes.", "In time it was accepted that the qasquakes threatened local lives and livelihoods (a clear sharable referent object even with those not directly threatened), that a reduction of extraction volumes was an effective (if not the only) solution, and most of all that the causal relationship between extraction, gasquakes and increasing damage was undisputable ([17] p. 74).", "On the other hand, the security of supply understanding has always been closely related to the existing gas infrastructure in the Netherlands.", "It is the existing infrastructure, including the conversion and extraction capacity and the gas fired boilers (with resulting demand for gas), and the expected rate of change of that infrastructure, which offers the boundaries of minimum extraction capacity and thereby the current boundary of the local safety claim.", "In other words, the debate highlights the need for security scholars to focus on the interaction between threat images, material events and especially the knowledge practices that mediate between these two.", "Contrary to the facilitating conditions that Securitization Theory offers, where material elements are subject to security ([14][14] p. 33), this paper places them in a flat relational ontology that equalizes them to security images.", "Such a flat relational ontology is proposed in the field which by lack of a better term is called ‘new materialism’ [19–21].", "Scholars in this broad field have long been uneasy with a sole focus on linguistic explanations in constructivist work, while not willing to refer back to more traditional realist and objectivist approaches as these lose the performative insight that our shared social understandings of the world help shape it.", "While I prefer the work of Barad and Foucault, for simplicity’s sake and the intent of this special issue I will use the indicative and very popular relational alternative offered by Actor-Network Theory (ANT) [22,23].", "This theory depicts the world to exist out of networks of nodes, which themselves are networks, and so on.", "These networks are irreducible and flat, meaning that one should not and cannot prefer one element over another.", "Whether that element is a social explanation (energy security), something material (a gas well), a law or a newspaper article, all these elements together form our current world.", "Importantly, as each node exists out of their own networks, theoretically, if any of these nodes is missing, the world is not the same.", "In other words, security claims and images are only one element out of many and they only have their specific meaning as part of a broader ‘security apparatus’([29]; c.f. [38]).", "As Latour summarizes the goal of ANT: ‘It simply means not to impose a priori some spurious asymmetry among human intentional action and a material world of causal relations ([22] p. 76).’", "Instead of assuming who acts (the speech actor) the idea is to actually study who acts by observing the traces that are left when relations change (the damage after an gasquake, a changed interpretation in reports, a new scientific model, etc.).", "Luckily, the core assumption is that networks are constantly changing and that stability is actually a product of hard work.", "In this sense, a security argument is the associative work done to create some semblance of stability in an ever-changing environment.", "The reading below shows how the local inhabitants, the Grunningers in their dialect, here represented by the Groninger Bodem Beweging (GBB, which translates as Groningen Ground Movement), have been increasingly successful in securitizing the gasquakes as a threat to their livelihood.", "They have managed to put the induced earthquakes on the political agenda with a sense of urgency, while achieving a reduction in gas extraction and an increase in monetary compensation.", "The lesson however is that they were not believed until the number of measurements increased – which only occurred after repeated security claims – nor granted their urgency until the materiality of the gasquakes became visible through those measurements and material effects.", "Just calling for the threat of the gasquakes had little effect, as shown by the duration of the debate, but including the scientific uncertainty in those safety arguments ultimately did.", "Especially, as the uncertainty led to more seismographs, which led to more measured gasquakes, thereby strengthening the claims of the local population.", "Simultaneously, one can witness within government and industry a debate geared towards the weighing of the benefits of natural gas extraction and the risk and consequences of these earthquakes.", "For these institutions, the debate itself hardly changed so much as the numbers and valuation behind them.", "Consequently, these assessments (the weighing of security of supply, safety and profit) and the uncertainty behind the scientific knowledge over these tremors lie at the core of the struggle between the local population and the gas-industrial complex (het Gasgebouw)6: the small and closely connected group of companies and institutions in charge of Dutch natural gas extraction.7", "With a score of 3.6 on the Richter scale, the Huizinge earthquake of 16 August 2012 was the strongest ever experienced in the Groningen area.8", "To understand its impact, it is necessary to place it in the ongoing debate on the potential relation between gas extraction and earthquakes.", "This includes the history that the Grunningers have with an increasing number of such earthquakes, which also have been of steadily higher magnitude (see Fig. 1), and their struggle for acknowledgement of these quakes.", "Moreover, it includes the public pressure that resulted a year later from the news that 2013 turned out to be a record year.", "Both in terms of an exceptionally high extraction volume (up to 54 billion Nm3: [39]), with subsequent revenues, and a record number of 119 tremors of which about 20 could be felt by inhabitants (out of 133 total in the Netherlands: [40]).", "Within the debate on the gasquakes in Groningen, the Huizinge earthquake thus acts as a turning point.", "Not only because its magnitude and subsequent public attention made the Grunningers start to protest in earnest (while allowing them a media platform), but also because it turned out to be the event that made officials acknowledge the need for a shift in their values.", "In this respect, the unsolicited report of the State Supervision of Mines (SSM) in response to the Huizinge earthquake [41] turned out especially important.", "As the official supervisory body, the SSM is responsible to ensure that any mining activities in the Netherlands are in accordance with the mining law [42].", "Even though the Huizinge earthquake fell below the maximum of 3.9 on the Richter scale that was calculated in earlier risk assessments, the SSM initiated a study in response to the growing unrest under the local population ([41]; see also [24] p. 66; [43]).", "In this unsolicited report, the SSM, until 2015 still part of the Ministry of Economic Affairs (and thus part of the gas-industrial complex), openly and strongly questions the constant adjustments and increases in the maximum magnitude that risk assessments have put forward since the early 1990s.", "The SSM combined this with earlier discussions on methodological uncertainties (see Section 6), and, for the first time, put these uncertainties upfront.", "Subsequently, it concluded that it is impossible to estimate a possible maximum magnitude for the induced earthquakes in Groningen, even going so far as to take a magnitude of 5.0 as a valid possibility.", "To prevent this, it advised the Minister of Economic Affairs to reduce the output of the Groningen gas field as quickly and drastically as possible.", "The SSM’s shift came as a surprise for the other parties within the gas-industrial complex ([24] p. 77) and its advise was not immediately accepted by the Ministry of Economic Affairs.", "Instead the Minister called for more research on the relation and effects of the earthquakes and the gas extraction (15 different studies in total, among them one on the minimal level needed to uphold security of supply, see: [44]).", "He did this, even though he acknowledged the chance for higher magnitude quakes and agreed with the NAM on a sum of 100 million euros for preventive construction measures [45–48].", "The SSM report meanwhile influenced the sub-national Province of Groningen to initiate its own study, which repeated the main conclusions of the SSM and thereby confirmed the local concerns.", "The local population, in turn, used these reports in their protests [45,49].", "Late 2013 the debate heated up once more following the range of reports requested by the Minister, which confirmed many of the concerns voiced up to that moment, as well as the news that 2013 turned out to be a record year [39,40,50–52].", "This time the government heeded the concerns and decided on a range of issues.", "These included, among others, the organization of an open dialogue among all affected private and public parties [53].", "The government also tasked the NAM to conduct a full-scale below ground survey (which was missing so far) and to reduce the extraction in the most effected clusters (while making up for the losses in other clusters).", "Simultaneously, it increased construction standards and preventative measures, while also improving the administrative procedures behind the compensation claims.", "And it offered the region an overall package to improve its economic and employment perspectives [54].", "These measures were reinforced in the winter of 2014–2015, at which point the Minister of Economic Affairs initiated a first provisional cap on the total gas extraction from the Groningen field, which has since been extended [55,56].", "Even though the Ministry of Economic Affairs reduced production at specific clusters and initiated a cap on the total extracted volume, there remains a strong political debate about the installation of permanent extraction quotas.", "Currently, the safety concerns of the Grunningers are acknowledged.", "However, the parties responsible for gas extraction are arguing that they are bound to produce whatever is needed in response to contractual and seasonal demand from the Dutch consumers and the European countries that have bought Groningen gas on long term contracts, and as such cannot limit themselves by installing a definite extraction cap [25,56].", "For instance, in its 2013 reaction to the reports from the SSM and other institutes on the Huizinge Earthquake the ministry argued that:", "In the near future, the Groningen gas extraction cannot be substituted by gas imports or other measures.", "A diminished availability of the Groningen natural gas will have serious consequences for the Dutch society and the societies in our surrounding countries ([46] p. 4).", "Likewise in 2015, after the initial decision to cap the volume, the ministry stated that:", "The consequences of long term gas extraction in Groningen have become increasingly clear in recent years.", "… Simultaneously, the gas extraction is of essential importance for the energy supply in the Netherlands.", "Both the mixture of the gas and the fact that the gas from Groningen, due to its size, can be used flexibly, makes that a reduction from the Groningen gas field could lead to problems with the heating of buildings or other usages.", "In addition, for multiple decades the gas extraction is an important source of income for the Dutch state ([57] p. 4).", "This position has since been confirmed – but simultaneously limited − with a ruling from the highest administrative court in the Netherlands [58,59].", "In its ruling, the court argued that in the assessment of the balance between the safety of the local population and security of supply (which the court defines as the low-calorific natural gas needed to comply with the demand for this type of gas) the Minister had not explained why he chose the demand from a harsh winter scenario that Gasunie Transport Services B.V. (GTS) calculated as the benchmark for the minimal supply needed [44].", "As such, the court considered that the lowest minimal production was not 30 bn.", "Nm3 as favored by Economic Affairs, but that it should follow a more average scenario of 27 bn.", "Nm3 (with upward allowances for harsh winters).", "This debate can be placed in an energy security context where the role of gas is already shifting for the Netherlands.", "Before the 1960s, the Dutch did not experience gas insecurity, simply because the level of gas consumption was minimal.", "Nowadays, the Netherlands is highly gas dependent, but not seen as gas insecure because most of its gas is extracted domestically.", "With the draining of the Groningen gas field and other Dutch natural gas reserves this is expected to change.", "In the near future, the Dutch will be gas insecure precisely because they have come to rely on it.", "Similarly, while the Netherlands is one of the least dependent European countries on Russian gas, it does import Russian gas and expects to increase its imports in the future to balance the reduction (and capping) of its emptying domestic fields – negatively influencing its security of supply position.", "To counter this position in the post-gas era, the Dutch state has launched an ambitious strategy to become the “gas roundabout” of North-Western Europe, combining its central position and the empty gas fields as its natural storage facilities [60].", "This gas roundabout idea aims to profit from the material (empty gas fields, pipelines and pumping and conversion capacity), legal (national and European long-term contract law and other regulations) and social (knowledgeable and influential gas elites) infrastructure that supports current gas extraction practices.", "It is especially the latter social framework and the practices resulting from it that the Dutch Safety Board describes as encumbering the incorporation of the everyday safety of individual citizens in the gas extraction decisions.", "The board describes the everyday decision-making of these organizations as driven by three main paradigms: (1) maximum profits and winnings, (2) an optimal and strategic use of the natural resources, and (3) a continuity of Dutch gas supplies for both citizens and industry ([24] pp. 70–71; comparable: [51] p. 4).", "All three are captured in the 1974 small fields policy that replaced the initial unstructured pumping of gas in the early 1960s with a more strategic and economic long term vision, based on an optimal development of new small gas fields by giving those fields priority on the Dutch gas market while using the Groningen field more sparingly as a swing field to fulfill the rest of demand [61].", "In addition, the board also concludes that ‘all efforts within the gas-industrial complex are aimed towards an imperceptible extraction of natural gas’ ([24]p. 74).", "Together these four maxims for a long time structured the everyday practices within the gas-industrial complex towards the Groningen natural gas field.", "Importantly, they exclude the safety and insecurity concerns of the locals, except as a condition to be met for the other goals ([24] p. 71; [62]).", "This, in turn, preconditioned the initial response from the organizations to claims of insecurity by locals, and it explains why these responses, for a long time, have been soothing instead of informative and why they only followed the scientific updates of the magnitude – without repeating the mentioned uncertainties and knowledge gaps ([24] pp. 81–82, 86).", "Up until the debate following the SSM report, the gasquakes were considered an externality of the gas extraction, to be paid off through damage payments.", "Security of supply arguments simply meant business as usual and an optimal utilization of the Dutch gas fields.", "With the SSM report and the increasing pressure of the Grunningers, media and other parties (like local governments, safety regions, environmental groupings, and so on), the discussion for the gas-industrial complex shifted to more extensive compensatory measures and mitigation practices, while opening up the question when precisely the Netherlands would be gas secure?", "Looking back, the gasquake protests and subsequent shift from the SSM and other knowledge institutes forced the government to look closer at its understanding of security of supply.", "In doing so, it renewed its security of supply considerations that structure the energy debates in the Netherlands, but this time interpreted it no longer in economic or strategic terms, but in terms of a minimal gas extraction.", "The minimal extraction that is required to fulfill expected demand within the current sociotechnical gas infrastructure including the technical capacity to deliver this through alternative means.", "The local inhabitants above the Groningen gas field do not primarily deal with energy security concerns, contractual obligations and other risk assessments.", "Instead, they deal with a steady increase in frequency and magnitude of induced earthquakes caused by the natural gas extraction, a reluctant acknowledgement of the causality between extraction, quakes and damages, the difficulties in getting their direct and indirect earthquake damages reimbursed, decreasing house prices, a soothing communication by the gas-industrial complex and the perceived unequal distribution of the gas benefits ([63–65], [66] pp. 7–9).", "These grievances and concerns are being voiced through media channels, legal procedures and letters of complaint to official institutions both regionally (like the safety region of Groningen and municipalities which in turn also started to petition) and nationally (the SSM, parliament, the Minister of Economic Affairs, and other regulatory and political institutions).", "In addition, the street protests grew in number and size to gatherings of hundreds of people late 2014 [67].", "These protests mainly focused on three claims.", "First, an overall demand for more attention and acceptance of the urgency of their problems with banners like ‘Groningen a ticking time bomb’ [68].", "Second, especially later in the debate, showcasing their distrust towards the gas-industrial complex and in particular the NAM and the Minister of Economic Affairs with banners like ‘Groningen tremors, but The Hague will quiver’ [69].", "And of course, the need to prevent further gasquakes by calling for a reduction of gas extraction, with banners reading ‘Kamp Gas Terug Nu’, calling on Kamp, the minister of Economic Affairs, to take his foot of the gas throttle [70].", "Obviously, some Grunningers have been aware of the gasquakes since the early 1990s and have tried to make themselves heard over time, either as individuals (see [71] on Van der Sluis; or more recent [72]) or through well-organized associations like the Groninger Ground Movement [63,73] or the more activist Shocking Groningen [74].", "Looking back earlier into the debate it is illustrative that it took 7 years and a large interdisciplinary study after the first earthquake in 1986 for the government and the NAM to officially acknowledge that the quakes were directly linked to the gas extraction [75].", "Until that study in 1993, the NAM ridiculed any claims from individuals and organizations that proposed such a link [71].", "Once recognized, it took another 20 years for the official parties to start taking the risks and potential consequences of these induced earthquakes serious enough to adapt their extraction volumes.", "In those 20 years, every couple of years NAM, government and knowledge institutes have been forced to increase their estimates on the frequency and magnitude of potential quakes.", "This eventful material reality and the decades of uncertain knowledge claims that accompany it (only reinforced by the slow response and delaying tactics of the gas-industrial complex since the Huizinge earthquake [76]) have led to feelings of insecurity and distrust [66].", "That it took until 2013 for the uncertainty behind the earthquakes to become widespread public knowledge, can be explained, so the argument goes, by the idea that the Grunningers were loyal and felt a sense of pride for helping the country to develop as whole ([45] p. 21).", "Something that slowly changed in time with the constant adjustment of the risk analyses and maximum magnitude of the earthquakes.", "In their search for answers, ever more people started to read the actual reports.", "They called on the uncertainty itself – in depth – in their letters of complaint to official institutions, for example to parliament (see [77]).", "The SSM report can be described as a turning point in this respect as well, as it not only informed the gas-industrial complex on the uncertainty in the analyses used so far, but also supported and legitimized earlier readings of the Grunningers.", "As Van der Voort & Vanclay argue: ‘[t]he publication of the SSM report was an impact in itself with people becoming more anxious about what will happen to them (2015, p. 8).’", "With the report and the media attention following it, a broader group of people learned that there was no certainty in store for them.", "What is more, the subsequent decision of the Minister of Economic Affairs to not directly follow the advice from the SSM gave the local population further ammunition and a clear focus for their grievances.", "One of the largest and most organized interest groups is the GBB, which is actively lobbying, securitizing and litigating against the gas-industrial complex.", "In terms of security of supply, the GBB has constantly maintained that ‘the extraction is reduced or halted until independent research shows at what level extraction can take place safely and securely ([78] p. 4).’", "More important, ‘if “safe extraction” is technical impossible, the GBB demands a total halt to the gas extraction ([63] p. 1)’.", "The GBB, together with other interested parties, made this tangible in their appeal to the highest administrative court in the Netherlands [79].", "Building on their earlier call that the Minister was not acting, here they argued that Economic Affairs had not sufficiently substantiated the decision to cap the gas extraction.", "It based its decisions on the certainties it had (the budgetary necessity of the gas benefits and security of supply), as opposed to the many uncertainties that accompany the gas extraction ([62] p. 7).", "During the hearing, they called for a preferred reduction to 12 bn.", "Nm3, which the SSM in 2013 argued was a safe extraction level [43], only accepting 21 bn.", "Nm3 if practical circumstances (read minimal security of supply and international contractual obligations) dictated otherwise in line with the bottom bandwidth of the GTS [44] report.", "Although they were unhappy with the legal ruling which confirmed the prevalence of security of supply concerns and set the level at 27 bn.", "Nm3 [80], the second options shows that the GBB is forced to accept security of supply as a primary consideration in the debate.", "In other words, while the local population does not primarily discuss the gasquakes in terms of security of supply, the governments’ security of supply considerations are a constant absent presence in their claims for recognition, safety and a reduction of natural gas extraction.", "Behind the debate on the minimal extraction volume, lies the lack of knowledge over the tremors; both in terms of the availability of seismographs to actually monitor them and in terms of the uncertainty of the scientific models that are used to analyze and predict the gasquakes.", "In this respect, the Groningen gas debate shows (1) that what is not monitored and measured cannot be known, (2) that researchers had little incentives to work on the uncertainties in their models, especially (3) when the institutions using and sponsoring the results are happy with the outcomes.", "For the argument of this paper, the debate shows that the decision to monitor is just as political as the decision to cap the extraction.", "Meaning that these decisions are heavily influenced by the interests and (energy) security positions of the parties involved, while simultaneously the parties feel justified in their positions by the subsequent outcomes of the studies.", "The only reason why the 1986 earthquake near Assen, a town just below the Province of Groningen with its own small gas field, was identified as an earthquake, contrary to other orally reported “air” tremors [65], was because it was strong enough to be picked up by the sensors of the Royal Netherlands Meteorological Institution (KNMI) in the middle of the country.", "One of the main results of the debate that followed was that the Dutch Parliament influenced the Minister of Economic Affairs to order the KNMI – for which the earthquake was unexplainable as it lacked any data – to install a number of seismographs around the Assen field in 1989 and around the Groningen field in 1992 [45,64,65,81].", "This brings up the question whether the start of Fig. 1 resulted from the first true Groninger gasquake or was actually a result of the capacity to monitor them.", "Either way, new waves of attention and research followed, among others in 1993, 1995, 1997, 2000, 2003, 2004, 2006 and 2009 due to both earthquakes and regulatory changes.", "Of these, 2004 is of interest on two accounts.", "First, because the NAM for the first time publicly acknowledged that the maximum magnitude could be corrected if necessary, thereby implying that these cannot be estimated upfront.", "And second, because the 2004 KNMI report not only increased the maximum magnitude to 3.9 after an update of its database, but also acknowledged that it was using static models for a situation that was not static (as the earthquakes result from shifting levels of gas extraction).", "However, it stated that it simply lacked the tools and sub-surface information to cope with the fluctuating gas extraction and its relation with the induced earthquakes ([82] ; see also [83]; for a similar study after the Huizinge earthquake; or the [75] report for a predecessor).", "It took until the Huizinge earthquake and the report by the SSM for the number of studies to increase dramatically (as did the number of seismographs and thereby the number of previously undetected gasquakes).", "Many of these reports were commissioned as part of the large interdisciplinary study on the gas chain by the Ministry of Economic Affairs, which was receiving conflicting advice from the parties involved.", "Before 2013, Economic Affairs relied on the reports of the KNMI and other knowledge institutes and it expected these reports to be paid for by the NAM after its legal obligation to take the necessary safety precautions.", "However, as the Dutch Safety Board concludes, the NAM, as the exploiter most knowledgeable of the gas fields, welcomed the results from these reports that the earthquakes would only have a minimum impact and hence saw no cause to order additional studies on the uncertainties mentioned in the reports ([24] pp. 65–66).", "It also took the semi-independent SSM until 2012 to put forward the uncertainties behind the scientific models and risk assessments.", "With the NAM unwilling to study the scientific uncertainties, the Ministry passively relying on advice and the SSM (and other expert councils) confirming the official reports, there was little incentive for the KNMI and other knowledge institutes to build alternative models.", "They tried, in part by looking at international research, but then quickly ran in to the unique material qualities of the Groningen gas field (see note 3; [24] p. 64).", "What remained were the static models and their outcomes, which ‘focused on the number, the estimated maximum magnitude ([24] p. 63).’", "In the end, this focus on the number worked both ways.", "It simplified matters for the gas-industrial complex as it constantly reinforced their position that the gasquakes did not pose a safety concern, but over time also fueled the distrust and uncertainty of the Grunningers for whom the message not to worry contrasted with the constant adjustments and their experience of the actual earthquakes.", "The 2013 SSM report in this sense is interesting as it shows the importance of reflexivity within an energy security and safety debate, in particular the acknowledgement that security of supply still needs a value judgement.", "Clearly, the Grunningers with their focus on safety (and their search for answers) judged this threshold differently than the gas-industrial complex that looked at the balance between costs, profits, legal obligations and security of supply.", "The Groningen gasquake debate offers an example of a security debate in a developed country that puts security of supply considerations in the context of human security arguments.", "The analysis builds on the interaction between three lines of enquiry.", "First, it takes seriously the material reality of the earthquakes, their impacts, but also the gas field itself and the infrastructure around it, which keeps the debate localized (no earthquakes outside Groningen) and situated in a Northern-European market through its pipelines at the same time.", "Second, it highlights the politics over these earthquakes in terms of their origin and their potential impact, as well as the knowledge politics related to the scientific uncertainty of the models behind the earthquakes and their future trends.", "Third, it illustrates the struggle by the Grunningers to attribute a sense of urgency to both the materiality of the earthquakes and their future uncertainty.", "While successful, the above also shows that the local population could not escape the assessments of the gas-industrial complex.", "In fact, in order to break with the conservative force stemming from an unreflective use of security of supply and in order to increase the audience acceptance of their safety claim, the local population needed, firstly (and ironically), the frequency and magnitude of the earthquakes as well as the visibility of their impact.", "Secondly, they helped speed this up by focusing on the scientific uncertainties in the reports, slowly convincing neighbors and local authorities and then media, semi-regulatory institutions, and so on.", "Thirdly, once the SSM accepted the claim and published the report that legitimized the safety claim, the focus shifted to the decisions of the Minister of Economic Affairs.", "Initially the decision not to act, thereafter to the decision about the level at which the natural gas extraction was capped.", "That said, while deciding to wait, the minister did already demand the report from the GTS on the minimal security of supply levels indicating an early acceptance to review the Dutch security of supply position.", "In reflection, this illustration highlights the importance of a contextual understanding of security of supply.", "Throughout the debate, the gas-industrial complex slowly increased its willingness to accept and compensate for the consequences of the gas extraction.", "It was not until it had compromised on all most all aspects of the gas supply chain that it was willing to consider a closer look at the meaning of security of supply and reduce the extracted volume for safety concerns.", "Similarly, the case shows that while the Grunningers call for a further reduction, they ultimately take a pragmatic legal stance towards a minimum extraction volume based on security of supply considerations.", "The debate thus centered on the meaning of security of supply as it relates to the low calorific natural gas of the Groningen gas field, the estimated demand and the available technical capacity to substitute the Groningen gas.", "From a security perspective, however, the local population through their resistance against the gas extraction practices helped reify the principle of security of supply.", "Even though their concerns fit a potential wider understanding of human energy security, they never really successfully questioned the security of supply principles behind the decision-making processes.", "Consequently, security of supply has been reproduced while it structured the debate by setting the outermost boundaries of acceptable actions and reductions.", "That said, it has not been reproduced the same: the gasquakes have challenged the Dutch notion of security of supply, with natural gas no longer seen as a silent and bountiful resource but as a necessity to which the country is addicted.", "From this we can draw three lessons.", "First, the need to study the use of security and threat images in their wider context.", "This should include the sociotechnical energy infrastructure, material causal events, and especially how security practices relate to other knowledge practices that are used to make sense of these events.", "Second, besides the insight that people who use security language are morally responsible for the distinctions they make, the agenda setting power they exert and the resources that are drawn from other options, there is the lesson that security is simultaneously about urgency and conservation.", "It is about protecting and conserving a certain situation, except that it always fails as security changes things itself.", "Protecting the existing gas extraction volumes had clear impacts, just as arguing for the safety and living standards had an impact on the actual gas extraction.", "Security is never static and the search for the definition of what energy security is can therefore only be described as an active political intervention itself.", "Energy security scholars should be aware of this and not only study “new” security threats or categorize old ones, but explicitly focus on existing energy security practices, their distinctions and the constant renegotiation and hard work that keep them stable." ]	[ "Introduces the Dutch debate on induced earthquakes following natural gas extraction.", "By placing securitization theory in a flat relational ontology derived from ANT.", "To study the knowledge practices mediating between security claims and materiality.", "As well as the challenge of a safety claim to a security of supply understanding.", "Which shifted the Dutch notion of security of supply towards minimal extraction." ]
Transmission blocking potency and immunogenicity of a plant-produced Pvs25-based subunit vaccine against Plasmodium vivax	Malaria transmission blocking (TB) vaccines (TBVs) directed against proteins expressed on the sexual stages of Plasmodium parasites are a potentially effective means to reduce transmission. Antibodies induced by TBVs block parasite development in the mosquito, and thus inhibit transmission to further human hosts. The ookinete surface protein P25 is a primary target for TBV development. Recently, transient expression in plants using hybrid viral vectors has demonstrated potential as a strategy for cost-effective and scalable production of recombinant vaccines. Using a plant virus-based expression system, we produced recombinant P25 protein of Plasmodium vivax (Pvs25) in Nicotiana benthamiana fused to a modified lichenase carrier protein. This candidate vaccine, Pvs25-FhCMB, was purified, characterized and evaluated for immunogenicity and efficacy using multiple adjuvants in a transgenic rodent model. An in vivo TB effect of up to a 65% reduction in intensity and 54% reduction in prevalence was observed using Abisco-100 adjuvant. The ability of this immunogen to induce a TB response was additionally combined with heterologous prime-boost vaccination with viral vectors expressing Pvs25. Significant blockade was observed when combining both platforms, achieving a 74% and 68% reduction in intensity and prevalence, respectively. This observation was confirmed by direct membrane feeding on field P. vivax samples, resulting in reductions in intensity/prevalence of 85.3% and 25.5%. These data demonstrate the potential of this vaccine candidate and support the feasibility of expressing Plasmodium antigens in a plant-based system for the production of TBVs, while demonstrating the potential advantages of combining multiple vaccine delivery systems to maximize efficacy.	[ "BIOC", "IMMU", "MEDI", "VETE" ]	[ "Malaria", "Plant-produced antigen", "Plasmodium", "Pvs25", "Subunit vaccine", "Transmission blocking vaccine" ]	[ "1313", "2400", "2725", "2739", "3400" ]	[ "Malaria is a global disease caused by parasites of the genus Plasmodium.", "An estimated 207 million cases of malaria are reported annually, causing approximately 627,000 deaths [1,2].", "Of the five species of malaria parasites that infect humans, Plasmodium falciparum and Plasmodium vivax lead to the greatest burden of disease.", "While P. falciparum is responsible for the majority of malaria-linked deaths, P. vivax can cause relapses months after the first infection [4] caused by hypnozoites, and require specialized treatment, e.g. primaquine [2–4].", "P. vivax is the most widely distributed human malaria parasite, with 2.5 billion people at risk of infection, and 80−300 million cases per annum [1,3].", "Multiple factors, including the appearance of chloroquine-resistant P. vivax [5], lack of practical alternatives to primaquine, combined with increasing global temperatures [6,7], have raised concerns related to increased risks of P. vivax-induced disease.", "No licensed vaccine for malarial prophylaxis is currently available [8,9].", "Chemotherapy is the only clinically available treatment for confirmed infection; however, recurring drug resistance reduces the efficiency of anti-malarials [4].", "The importance of vaccines against P. vivax is well understood, but a lack of long-term in vitro culture systems and suitable animal models have hindered developmental advances.", "Malaria transmission-blocking (TB) vaccines (TBVs) show promise as a method to reduce transmission.", "Briefly, antibodies produced within an individual in response to vaccination are ingested by the mosquito along with gametocytes, during a bloodmeal.", "These antibodies prevent parasite development in the mosquito midgut by binding to surface proteins of the sexual stages, impeding further transmission [10,11].", "One of the primary targets for TBV development is the P25 protein, expressed predominantly on the surface of the Plasmodium zygote and ookinete [12].", "P25 is characterized by the presence of epidermal growth factor (EGF)-like motifs, multiple cysteine residues and a complex tertiary structure [13], making it challenging to produce recombinant protein with appropriate conformation.", "Previous studies that have successfully expressed P. vivax P25 (Pvs25) in native conformation have been limited to small-scale studies, with “classical” recombinant expression systems [15–17] or using multiple viral delivery systems [18,19,39].", "Previously, studies have examined the potential of utilizing plants as a cost-effective and scalable platform for vaccine production [20,21].", "Nicotiana benthamiana has been used in conjunction with a hybrid plant virus vector-based expression system [22] to produce subunit vaccine candidates against influenza, plague and anthrax [23–28].", "In related studies, this system has produced soluble versions of P. falciparum P25 (Pfs25), either as stand-alone proteins or as fusions to the modified lichenase carrier molecule (LicKM).", "In mice and rabbits, fusion and non-fusion versions of plant-produced Pfs25 elicited high titers of anti-Pfs25 antibodies when administered with Alhydrogel as an adjuvant.", "These antibodies demonstrated potent TB activity [29,30].", "Here, a Pvs25-LicKM fusion protein (Pvs25-FhCMB) was produced using N. benthamiana as an expression host.", "We examined efficacy of this candidate vaccine by performing a head-to-head comparison of induced TB potency following mouse immunization with recombinant Pvs25-FhCMB in the presence of two clinically relevant adjuvants: Alhydrogel, a common aluminium hydroxide wet gel suspension, and Abisco-100, a non-toxic saponin-based adjuvant, and compared these to immunization with a lead adenoviral vaccine platform.", "Recently, the development of viral vectored blood-stage malaria vaccines has shown that high-level antibody responses can be induced by the adenovirus in mice [31,32], rabbits [33,34] and rhesus macaques [35].", "Studies on vaccine candidates AMA1 and MSP have demonstrated that this regimen is safe and immunogenic [36,37].", "Previous experiments involving immunization of mice with adenovirus expressing Pfs25 or Pvs25 have led to antibodies exhibiting TB efficacy [38,39,49].", "We show that immunization of mice with Pvs25-FhCMB elicited effective Pvs25-specific humoral immune responses and significant TB activity.", "The ability of antiserum generated from each immunization regime to recognize native Pvs25 was examined by immunofluorescence assay (IFA).", "TB activity was assessed in vivo, using a transgenic rodent malaria parasite P. berghei (Pvs25DR3) expressing Pvs25, enabling rapid, safe and cost-effective examination of anti-Pvs25 responses [18,19,40].", "We additionally examined the benefit of a heterologous prime/boost regimen by priming animals with Pvs25-FhCMB followed by boosting with recombinant chimpanzee adenovirus expressing Pvs25 (ChAd63-Pvs25).", "Efficacy was examined by direct feeding assay (DFA), and in direct membrane feeding assay (DMFA) against P. vivax field samples.", "Maximal efficacy was observed when combining adenoviral and plant-derived immunogens in a single regime.", "Our data indicate that Nicotiana-produced Pvs25 antigen can induce significant TB responses in vivo and ex vivo, and that this technology could be used as part of a successful TBV immunization regime, either alone, or in combination with other delivery systems.", "Pvs25 protein sequence corresponding to Ala23-Leu195 (NCBI accession XP_001608460) was codon optimized for plants expression (GeneArt®, Germany).", "The sequence was fused to the C-terminus of LicKM (NCBI accession number ABG78599) [22], containing pathogen-related-protein 1a signal peptide (PR-1a) [(NCBI accession BAA14220)] at the N-terminus, the hexa-histidine (6xHis) tag and the endoplasmic reticulum retention signal at the C-terminus, to yield Pvs25–FhCMB (Fig. 1a).", "The gene was cloned into the TMV-based hybrid expression vector pGRD4 [22,27].", "Vector was introduced into Agrobacterium tumefaciens GV3101 by electroporation, and vacuum infiltrated into leaves of six-week-old hydroponically grown N. benthamiana as described previously [22,27].", "N. benthamiana plants expressing Pvs25-FhCMB were harvested at 6 dpi and stored at ≤-60 °C.", "Plants were homogenized, extracted with Triton X-100 (0.5%), centrifuged (16,000 × g, 15 min, 4 °C) and filtrated (Sartopore 2, 0.45/0.2 μm) prior to loading onto IMAC resin (Ni-Sepharose Fast-Flow, GE Healthcare).", "Proteins was eluted and loaded onto a CaptoQ column (GE Healthcare).", "Eluted fraction was concentrated 10-fold by centrifugation (Centricon-70, 30 kDa MWCO) and aliquots were frozen at ≤-60 °C.", "LicKM was expressed as described previously [29,30].", "SDS-PAGE was performed on a 10% acrylamide gel and Coomassie stained.", "For Western blot, samples were blocked with I-Block (Applied Biosystems).", "Blots were developed using anti-4xHis mAb (Qiagen).", "Protein concentration of Pvs25-FhCMB was determined by UV−vis spectrometry using the denaturing method of Edelhoch (1967) with the theoretical extinction coefficient of 73,960 M−1 cm−1.", "Analytical size exclusion chromatography was performed on a Zenix300 HPLC column (Sepax Technologies, Inc.).", "Antigen sequence (excluding the glycosylphosphatidylinositol (GPI) anchor addition site), i.e. Ala23-Leu195 of Pvs25 (Sal I strain), was obtained from the NCBI protein database, codon optimized for expression in Homo sapiens (GeneArt®, Life Technologies, Germany), and the predicted native signal peptide replaced with the human tissue plasminogen activator signal peptide.", "Recombinant ChAd63, MVA vaccines and controls were generated [31,38,42] .", "Pvs25 antigen sequence was codon optimized for expression in Homo sapiens, subcloned into modified pENTR4-LPTOS TM [48] plasmid between a tPA leader and an affinity sequence encoding a biotin-acceptor peptide and a hexahistidine tag.", "Plasmids were transfected into 2 × 106 cells/mL HEK293E cells.", "Soluble recombinant protein (r-Pvs25) was affinity-purified using a HiTrap HP nickel column (GE Healthcare, USA), followed by size exclusion chromatography using a Superdex 200 pg gel filtration column (GE Healthcare, UK) and confirmed by SDS-PAGE.", "Eighty mice (Female BALB/c, 6–8 weeks of age (Harlan, UK)) were divided into groups of 10 mice, each group used for an individual immunization regime (Fig. 2).", "Regimes 1−4 were experimental whereas regimes 5−8 were immunized (i.m.) with control immunogens.", "In each group, five mice were challenged with Pvs25DR3 [40], and five were challenged with wild-type (WT) P. berghei (2.34).", "Transmission blockade observed with 2.34 was considered non-specific and not attributed to Pvs25 immunization.", "For vaccination with Alhydrogel, 10 doses (50 μL per mouse) were prepared by combining 85 μL of Alhydrogel™ containing 850 μg of aluminium in Eppendorf tubes under aseptic conditions with 165 μL of TRIS buffer (20 mM Tris, pH 7.4, 0.9% NaCl).", "After 15 min, 250 μL of antigen solution was added to corresponding tubes, which were vortexed and incubated for 1 h. For Abisco-100™ adjuvant, recombinant protein was mixed with adjuvant, vortexed for 10 s and injected immediately (5 μL Abisco-100 per mouse in total vaccination volume).", "Viral-vectored vaccines were prepared in sterile, endotoxin-free PBS prior to immunization, with doses of 1 × 1010 viral particles of ChAd63 vaccines at week 0 and 1 × 107 plaque forming units of MVA vaccines at week 8.", "Control mice were immunized with ChAd63 and MVA expressing GFP as an irrelevant antigen in place of Pvs25 (Ad-MVA GFP) [42].", "For regimens where Pvs25-FhCMB protein was used for immunization and boost, sera were collected at days 0, 21 and 42.", "When initial immunization was performed with ChAd63, sera were collected at days 0, 56 and 70.", "Sera from mice were collected by tail-bleed prior to boost and challenge.", "Nunc-Immuno Maxisorp 96-well plates (NUNC) were coated with 100 ng recombinant protein (Pvs25FhCMB or r-Pvs25) overnight.", "Test sera was incubated for 2 h followed by goat anti-mouse IgG-AP secondary (Sigma), 1:3000.", "Plates were developed using pNPP and read at OD405 until end-point detection set for each antigen.", "For r-Pvs25 ELISA, control regime titres were subtracted from mean end-point titres.", "The cut-off for determining end-point titer was the mean OD value for pre-immune sera + three times the standard deviation.", "Routine maintenance of P. berghei was carried out as described previously [40,41].", "Prior to challenge, mice were PHz treated, and 3 days later infected i.p. with 106 P. berghei ANKA 2.34 or P. berghei Pvs25DR3 [19,40].", "Three days post-infection, DFA was performed as described in Blagborough et al. [19], with >50 Anopheles stephensi mosquitoes allowed to feed on each mouse.", "Twelve days later, midguts were dissected, and oocyst prevalence and intensity recorded and inhibition, these values were compared to intensity and prevalence in mice immunized with appropriate controls.", "Peripheral blood was collected from P. vivax infected Thai volunteer patients.", "Ninety microliter of pooled serum from mice immunized with ChAd63u-Pvs25/Pvs25-FhCMB boost (regime 4) or mice immunized with ChAd63-GFP/LicKM boost (regime 8) were mixed with 90 μL of heat-inactivated AB serum from naive donors.", "Serum was mixed with P. vivax-infected blood cells (1:1 v/v ratio).", "The mixture was placed in a membrane feeder, 37 °C and Anopheles dirus A mosquitoes (Bangkok colony, Armed Forces Research Institute of Medical Sciences) were allowed to feed for 30 min.", "Mosquito maintenance and dissection were performed as described in Blagborough et al. [19].", "Reductions in intensity and prevalence were assessed with respect to the relevant controls.", "The ability of harvested sera to recognize native Pvs25 was assessed by IFA on Pvs25DR3 ookinetes as described in Sinden et al. [41].", "All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act (PPL 70/7185) and approved by the Imperial College AWERB.", "The Office of Laboratory Animal Welfare Assurance for Imperial College covers all Public Health Service supported activities involving live vertebrates in the US (no. A5634-01).", "Protocol for blood collection from Thai patients (protocol # TMEC-11-033) was approved by Ethical Committees from the Faculty of Tropical Medicine, Mahidol University and Ministry of Public Health, Thailand.", "Statistical analysis was performed using Graphpad Prism.", "For DFA and DMFA, significance was assessed using Mann–Whitney U (to examine differences in intensity) and Fisher's exact probability (to examine differences in prevalence).", "Parametric ELISA tests were assessed using t-test.", "P-values < 0.05 were considered statistically significant.", "Pvs25-FhCMB expression was evaluated by Western blot of clarified extract (Fig. 1b).", "Solubility was examined using an anti-4xHis mAb by Western blot of total unclarified protein homogenate (H), total soluble protein (S) and total soluble protein extracted with 0.5% Triton X-100 (dS) (Fig. 1b).", "Densitometry analysis of Western blots indicated that Pvs25-FhCMB was ∼100% soluble.", "Pvs25-FhCMB was recovered to >90% purity of TP using a two-column chromatography approach consisting of IMAC followed by anion exchange chromatography (Capto Q).", "SDS-PAGE indicates a single prominent band at approximately 55 kDa (Fig. 1c).", "When Pfs25-FhCMB was run in non-reducing buffer, a single band, migrating at a similar molecular weight is observed.", "Analysis by analytical SEC indicates a single prominent peak eluting at a size equivalent to a 45 kDa standard (Fig. 1d), in good agreement with the theoretical molecular weight of 46.4 kDa.", "Both the non-reduced SDS-PAGE and the single SEC peak indicate Pfs25-FhCMB is a non-aggregated, monomeric protein.", "To examine immunogenicity, pre-boost and end-point sera were analyzed by ELISA against recombinant Pvs25-FhCMB (comprising LicKM) following immunization as outlined in Fig. 2.", "Sera were taken from individual mice (five per group) and analyzed for Pvs25-FhCMB-specific responses.", "No antibody responses against Pvs25-FhCMB were detected in day 0 sera.", "Analysis of responses following Pvs25-based immunization detected anti-Pvs25 antibody both pre- and post-boost for all regimes tested, with higher end-point titers detected post-boost (Fig. 3a).", "There was a significant increase in titers following booster immunizations in regimes 1, 2 and 4 (p < 0.05), with the most pronounced increase detected in regime 4, and a non-significant increase in titers following boost in regime 3 (p = 0.145).", "The highest mean titer was observed in regime 1 (Pvs25-FhCMB + Abisco-100).", "The lowest anti-Pvs25-FhCMB end-point titers were exhibited with regime 3 (Pvs25-ChAd63 + MVA).", "Differences detected between mean end-point titers from regimes 1, 2 and 4 were not statistically significant (p > 0.05).", "End-point titers resulting from immunization with Pvs25-ChAd63/MVA (regime 3) were significantly lower than those detected in all other groups (compared to regime 1: p < 0.0001; compared to 2 (p = 0.0163); compared to 4 (p = 0.0003).", "To assess end-point response to Pvs25 per se, as opposed to Pvs25-LicKM conjugate, ELISA was carried out against recombinant r-Pvs25 (Fig. 3b).", "The lowest mean end-point titers were observed against serum from regime 2, whereas the highest was observed from regime 4; however, differences detected between end-point titers from all regimes were not statistically significant (p > 0.05).", "To confirm the induction of Pvs25-specific antibodies that recognize native protein, we performed IFA on P. berghei Pvs25DR3 ookinetes using pooled end-point serum.", "Specific-specific staining was observed using sera from immunization regimes 1, 2, 3 and 4 (Fig. 3c), confirming the ability of generated serum to recognize native Pvs25.", "Staining was not observed with sera from any regime against WT ookinetes, or with sera derived from control regimes (i.e. regimes 5, 6, 7 and 8).", "For examination of transmission blockade in vivo, five immunized mice per challenge group were infected with Pvs25DR3 [40], and five mice infected with WT P. berghei.", "Three days later, DFA was performed (Table 1a).", "Transmission blockade was assessed as reduction in intensity and prevalence with respect to the relevant non-Pvs25 control regimes (i.e. regime 1 is controlled by regime 5, regime 2 by regime 6, regime 3 by regime 7 and regime 4 by regime 8–Table 1b).", "For each Pvs25-derived regime, a TB effect was observed following infection with P. berghei Pvs25DR3.", "Significant reduction in intensity was observed with regimes 1, 2 and 4, and significant reduction in prevalence was observed with all Pvs25-regimes.", "The highest efficacy in terms of both intensity and prevalence was observed following immunization with Pvs25-ChAd63, boosted with Pvs25F3E-LicKM/Abisco-100 (regime 4), where a mean reduction in intensity/prevalence of 74.5%/68.3%, respectively was achieved.", "Direct correlation between anti-Pvs25 titer and transmission-blocking efficacy in individual mice was not observed with any significance.", "As previously demonstrated [18,19,40], challenge with WT P. berghei gives rise to higher oocyst numbers than observed with Pvs25DR3.", "There was no detectable TB effect following WT P. berghei infection, indicating specific anti-Pvs25-derived transmission blockade.", "Serum collected from mice primed with Pvs25-ChAd63 and boosted with Pvs25-FhCMB/Abisco-100 (regime 4), was pooled and evaluated in DMFA.", "Results showed that the intensity and prevalence of oocyst infection on the mosquito midgut was profoundly reduced in comparison with serum collected from the control group immunized with GFP-ChAd63 and boosted with LicKM/Abisco-100 (regime 8) (Fig. 4).", "Over two replicate experiments, an overall 85.3% reduction in infection intensity and a 25.6% reduction in prevalence were observed.", "Reduction in both intensity and prevalence were significant compared to control feeds (p = 0.0001 and p = 0.0033, respectively).", "Future control of malaria will require a multidimensional approach.", "Both the WHO and the Malaria Eradication Research Agenda have previously set as a core goal for any malaria vaccine program the need to reduce transmission and morbidity [43].", "TBVs are one potential component of this approach.", "Anti-malarial TB immunity can be mediated by antibodies that inhibit parasite development in the mosquito midgut [43–47].", "We have previously reported on the production of Pfs25 variants in N. benthamiana [29].", "In that study, the presence of N-linked glycans impaired the induction of TB activity by the recombinant non-fusion version of Pfs25.", "To increase immunogenicity, Pfs25 lacking N-glycosylation sites was fused to modified lichenase carrier molecule, LicKM [30].", "The resulting protein induced high titers with long-lasting TB activity in mice.", "These results indicated the utility of plant-derived Pfs25 as a TBV, and the importance of appropriate carrier molecules to enhance immunogenicity.", "Here, in a parallel study, we describe the production and purification of soluble, Pvs25 recombinant antigen (Pvs25-FhCMB), using a viral plant-based system.", "Similar to Pfs25, the Pvs25 sequence contains 22 cysteines, but unlike the Pfs25 sequence Pvs25 does not contain any putative N-linked glycosylation sites.", "Induction of functional TB antibody responses against Pvs25 was assessed, following immunization with four different regimes: 1).", "Pvs25-FhCMB with Abisco-100 adjuvant; 2).", "Pvs25-FhCMB with Alhydrogel; 3).", "Viral delivery of Pvs25 (Pvs25-ChAd63) with an MVA boost (i.e. classical Adeno-MVA ‘prime-boost’); and 4).", "A combination of adenoviral delivery (Pvs25-ChAd63) with a Pvs25-FhCMB recombinant protein boost.", "Ability of each regime to produce antibody capable of recognizing native protein and exhibit TB efficacy (in vivo and ex vivo, with field samples) was examined.", "Immunization resulted in immunogen-specific responses against recombinant Pvs25-FhCMB and r-Pvs25 in all immunization regimes tested.", "As expected, a booster dose significantly increased the antibody titer against Pvs25-FhCMB in each regime.", "The highest mean titer was observed with regime 1 (prime/boost with Pvs25-FhCMB/Abisco-100), whereas the lowest anti-Pvs25-FhCMB end-point titers were elicited by regime 3 (Pvs25-ChAd63/MVA).", "Recognizing that ELISAs against the Pvs25-FhCMB recombinant protein included significant responses to LicKM (Fig. 3a outlines responses to Pvs25-FhCMB, comprising LickM), we examined responses against Pvs25 specifically (Fig. 3b).", "Under these conditions, the highest titer was observed with regime 4 (Pvs25-ChAd63/Pvs25-FhCMB/Abisco-100), and the lowest with regime 2 (Pvs25-FhCMB/Alhydrogel).", "However, when objectively comparing differences in responses, it should be noted that no significant difference could be detected between regimes.", "It should be stated that no detectable correlation between anti-Pvs25 titer and transmission-blocking efficacy in individual mice was observed, supporting the hypothesis that antibody titer is not necessarily the key parameter for predicting TBV efficacy.", "It is likely that other additional qualitative changes, such as isotype switching and affinity maturation, will significantly impact the biological activity and effector functions of any humoral immune response post-immunization.", "When examining the ability of serum to recognize native Pvs25 by IFA, sera from all regimes resulted in surface staining, corresponding to surface localization of Pvs25 [18,40].", "Staining was not observed with sera collected from mice immunized with control regimes (regimes 5, 6, 7 and 8), or when IFAs were conducted on non-Pvs25-expressing (WT 2.34) ookinetes.", "Although high titers are desirable, the functional effect of immunization on intensity and prevalence remains the key readout to measure TBV effectiveness.", "These experiments employed a proven transgenic model [18,19,40] to compare the ability of immunization to reduce oocyst development in the mosquito, giving in vivo data to examine the comparative potency of potential Pvs25-targeted TBVs.", "Every Pvs25-derived immunization regime examined induced a TB response, with varying efficacy.", "In this study, immunization with the Pvs25-ChAd63/MVA platform (regime 3) led to the lowest observed TB effect (30.0% reduction in intensity/35.6% reduction in prevalence).", "Substitution of MVA boost at 8 weeks with 50 μg of Pvs25-FhCMB plus Abisco-100 adjuvant not only increased the end-point anti-Pvs25 antibody titer (against both Pvs25-FhCMB and r-Pvs25), but also increased the efficacy of the regime, to a 74.5% reduction in intensity/68.3% reduction in infection prevalence.", "The TB responses generated following this regime are the highest induced within this comparative study, clearly demonstrating the attractiveness of combining adenoviral delivery with a protein antigen.", "In the absence of adenoviral delivery, a potent response is still induced by regime 1, prime/boost with Pvs25-FhCMB/Abisco-100, generating the second highest titers and TB responses (65.25% reduction in intensity/54.1% reduction in prevalence).", "When using Alhydrogel as adjuvant, a 56.58% reduction in intensity/56.82% reduction in prevalence was recorded.", "Significant differences in antibody titers or TB efficacy were not recorded when using Alhydrogel or Abisco-100 as an adjuvant with Pvs25-FhCMB.", "None of the regimes generated a non-specific TB effect following challenge with P. berghei 2.34, suggesting a specific response against Pvs25, and a lack of non-specific anti-P.", "berghei TB activity.", "The TB potential of regime 4 (Pvs25-ChAd63/Pvs25-FhCMB – the highest performing regime in the DFA), was further evaluated by DMFA, resulting in a mean 85.3%/25.5% reduction in intensity and prevalence respectively, confirming the ability of antibodies generated by this regime to block transmission in field relevant assays.", "These results are broadly comparable to results observed using serum generated from i.m. delivery of adenoviral-vectored Pvs25 in mice, described in Miyata et al. [39], where DMFA (carried out in identical conditions to those described here) resulted in reported reductions in oocyst intensity and prevalence of 84.1% and 41.7%, respectively.", "These promising results were achieved with an immunization of 50 μg of Pvs25-FhCMB per mouse.", "Obviously, further dose ranging studies examining proportional immunizations will need to be conducted before perusing product development in human subjects.", "Immunization with the regimes outlined here can be compared to previously reported studies where the transgenic chimeric parasite Pvs25DR3 was used to determine in vivo potency of candidate TBVs.", "Pvs25 produced using an experimental baculovirus expression system was shown to induce a strong TB effect of 83.8% (intensity) and 88.4% (prevalence) in DFA following i.m. immunization (mean control intensity of 2.16 oocysts/midgut) [19].", "These results are comparable to the 74.5% (intensity)/68.3% (prevalence) reduction seen using regime 4 (Pvs25-ChAd63/Pvs25-FhCMB boost), but are higher than the efficacy observed with other immunogens and adjuvants used within this study.", "Crucially, the Baculovirus-Dual-Expression system (BDES) described in Blagborough et al. [19] is not currently approved for use in human trials, whereas both adenoviral and Nicotiana-based expression systems have an extensive track record for the production of antigens to be used in human trials.", "Additionally, a similar approach has previously been taken to examine the efficacy of adenoviral anti-Pfs25 (P. falciparum homolog of Pvs25) vaccines [38].", "TB efficacy was assessed using a homologous chimeric parasite (Pfs25DR3) generated identically to Pvs25DR3.", "Cohorts of mice were immunized with AdHu5/MVA Pfs25 in a heterologous prime boost regime, and subjected to DFA.", "A 67% reduction in intensity and a 28% reduction in prevalence were reported.", "This translated to a 96% reduction in intensity and 78% reduction in prevalence at sera dilutions of one in five in SMFA.", "The ability of the plant-derived antigens tested in this study to induce in vivo and ex vivo efficacy similar to previously examined P25-based immunogens lends weight to their continued development within the TBV pipeline.", "This study gives a clear indication as to the value of Nicotiana and adenoviral-derived immunogens to induce a TB response.", "The efficient plant-based production of Pvs25, generation of high titers and induction of a TB response demonstrates the capability of these approaches to produce anti-malarial TBVs.", "Crucially, this study also demonstrates that combining this with other vaccine platforms (e.g. adenoviral delivery) can elicit maximal potency.", "The authors are not aware of any conflicts of interest arising from this work." ]	[]
Dataset on transcriptome profiling of corneal endothelium from patients with Fuchs endothelial corneal dystrophy	Fuchs endothelial corneal dystrophy (FECD) is a bilateral inherited eye disease with advanced forms only treatable by corneal transplantation. The pathogenesis of FECD has not been worked out yet, however, trinucleotide repeat polymorphism CTG18.1 in the TCF4 gene has recently been associated with late-onset FECD. Gene expression profiling of corneal endothelium with and without this expansion can help elucidate molecular mechanisms of the disease development. Current data article represents whole transcriptome profiles of corneal endothelium obtained from 12 patients with FECD and 6 control tissues from eye bank donors. RNA sequencing data is available at NCBI Sequence Read Archive under Accession No. PRJNA524323. In addition, each patient and donor were genotyped for CTG18.1 expansion and the corresponding numbers of CTG repeats in the TCF4 gene are provided within this article. The dataset includes samples from FECD patients both with and without CTG18.1 expansion.	[ "MULT" ]	[ "Fuchs endothelial corneal dystrophy", "RNA-Seq", "Transcriptome" ]	[ "1000" ]	[ "The dataset contains raw sequencing data obtained through the transcriptome sequencing of corneal endothelium from 12 patients with FECD and 6 donors.", "The data files (reads in FASTQ format) were deposited at NCBI SRA database under project accession No. PRJNA524323.", "Information about tissue samples collected from patients with FECD and control samples from donors is presented in Table 1 and Table 2, respectively.", "All patients and donors were also characterized by genotyping for trinucleotide repeat polymorphism CTG18.1 in the TCF4 gene (Table 3).", "This study was approved by the Institutional Review Board of The S. Fyodorov Eye Microsurgery Federal State Institution and was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).", "Samples of corneal endothelium were obtained from patients diagnosed with FECD and undergoing corneal transplantation at The S. Fyodorov Eye Microsurgery Federal State Institution.", "All patients included in this study had signed an informed consent form.", "Table 2 summarizes information about tissue samples collected from patients with FECD.", "Disease stage was identified according to the Volkov and Dronov classification as described in Ref. [1].", "During the endothelial keratoplasty procedure, circular descemetorhexis with a diameter of 7–8 mm was performed using a special hook to obtain samples of corneal endothelium/Descemet's membrane (CEC-DM) complex.", "RNA in CEC-DM complex was stabilized using RNAlater solution (ThermoFisher Scientific, USA) according to the manufacturer's instructions.", "Venous blood (4–6 mL) was collected from each patient into vacutainer tubes with EDTA (Becton Dickinson, USA).", "Blood samples were stored at −20 °C prior to DNA extraction.", "Control samples of corneal endothelium were collected at the Eye Bank of The S. Fyodorov Eye Microsurgery Federal State Institution.", "Donor samples were selected from those not suitable for corneal transplantation.", "Information about control tissue samples collected from donors is presented in Table 3.", "The eyeball was stored for a maximum of 1 day before extraction of the corneoscleral disc which then was kept in a preservative medium for the period of up to 12 hours.", "The preservative medium contained: 25% of medium 199, 25% of F-10 medium, 45.3% of DMEM, 2% dextran, 2.7% chondroitin sulfate, gentamicin sulfate 0.00014%, amphotericin B 0.00015%.", "Afterward, the corneoscleral disc was placed in an artificial anterior chamber endothelial side up.", "Circular excision of CEC-DM complex 7–8 mm in diameter was performed using spatula and tweezers.", "After isolation, the donor CEC-DM complexes were immediately immersed in the RNAlater solution (ThermoFisher Scientific, USA).", "Iris samples were also collected from donors for the TCF4 repeats expansion genotyping.", "The density of corneal endothelial cells was measured using Tomey EM-3000 Specular Microscope (Tomey, USA).", "The shooting method was non-contact, the measurement mode was manual/automatic, the shooting area was 0.25 mm × 0.54 mm with 7 capture points (central + 6 points on the periphery).", "The accuracy of corneal thickness measurement was ±10nm \".", "CEC density was evaluated if at least one of the capture points had enough transparency for the analysis.", "DNA was isolated from thawed blood samples with Gentra Puregene Blood Kit (Qiagen, Germany) according to the manufacturer's protocol.", "DNA was resuspended in a low TE buffer to a final concentration of 10 ng/μl.", "DNA from iris samples was extracted using ZR Genomic DNA Tissue MiniPrep (Zymo Research, USA).", "Identification of the number of CTG repeats within the CTG18.1 allele in the TCF4 gene was carried out using short tandem repeat (STR) and triplet primed PCR (TP-PCR) techniques exactly according to the procedure described in Ref. [1].", "The TCF4 allele was classified as expanded if the number of CTG repeats was ≥40 according to previously reported literature [2,3].", "Disruption and homogenization of tissue samples were performed with TissueRuptor II (Qiagen, Germany).", "The time of homogenization was 20 seconds.", "Total RNA was isolated using RNeasy Micro Kit (Qiagen) following the manufacturer's protocol.", "Traces of DNA were removed with TURBO DNA-free Kit (ThermoFisher Scientific).", "RNA concentration was assessed using the Qubit 2 instrument (Invitrogen, USA) with Qubit HS RNA Assay Kit (ThermoFisher Scientific, USA).", "The quality of total RNA expressed as RNA Integrity Number (RIN) was determined with Bioanalyzer 2100 instrument (Agilent, USA) using an Agilent RNA Pico 6000 Kit (Agilent, USA) [4].", "On average, the RIN value of 6.5 was obtained, which is typical for RNA samples isolated from tissues [5].", "Ribosomal RNA was depleted using NebNext rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, USA).", "Transcriptome libraries were constructed with NEBNext Ultra II Directional Library Prep Kit for Illumina (New England Biolabs, USA) and Multiplex Oligos for Illumina (96 Indexes) (New England Biolabs, USA).", "The resulting paired-end libraries were sequenced on the Illumina HiSeq 2500 instrument with 2 × 125 cycles using HiSeq SBS Kit v4 (Illumina, USA)." ]	[]
Cognitive reserve and cognitive performance of patients with focal frontal lesions	The Cognitive reserve (CR) hypothesis was put forward to account for the variability in cognitive performance of patients with similar degrees of brain pathology. Compensatory neural activity within the frontal lobes has often been associated with CR. For the first time we investigated the independent effects of two CR proxies, education and NART IQ, on measures of executive function, fluid intelligence, speed of information processing, verbal short term memory (vSTM), naming, and perception in a sample of 86 patients with focal, unilateral frontal lesions and 142 healthy controls. We fitted multiple linear regression models for each of the cognitive measures and found that only NART IQ predicted executive and naming performance. Neither education nor NART IQ predicted performance on fluid intelligence, processing speed, vSTM or perceptual abilities. Education and NART IQ did not modify the effect of lesion severity on cognitive impairment. We also found that age significantly predicted performance on executive tests and the majority of our other cognitive measures, except vSTM and GNT. Age was the only predictor for fluid intelligence. This latter finding suggests that age plays a role in executive performance over and above the contribution of CR proxies in patients with focal frontal lesions. Overall, our results suggest that the CR proxies do not appear to modify the relationship between cognitive impairment and frontal lesions.	[ "NEUR", "PSYC" ]	[ "Age", "Cognitive performance", "Cognitive reserve", "Education", "Frontal lesions", "Literacy attainment" ]	[ "2802", "2805", "3205" ]	[ "It is well known that the cognitive response to brain damage caused by stroke, tumour, trauma, dementia and/or age-related changes can vary across individuals to a considerable degree (e.g., Stern, 2009; Lindenberger et al., 2013).", "The Cognitive Reserve (CR) hypothesis was put forward to account for some of the reported variability in cognitive performance and suggests that the effects of age-related changes or brain damage can be mitigated by the premorbid efficiency, capacity and flexibility of cognitive processing (e.g., Stern, 2002; Jones et al., 2011; Barulli and Stern, 2013; Levi et al., 2013).", "It has also been proposed that the effectiveness of cognitive processing can be shaped by life experiences (Stern, 2012).", "Several proxies have been used to estimate CR.", "These are thought to ‘protect’ against the impact of brain damage and include: education, socio-economic status, occupational achievement and engagement in cognitively and socially stimulating activities (Suchy et al., 2011; Levi et al., 2013; Liu et al., 2013; Okonkwo et al., 2014; for a review see Arenaza-Urquijo et al., 2015).", "The most commonly used proxy of CR is education (e.g., Bennett et al., 2003; Farfel et al., 2013; Sumowski et al., 2013).", "Education encompasses the accumulated knowledge and skills gained through formal schooling.", "It is easy to quantify and has been shown to be a good predictor of healthier lifestyle and economic prosperity (e.g., Mirowsky and Ross, 2005).", "In patients with similar levels of pathology, high education has been associated with less cognitive impairment than in patients with low education (e.g., Bennett et al., 2003; Ngandu et al., 2007; Roe et al., 2007).", "Another proxy of CR is literacy attainment, often assessed using the National Adult Reading Test (NART; Nelson and Willison, 1991) or other single word reading tests, as they are assumed to reflect premorbid intelligence (Wiens et al., 1993).", "Higher NART IQ has been associated with greater CR capacity (Tucker and Stern, 2011; Stern, 2012).", "The concept of CR has been investigated primarily in neurodegenerative disorders, traumatic brain injury and healthy aging.", "Several studies have reported that individuals with similar brain pathology demonstrate differences in their cognitive impairment, depending on whether they have higher or lower levels of education and/or NART IQ (e.g., Grafman et al., 1986; Bennett et al., 2003; Stern, 2006; Singh‐Manoux et al., 2011; Serra et al., 2014; Bozzali et al., 2015).", "For example, low education increases the risk of dementia whilst high education and NART IQ protects against dementia, particularly the onset of Alzheimer's disease (AD, Schmand et al., 1997; Meng and D’Arcy, 2012; Lo, and Jagust, 2013; see for a review Xu et al., 2015).", "High education has been shown to attenuate the decline in attention, speed and memory performance in patients with multiple sclerosis (Sumowski et al., 2014).", "Raymont et al. (2008) reported that higher pre-morbid intelligence, assessed using the armed forces qualification test, was the strongest deterrent against cognitive decline in patients with penetrating head injury.", "However, few studies have investigated the effects of CR on cognitive performance in aetiologies such as stroke (for a review see Nunnari et al., 2014) or brain tumour where the lesions are focal compared to the diffuse lesions associated with slow progressive diseases such as Alzheimer's disease.", "In stroke, it has been reported that education protects against global cognitive decline (e.g., Sachdev et al., 2004; Elkins et al., 2006; Zieren et al., 2013) or severity of aphasia (González-Fernández et al., 2011).", "In brain tumour, although data have not been reported to suggest that education can attenuate cognitive impairment, age and tumour location (frontal) have been shown to predict cognitive outcome on speed, executive and working memory tasks (Kaleita et al., 2004).", "Relatively little is known about whether CR may differentially affect performance on different cognitive measures.", "There is some preliminary evidence suggesting that certain cognitive abilities may be more susceptible than others to the mitigating effects of CR.", "High education has been associated with better performance in stroke patients on tests of language, perception and memory, but not executive functioning, once white matter integrity had been taken into account (Ojala-Oksala et al., 2012).", "Higher rates of decline in AD patients with lower education have been reported on memory and executive tasks but not abstract reasoning, visual-spatial skills or language (Scarmeas et al., 2006).", "Importantly, the relationship between CR and different cognitive measures may also be dependent on the proxy used to estimate CR.", "In a large cohort study including healthy older adults and patients with possible dementia, Jefferson et al. (2011) found that education was primarily related to performance on global cognition, episodic and semantic memory, and perception.", "In contrast, NART IQ was found most strongly associated with working memory but also global cognition and episodic memory.", "Siedlecki et al. (2009) found no significant correlation between education and NART IQ in healthy controls and suggested that these two proxies may account for different elements of the variance in cognitive performance.", "Using path analysis on data from the MRC National Survey of Health and Development cohort, Richards and Sacker (2003) demonstrated three independent paths from childhood cognition, and educational and occupational attainment to CR using the NART as an index.", "Furthermore, when examining the relationships between brain volume and CR proxies in healthy adults over a two-year period, Persson et al. (2016) reported that larger baseline brain volumes predict greater increases in fluid intelligence.", "In contrast, no relationships between literacy attainment and brain volumes were found.", "Researchers have argued that more than one CR proxy measure should be considered, as CR is the result of a combination of life experiences and activities (e.g., Stern, 2009; Tucker and Stern, 2011).", "One CR proxy is unlikely to provide an absolute measure of CR.", "A recent meta-analysis examining the influence of education, occupational attainment, and involvement in cognitively stimulating activities (e.g., crosswords, playing bridge) in healthy individuals demonstrated that while different CR proxies are associated with one another, they also offer a unique contribution to CR (Opdebeeck et al., 2016).", "The effects of CR may also be dependent on the specific brain regions that are damaged.", "For example, Robertson (2014) suggested that the structural and functional integrity of the lateral surface of the right prefrontal cortex and/or the right inferior parietal cortex may play a crucial role in CR.", "Higher levels of CR have also been associated with the concept of scaffolding, a lifelong process that involves the use and development of complementary, alternative neural circuits to achieve a particular cognitive goal (Alexander et al., 1997; Perneczky et al., 2006).", "The concept of scaffolding has been developed in the theoretical context of the Scaffolding Theory of Aging and Cognition (STAC) suggesting that, in healthy aging and in pathological brain damage, scaffolding can compensate for cognitive decline (Reuter-Lorenz and Park, 2014).", "Importantly, Park and Reuter-Lorenz (2009) proposed that scaffolding processes largely reside in the prefrontal cortex.", "Thus, damage to the prefrontal cortex may have a detrimental effect on the compensation provided by CR.", "Surprisingly, the studies conducted so far have not investigated whether there are specific brain areas critical for CR.", "The non-specific nature of brain-related changes in neurodegenerative diseases, traumatic brain injury and healthy aging limits our ability to draw conclusions as to which specific brain areas may contribute to CR.", "Examination of patients with more focal brain lesions, such as those resulting from stroke and tumour, may overcome such limitations.", "However, the stroke and tumour studies conducted so far have included patients with lesions not restricted to specific cortical areas.", "Thus, the high degree of variability in the patients’ cognitive performance inevitably reduces one's ability to draw conclusions regarding the interaction between CR and brain lesion.", "To our knowledge, our study is the first to investigate the independent effects of education and NART IQ on measures of executive function, fluid intelligence, speed of information processing, verbal short term memory (vSTM), naming and perception in a large sample of patients with focal, unilateral frontal lesions.", "Our study also investigated for the first time whether CR might safeguard against focal neuropathology and moderate cognitive impairment across these various cognitive measures.", "Data from 164 patients who had attended the Neuropsychology Department of the National Hospital for Neurology and Neurosurgery, Queen Square, London, were retrospectively screened for study eligibility.", "All patients had a unilateral lesion confined to the frontal region resulting from stroke or brain tumour.", "All tumour patients had undergone lesion resection prior to neuropsychological assessment.", "Our exclusion criteria were: i) age at the time of cognitive testing ≥80 years due to the availability of age-matched healthy control data and standardised age norms; ii) current or previous psychiatric disorders; iii) previous neurological disorders including strokes or tumours; iv) presence of metastatic tumours; v) previous chemotherapy; vi) gross visual (i.e., cortical blindness), perceptual (i.e., neglect; agnosia), motor (i.e., hemiplegia) or language (i.e., dysphasia) impairment; vii) previous head trauma; viii) history of alcohol or drug abuse; ix) no MRI or CT scan results available; x) no or limited neuropsychological data available; xi) a score <5th percentile on a test of general intelligence (WAIS-III, Wechsler, 1997; WAIS-R, Wechsler, 1981; or Raven's Advanced Progressive Matrices, RAPM, Raven, 1976); xi) a score of <2 on a measure of frontal lesion severity (see below, ‘Frontal lesion severity’ section).", "Non-native English speakers were only included in the study if they obtained a score ≥25th%ile on the National Adult Reading Test (NART IQ, Nelson, 1982).", "This was to ensure that their English abilities were sufficient to cope with task demands.", "Application of our exclusion criteria resulted in the data of 86 frontal patients being included in the study (stroke, n=22; high grade tumours, n=18; low grade tumours; n=22; meningioma n=24).", "Some clinical and cognitive aspects of these patients have been previously reported (MacPherson et al., 2010, 2016; Robinson et al., 2012, 2015; Murphy et al., 2013; Cipolotti et al., 2015a).", "Data from a group of 142 healthy controls who did not significantly differ in terms of age, gender, NART IQ and years of full-time education to the frontal group were also reviewed (see below).", "The study was approved by the National Hospital for Neurology and Neurosurgery and the Institute of Neurology Joint Research Ethics Committee (UK).", "MRI data were available for 76 of the frontal patients and CT data for the remaining 10.", "Hard copies or digital records of all scans were reviewed by two independent neurologists (MB and BS) who were blind to the experimental results.", "Digital brain MRI scans were obtained on systems operated at 0.5, 1.5 or 3 T and included the acquisition of an axial dual-echo and an axial and coronal T1-weighted scan.", "CT scans were obtained using spiral CT systems.", "Axial images were collected with an effective slice thickness of 5 mm and pitch of 1.5.", "Only T1-weighted MRI scans (or CT scans when MRI was not available) were used for the assessment of frontal lesions.", "We conducted an analysis of the total frontal lesion volumes only for a subset of patients (N=43).", "These were the patients for whom we obtained the largest number of MRI scans at the same magnetic strength (1.5T).", "The lesion localization method adopted has been described in detail in our previous work (e.g., MacPherson et al., 2010; Murphy et al., 2013; Cipolotti et al., 2015a, 2015b; Robinson et al., 2015).", "The exclusion criteria and lesion assessment guidelines were based on detailed anatomical localization using standard atlases (Duvernoy et al., 1991).", "Briefly, each frontal patient was coded for the presence of lesion and oedema in nine left and nine right frontal subregions (18 subregions in total).", "A subregion was coded as damaged if at least 25% was affected.", "Lesions were distributed throughout the prefrontal cortex.", "A measure of the severity of frontal lesions was obtained for each patient, using T1-MRI or CT scans.", "Lesion severity was assessed by visual rating (MB and BS) of each of the 18 frontal subregions based on a scale whose scores ranged from 0 to 4 (0=absence of lesion; 1=minimal damage; 2=moderate damage; 3=severe damage; 4=very severe damage).", "We summed the severity scores for each of the 18 frontal subregions to produce a total frontal lesion severity score (out of a possible 72); the higher the score indicated the greater the frontal lesion severity (see Fig. 1 for examples).", "A volumetric lesion analysis was also conducted on a subsample of 43 frontal patients who had a digital MRI scan (stroke=11, low grade tumours=12, high grade tumours=9 and meningioma=11) using a semi-automated local thresholding contouring technique (Jim 5.0, Xinapse System, Leicester, UK, http://www.xinapse.com/).", "The frontal lesion volume was then calculated for each patient.", "All frontal patients and healthy controls had previously taken part in a single neuropsychological assessment and their cognitive performance on well-known clinical tests with published standardised normative data was retrospectively considered.", "While a single test was administered to assess fluid intelligence, speed of information processing, naming, verbal short-term memory (vSTM), and visual perception (see Table 1 for the tests administered), the data from two tests assessing executive abilities were available (Phonemic fluency ‘S’, Tombaugh et al., 1999 and the Stroop Colour-Word Test, Trenerry et al., 1989).", "Due to the retrospective nature of our study, certain data were not available for some participants.", "The number of frontal patients who had data for each cognitive measure were as follows: executive function: Phonemic fluency: N=80; and Stroop Colour-Word Test: N=53; fluid intelligence: N=55; speed of information processing: N=55; naming: N=83; vSTM: N=36; and visual perception: N=69.", "The number of healthy controls who had data for each cognitive measure were as follows: executive function: phonemic fluency: N =45; and Stroop Test: N=60; fluid intelligence: N=82; speed of information processing: N=86; naming: N=139; vSTM: N=16; and visual perception: N=25.", "A pairwise deletion method was used with no substitutions made to the dependent variables.", "Education and literacy attainment were adopted as our two proxies of CR, with these data available for all frontal patients and healthy controls.", "We operationally defined educational attainment as the number of full-time years in formal education.", "In keeping with previous studies, a proxy of literacy attainment was obtained using a test of single word reading (e.g., Scarmeas et al., 2006; Stern et al., 2008).", "This was based on the corresponding IQ score on the NART.", "Unless stated otherwise, the statistical analyses were carried out using IBM SPSS Statistics 21 (http://www01.ibm.com/software/analytics/spss/).", "A Pearson's product moment correlational analysis was conducted to examine whether there was a significant relationship between our frontal lesion severity score based on visual ratings and our volumetric lesion analysis on a subsample of 43 frontal patients.", "Independent samples t-tests investigated whether the frontal patients and healthy controls, significantly differed in terms of age, years of education and NART IQ.", "Levene's tests were used to assure equality of variances.", "NART IQ and time between damage and assessment violated this assumption.", "Hence we used the results which did not assume equality of variances.", "Analyses of covariance (ANCOVAs) with age, years of education and NART IQ as covariates were conducted to investigate whether performance on our cognitive measures significantly differed between the frontal group and healthy controls.", "To examine the effect of lesion severity on cognitive performance, frontal patients were subdivided into high and low lesion severity groups and their performance was compared using independent samples t-tests.", "To investigate the effect of our two CR proxies on performance on the cognitive measures in our frontal patients, we fitted multiple linear regression models for each of the cognitive measures using R function ‘lm’.", "In the first three steps of the analysis, chronicity (step 1), age (step 2) and severity of lesion (step 3) were each separately entered as continuous covariates.", "In the fourth step, either education (step 4a) or NART IQ (step 4b) was entered as a predictor variable.", "This fourth step allowed us to examine the contributions of our CR proxies, education and NART IQ, to cognitive performance, over and above any effect of chronicity, age and lesion severity.", "In step 5, education and NART IQ were entered into the same model to examine whether they independently contributed to cognitive performance.", "Lastly, in step 6, either the interaction term between lesion severity and education (step 6a) or lesion severity and NART IQ (step 6b) was added.", "This was added to determine whether any association between lesion severity and cognitive performance differed depending on whether education or NART IQ was high or low.", "For all models, the contribution and significance of each predictor was estimated at each step.", "Throughout the results section, we report standardized beta values.", "The p-value was Bonferroni corrected for multiple comparison (0.05/7=0.007) as seven cognitive measures were considered.", "Missing data for our chronicity and lesion severity covariates were treated by a simple two-stage method of imputation and sensitivity analysis (Allerhand et al., 2014).", "In the first stage, each independent variable was regressed onto the other independent or auxiliary variables that provided coverage.", "The regression model for each variable was used to predict a value for each case where data were missing.", "The value that was imputed was then the most plausible in the sense of the most expected by the other variables.", "Values were not imputed for the dependent variables as imputation of these missing data is not considered to provide additional information, and may introduce additional error (von Hippel, 2007).", "In the second stage, the results of the final model were assessed for sensitivity to the imputed values.", "A set of 100 samples were generated in which each value to be imputed was randomly drawn from a uniform distribution bounded by the prediction interval around the value predicted in the first stage.", "These data were all analyzed in the same way and the distribution of each estimated regression coefficient was compared against the confidence intervals of its original estimate.", "Lesion definition was in an excellent range of inter- and intra- rater reliability (inter-class correlation=0.967; intra-class correlation=1.000).", "We first assessed whether scores of frontal lesion severity (based on visual ratings) were correlated with lesion volumes in the subsample of 43 patients with digital MRI data.", "As expected, our frontal lesion severity measure correlated highly with our volumetric frontal lesion analysis (r=0.676, p<0.001), indicating that a higher frontal severity score was associated with higher frontal lesion damage in our subgroup.", "The frontal group and healthy controls did not significantly differ in terms of age (t(226)=0.298, p=0.766), years of full-time education (t(226)=0.867, p=0.387) and NART IQ (t(136.706)=−0.678, p=0.499; See Table 2a).", "In our frontal patients, age did not significantly correlate with education (r=−0.17, p=0.12) or NART IQ (r=0.04, p=0.69).", "However, education and NART IQ correlated highly with one another (r=0.48, p<0.001).", "Frontal patients significantly differed from healthy controls on all neuropsychological measures except the Stroop Test, as shown using ANCOVAs with age, years of education and NART IQ as covariates (executive function: fluency F(1,120)=13.158, p<0.0001; and Stroop F(1,108)=0.708, p=0.410; fluid intelligence F(1,142)=13.408, p<0.001; speed of information processing F(1,136)=6.806, p=0.010; naming F(1,217)=9.584, p=0.002; vSTM F(1,47)=4.375, p=0.042 and perception (F(1,89)=25.637, p<0.001).", "For all significant differences, with the exception of perception, frontal patients performed significantly poorer than healthy controls.", "For perception, frontal patients obtained a better score than healthy controls, although scores were close to ceiling for both samples (see Table 2b).", "Qualitatively, 60 of the 78 of the patients that could not be included in our retrospective study had limited or no neuropsychological data available because they were not able to cope with the demands of our cognitive battery.", "The largest majority of these patients had very large frontal lesions, suggesting that severity of lesion is a powerful determinant of cognitive performance.", "The sample of frontal patients who were included in our retrospective study had lesion severity scores ranging from 2 to 66 out of a possible 72.", "Noticeably, we had few patients with the most severe scores.", "To investigate formally the effect of lesion severity on cognitive performance, we subdivided into two groups: patients with a high lesion severity score (≥25); and patients with a low lesion severity score (≤5).", "The means and standard deviations for these two groups are demonstrated in Tables 2a and 2b.", "We found no significant differences between these two patient groups in terms of age (t(43)=1.079, p=0.29), time between damage and assessment (t(12.45)=1.287, p=0.22), education (t(20.90)=−0.603, p=0.55) or NART IQ (t(43)=−1.711, p=0.09).", "Independent samples t-tests revealed that the frontal patients with high lesion severity performed significantly more poorly than the frontal patients with low lesion severity on fluency (t(38)=−2.473, p<0.05) and speed of information processing (t(25)=2.290, p<0.05) tasks.", "No significant differences were found for fluid intelligence (t(35)=−1.156, p=0.26), naming (t(42)=−0.154, p=0.61) or perception (t(33)=0.496, p=0.62).", "Due to the relatively small sample sizes for Stroop (high severity: N=9 and low severity: N=15) and vSTM (high severity: N=3 and low severity: N=15), no formal statistical analyses could be carried out for these cognitive measures.", "Importantly, the patients with high lesion severity scores obtained scores that were numerically lower.", "For each of our linear regression models, we examined multicollinearity using the variance inflation factor (VIF).", "In all instances, VIF was below 2, suggesting that there were not high intercorrelations among our predictor variables.", "Table 3 shows the results of the multiple linear regression models testing for the effects of education and NART IQ on each cognitive test with chronicity, age and frontal lesion severity as covariates; including education only (step 4a); NART IQ only (step 4b); education and NART IQ (step 5); and adding the interaction term between lesion severity and education (step 6a) or NART IQ (step 6b).", "In these analyses, lesion severity was a continuous variable.", "For both executive measures, age and NART IQ independently contributed to the fit of the model.", "In contrast, chronicity or frontal lesion severity made no significant contribution to the model either as main effects or as interaction terms with NART IQ or education.", "In the case of fluency, the model including NART IQ explained 27% of the variance compared to 16% before NART IQ was entered.", "For the Stroop test, the model including NART IQ explained 54% of the variance compared to 10% before NART IQ was entered.", "Education (step 4a) and NART IQ (step 4b) contributed to the fit of the model.", "However, when both education and NART IQ were entered into the same model (step 6), neither cognitive proxy continued to predict performance, leaving age as the only significant predictor.", "The final model explained 36% of the variance with 12% of the variance explained by age.", "Chronicity and frontal lesion severity did not significantly contribute to any of the models as main effects or interacting with education or NART IQ.", "Only age significantly contributed to the fit of the model explaining 40% of the variance on TMT-A.", "In contrast, chronicity, frontal lesion severity, education and NART IQ made no significant contributions to the model at any stage.", "None of our covariates (chronicity, age and frontal lesion severity) or predictors (education or NART IQ) made significant contributions to performance on the digit span task.", "Both education and NART IQ significantly contributed to performance on the GNT.", "However, their contributions were not independent of one another, as entering both education and NART IQ into the same model removed the association between education and GNT performance, leaving only NART IQ as a significant predictor.", "Chronicity, age and lesion severity did not contribute to the model as main effects or interaction terms with education or NART IQ.", "NART IQ accounted for 48% of the variance on the GNT.", "Only age significantly predicted performance on the Object Decision test, accounting for 9% of the variance, with no significant contributions to the model from chronicity, frontal lesion severity, education or NART IQ.", "In Fig. 2, we report the percentage of the total variance R2 accounted for by each of the five steps entering chronicity, age, frontal lesion severity, education, NART IQ and finally education and NART IQ in the regression models for all cognitive measures.", "All the significant associations were in the expected direction with better cognitive performance associated with younger age and higher education and NART IQ.", "Our sensitivity analyses were conducted on our final full models.", "They showed that none of the estimates using randomly imputed values were significantly different from the estimate obtained using the most plausible value.", "We retrospectively investigated the effects of years of education and literacy attainment based on NART IQ on the cognitive performance of a large sample of patients with focal unilateral frontal lesions.", "Our unilateral frontal patients were significantly impaired on measures of executive function, fluid intelligence, speed of information processing, vSTM and naming compared to healthy controls.", "Our investigation of the contributions of education and NART IQ, after adjusting for chronicity, age and frontal lesion severity, revealed that the two variables do not represent the same proxy measures of CR.", "When both education and NART IQ were entered into the same model, we found that NART predicted executive and naming performance.", "Neither education nor NART IQ predicted performance on fluid intelligence, processing speed, vSTM or perceptual abilities.", "These findings support the suggestion that CR is a multidimensional construct with different proxies offering distinct contributions to performance on different cognitive measures (Siedlecki et al., 2009; Opdebeeck et al., 2016).", "However, it remains inconclusive which specific cognitive domains are related to specific CR.", "For example, studies have reported that education and NART IQ are strongly associated with fluid intelligence (e.g., Ritchie et al., 2013; Deary and Brett, 2015) and naming (Vemuri et al., 2011) when considered separately.", "Robertson (2014) suggested that CR predicts performance on frontal executive tasks.", "However, literacy attainment has been shown to predict performance on speed of information processing in patients with multiple sclerosis (Benedict et al., 2010) and a latent executive variable in traumatic brain injury (Green et al., 2008).", "Educational attainment in healthy aging has been found to predict performance on executive abilities and working memory (e.g., Meguro et al., 2001) and memory and perception (Jefferson et al., 2011).", "Education has been shown to predict performance on executive tasks and memory in AD (Scarmeas et al., 2006) but not in stroke (Ojala-Oksala et al., 2012).", "Moreover, our retrospective study has the disadvantage of having relatively smaller sample sizes for certain cognitive measures (e.g., vSTM N=36) compared to other measures.", "Therefore, caution should be taken when concluding that NART and education are selectively unrelated to vSTM.", "Future prospective work is needed to examine further the relationship between CR proxies and cognition.", "We found that age significantly predicted performance on executive tests and on the majority of our other cognitive measures, exceptions being vSTM and GNT.", "For fluid intelligence, when both education and NART were entered together in the model, only age remained a significant predictor.", "We have previously reported that age and NART IQ significantly predicted performance on a variety of cognitive measures in patients with frontal lesions, some of whom have participated in the current study (Cipolotti et al., 2015a).", "Previous research has reported significant age-related decline in fluid intelligence performance in healthy adults (Raz et al., 2007; Elderkin-Thompson et al., 2008).", "We have also previously documented that age in frontal patients significantly predicts the magnitude of the impairment in fluid intelligence (RAPM, Cipolotti et al., 2015b).", "Our current findings further confirm the important role of age in executive performance in the context of frontal lesions over and above the contribution of CR proxies.", "Interestingly, in our current study, our regression analyses suggest that the severity of our patients’ frontal lesions does not appear to play a critical role on their executive and fluid intelligence performance.", "However, more broadly, frontal lesion severity appears to contribute to cognitive performance.", "Qualitatively, we noted that the large number of patients who were unable to cope with the demands of our cognitive battery, and hence were not included in our retrospective study, tended to have rather large frontal lesions.", "Moreover, when we compared the patients with high and low lesion severity, we found that patients with high lesion severity performed significantly more poorly on fluency and speed of processing tasks than patients with low lesion severity.", "They also obtained poorer scores on the other cognitive tests, the only exception been perception.", "Importantly, the two groups of patients were matched on education and NART IQ.", "Future prospective studies are necessary to examine the role of lesion severity on cognition.", "Our results also revealed that the effects of education and/or NART IQ on our cognitive measures did not interact with lesion severity.", "Thus, we found no evidence that the effect of lesion severity on cognitive impairment is altered by either CR proxy in our frontal patients.", "It seems unlikely that these lack of interactions simply reflect the fact that lesion severity is irrelevant and they may reflect non-linear effects when lesion severity is considered as a continuous variable.", "As reported above when considered a dichotomous variable, lesion severity may play a role in the cognitive performance of frontal patients.", "While this was not captured by our regression analyses, we had fewer patients with the most severe scores.", "Stern (2009) suggested that higher CR individuals are able to tolerate a greater degree of pathology for a longer time before cognitive decline presents behaviourally.", "However, there is also a growing body of longitudinal evidence suggesting that CR does not alter the slope of cognitive decline (Tucker-Drob et al., 2009; Singh‐Manoux et al., 2011; Zahodne et al., 2011).", "Our cross-sectional study has the disadvantage of not allowing us to examine the effects of our CR proxies on the rate and magnitude of cognitive decline over time.", "However, our results suggest that the CR proxies do not modify the relationship between cognitive impairment and frontal lesions.", "It might be argued that these inconsistent results are due to the type of CR proxy measures used.", "The two measures we used, NART IQ and education, have been commonly adopted in the literature (Valenzuela and Sachdev, 2006a, 2006b).", "However, education has been criticised as a CR proxy given that the quality, availability and subjects taught varies across different countries and social groups (e.g., Jones et al., 2011).", "Some authors have suggested that literacy attainment may be a better marker of educational attainment (e.g., Manly et al., 2003).", "However, dyslexia and other learning difficulties are detrimental to literacy attainment and result in an inaccurate estimate of CR.", "Hence, some authors suggest that education is the better proxy (e.g., Ikanga et al., 2016).", "Alternatively, while CR allows individuals to cope more successfully with healthy and pathological age-related changes in the brain (Stern, 2009), it may not have the same neuroprotective benefit in the context of focal brain damage due to brain tumour or stroke, as in the case of our patients.", "Perhaps a critical difference is that there may be more plasticity and functional reorganization in healthy aging and in the neurodegenerative conditions due to their slow progressive nature (Morris, 2005; Ryan and Rossor, 2011).", "It may be that the effect of the CR proxies can facilitate functional reorganization to compensate for brain damage, but only when the disease process is slow (Stern, 2006).", "Instead, stroke and tumour are usually associated with a much more rapid disease process, hence the limited effect of the CR proxies.", "It is also possible that our patients’ frontal damage hindered the degree of compensation provided by our two proxies.", "This notion is broadly in keeping with the view that the prefrontal cortex compensates for declines in cognitive functioning (Park and Reuter-Lorenz, 2009) and that the right prefrontal cortex, in association with other areas, mediate some the CR effects (Robertson, 2014).", "Unfortunately, we did not have the opportunity to investigate the effects of our two CR proxies in patients with non-frontal lesions.", "This would have allowed us to directly compare whether the degree of variance accounted for by education and NART IQ is reduced in frontal patients when compared to non-frontal patients, as suggested by the STAC model.", "We acknowledge a number of limitations of our retrospective study.", "We were unable to examine the influence of CR on memory abilities due to insufficient data.", "We could not assess our patients’ lifestyle factors which may also mitigate the occurrence of cognitive decline and differentially modify individuals’ performance on different cognitive measures.", "Additionally, given the heterogeneous neuroimaging data available, we could not investigate parameters such as white matter intensities and cortical atrophy, known to associate with CR and cognitive performance (e.g., Brickman et al., 2011; Murray et al., 2011).", "However, a major strength of our study is that, to our knowledge, it is the first study to systematically investigate the effects of two CR proxies on the performance of patients with focal unilateral frontal lesions across several cognitive measures.", "Overall, our data suggest that in the context of prefrontal lesions, the protective effects of education and NART IQ are limited to specific cognitive measures.", "Unlike previous studies reporting that CR can compensate for brain pathology due to healthy aging or neurodegenerative diseases, our data suggest that CR does not show the same ‘protection’ against the impact of focal brain damage involving the frontal lobes.", "Future work is needed to examine further the complex relationship between CR, age and the presence of a frontal lesion when attempting to understand the resulting impairments in executive tasks." ]	[ "Effect of cognitive reserve (CR) proxies in focal unilateral lesions.", "NART IQ accounts for a larger proportion of variance in executive and naming skills.", "Age predicts performance on executive, fluid intelligence, speed and perception tests.", "Education and NART IQ did not modify the effect of lesion severity on cognitive impairment." ]
Impaired retrieval processes evident during visual working memory in schizophrenia	Prominent working memory (WM) deficits have been observed in people with schizophrenia (PSZ) across multiple sensory modalities, including the visuospatial realm. Electrophysiological abnormalities noted during early visual processing as well as later cognitive functions in PSZ may underlie deficiencies in WM ability, though the mechanisms linking behavior to neural responses are not well understood. WM dysfunction has also been observed in biological relatives of PSZ (REL) and therefore may be a manifestation of genetic liability for the disorder. We administered a delayed response visuospatial WM task to 23 PSZ, 30 of their REL, and 37 healthy controls (CTRL) to better understand the contributions of neural abnormalities to WM performance deficits associated with schizophrenia. PSZ performed more poorly on the WM task and failed to effectively process distractor stimuli as well as CTRL and REL. N1 electrophysiological responses to probes during retrieval differentiated the type and locations of stimuli presented during encoding in CTRL. Retrieval N1 responses in PSZ, however, failed to do so, while retrieval responses in REL showed more pronounced differentiation of stimulus features during encoding. Furthermore, neural responses during retrieval predicted behavioral performance in PSZ and REL, but not CTRL. These results suggest that retrieval processes are particularly important to efficient visuospatial WM function in PSZ and REL, and support further investigation of WM retrieval as a potential target for improving overall WM function through clinical intervention.	[ "MEDI", "NEUR" ]	[ "Endophenotype", "Event-related potential", "Relatives", "Schizophrenia", "Working memory" ]	[ "2738", "2805" ]	[ "Working memory (WM) dysfunction in people with schizophrenia (PSZ) has been demonstrated across various sensory modalities (Fleming et al., 1997; Haenschel et al., 2007; Lee and Park, 2005).", "WM deficits have likewise been observed in the unaffected first-degree relatives of PSZ (Conklin et al., 2000; Park et al., 1995; Pirkola et al., 2005; Seidman et al., 2012), suggesting that WM impairment may represent an endophenotypic marker for schizophrenia (Gottesman and Gould, 2003; Haenschel and Linden, 2011).", "In addition to WM performance deficits, related neurophysiological abnormalities have been demonstrated in PSZ and their unaffected relatives.", "Deficient early visual processes have been repeatedly observed in PSZ during WM tasks (Dias et al., 2011; Haenschel et al., 2007; Zhao et al., 2011), and related deficits have been observed in unaffected relatives who presumably carry genetic liability for the disorder (Yeap et al., 2006).", "Electrophysiological correlates of later cognition, including WM functions (Haenschel et al., 2007; Zhao et al., 2011), have likewise been shown to be abnormal in PSZ.", "Some abnormalities in later processes have similarly been reported in unaffected relatives (Lee et al., 2010; Sponheim et al., 2006).", "Recent work in WM has focused on the role of distracting stimuli in preventing efficient encoding which may compromise the amount or content of material in WM in PSZ (see Eich et al., 2014; Erickson et al., 2014).", "However, researchers have yet to understand the mechanisms linking these neural abnormalities to observed behavioral deficits during WM in PSZ and their unaffected relatives.", "We analyzed event-related potentials (ERPs) elicited during WM encoding and retrieval from PSZ, their unaffected relatives, and nonpsychiatric controls to better understand the contribution of neural responses to WM dysfunction associated with the disorder.", "To understand neural mechanisms associated with WM performance deficits, we examined electrophysiological responses to task manipulations related to distracting stimuli, amount of material (i.e., load), and the location of the probe stimulus.", "If, for example, PSZ and/or relatives showed abnormal modulation of neural responses to distractors versus target stimuli, this would support the notion that distracting stimuli may be particularly important in explaining WM deficits in these populations.", "Similarly, examination of responses during encoding and retrieval would allow for isolation of neural deficits to a particular component of WM. We expected to see ERP abnormalities in PSZ and their first-degree relatives as compared to controls, as have been previously observed in studies outside the realm of WM and only scarcely investigated in visuospatial WM, especially in unaffected relatives.", "Specifically, we hypothesized that PSZ alone would show increased late potential amplitudes, thought to index WM load, for distractor stimuli during encoding, suggesting that PSZ were encoding task-irrelevant information.", "In addition, we hypothesized that REL would show stronger abnormalities in neural indices than behavioral indices.", "Participants (n = 90) were 23 PSZ, 30 first-degree biological relatives of PSZ (REL), and 37 healthy controls (CTRL; Table 1).", "They were enrolled as part of a family study of severe psychopathology based at the Minneapolis Veterans Affairs Medical Center.", "PSZ were recruited through a mental health clinic and past research rosters, other current studies of severe psychopathology, referrals from physicians, as well as community-based mental health facilities and the medical center.", "REL were recruited using contact information provided by PSZ, and CTRL were recruited primarily through advertisement in the medical center and community, as well as from past research rosters.", "Enrolled participants underwent clinical assessments, the results of which were subjected to a consensus diagnosis process in which two or more Ph.D. clinicians or advanced doctoral students reviewed participants' study materials to form jointly agreed upon diagnoses.", "Full inclusion and exclusion criteria and clinical assessments are described in the supplementary materials.", "Participants were administered a spatial WM task derived from Park's (1997) delayed response task; see Fig. 1 for further description.", "EEG was recorded using a BioSemi Active-Two AgCl electrode system (BioSemi Inc., Amsterdam, The Netherlands).", "Recordings utilized a 128-channel, full scalp dense array sampled at 1024 Hz.", "Recordings were down-sampled offline to 512 Hz, high-pass filtered at 0.5 Hz, and transformed to a linked earlobe reference.", "Data were preprocessed using a custom independent component analysis (ICA) based method for ocular, muscular and cardiac artifact removal; see supplementary materials for details.", "To investigate whether memory stimuli were differentially processed during encoding, ERPs were computed for stimulus type (target vs. distractor).", "To examine neural processes associated with increasing WM load during encoding, we computed ERPs for order of stimulus presentation (first vs. second vs. third).", "Finally, to study neural processes associated with retrieval as modulated by encoding manipulations, we computed ERPs for probe location based on the type of encoding stimulus that appeared in the same location (“encoded type”: probe at previous target vs. previous distractor location vs. elsewhere), as well as probe location based on the sequential position of the encoding stimulus (“encoded order”: probe at first vs. second vs. third stimulus position vs. elsewhere).", "ERPs were time-locked to the relevant stimulus and epoched from −150 ms to 850 ms with stimulus onset designated as 0 ms; subject averages were low-pass filtered at 20 Hz for ERP component analysis.", "ERP components of interest included the P1, N1, and a late positive potential (LPP) encompassing the P300 but extending as far as 850 ms after stimulus onset.", "P1 and N1 were measured by computing peak amplitudes between 100 and 175 ms and 125–225 ms respectively.", "The LPP was assessed by computing mean amplitudes within 50 ms time windows between 200 and 850 ms after stimulus onset.", "For each independent variable (stimulus type, order of stimulus presentation, probe location/stimulus type and probe location/presentation order), P1 and N1 were measured at electrode sites PO4 and PO8, and the LPP was measured at sites FC1, C2, and CP1.", "All reported findings are for these electrode sites because they contained the greatest component amplitudes.", "To examine the effects of group status and task manipulations on participants' performance of the WM task, separate repeated measures ANOVAs were run for independent variables of number of trial stimuli (two vs. three), trial type (with vs. without distractor), probe location based on encoded stimulus type (probe at previous target vs. distractor location vs. elsewhere), and probe location based on presentation order of encoded stimulus (probe at first vs. second vs. third stimulus location vs. elsewhere); diagnostic group (CTRL, PSZ or REL) was included in each ANOVA as an additional factor.", "ERP measures were analyzed using mixed model ANCOVAs.", "Separate ANCOVAs were run for independent variables of stimulus type, order of stimulus presentation, probe type, and probe order.", "Each model included as fixed factors the relevant independent variable, electrode site, diagnostic group (PSZ, REL or CTRL), and for the LPP, time window.", "Subject was included as a random factor; age and gender were included as covariates.", "Nearly all effects of electrode and time window were significant; those associated with group status and task manipulations are reported and with Greenhouse–Geisser (G-G) correction where appropriate.", "Post-hoc testing of significant ANCOVAs used Tukey's Honestly Significant Difference tests.", "In order to determine the relationship between ERP measures and behavioral performance, Pearson's correlations were computed for measures showing differences between CTRL and PSZ.", "Behavioral results for the spatial WM task are presented in Table 2.", "ANOVAs revealed that groups differed in accuracy across task manipulations (number of stimuli: F(2,87) = 6.06, p = .003, η2p = .12; trial type: F(2,87) = 7.17, p = .001, b = .14; probe location, encoded stimulus type: F(2,87) = 7.84, p = .001, η2p = .15; probe location, presentation order of encoded stimulus: F(2,87) = 7.15, p = .001, η2p = .14).", "In all cases, PSZ performed worse than CTRL (ps < .012, ds ≥ .71) and REL (ps < .005, ds ≥ .77); REL failed to differ from CTRL on behavioral indices.", "Effects of task manipulations on performance did not differ across groups, except for an observed interaction between group and trial type, F(2,87) = 3.44, p = .036, η2p = .07.", "Here, PSZ benefitted markedly less from the presence of a distractor (p = .02, d = .40) than did CTRL and REL (ps < .001, ds ≥ 1.03; Fig. 2a).", "Examination of reaction times to probes revealed an interaction between group and probe location, F(3.56,149.70) = 2.51, p = .05, η2p = .06.", "CTRL and REL were quicker to react to probes in previous distractor positions as well as previous targets (ps < .001, ds > .62), whereas PSZ showed faster reactions to probes in previous target positions (p = .005, d = .32) but only a trend for probes in previous distractor positions (p = .08, d = .20; Fig. 2b).", "Thus, CTRL and REL performed better than PSZ across all task conditions, and PSZ gained a lesser performance advantage on trials with distractors than did CTRL and REL.", "ERPs were computed for target and distractor stimuli in an effort to investigate whether the relevance of stimuli affected neural responses.", "Early P1 responses over occipital brain regions showed a main effect of stimulus type (F(1,87) = 38.11, p < .001, η2p = .30), demonstrating that target and distractor stimuli were differently encoded.", "Furthermore, LPP responses showed a significant interaction between stimulus type and time window, F(7.55,656.58) = 9.45, p < .001, η2p = .10, suggesting temporally isolated discrimination between the stimulus types.", "P1 and LPP amplitudes were greater in response to distractors than targets (ps < .03, ds > .29).", "There was no main effect of group on encoding ERPs, and stimulus type failed to interact with group for the ERP amplitudes.", "Thus, P1 and LPP responses suggest successful discrimination of target versus distractor stimuli across all diagnostic groupings.", "ERPs to stimuli were averaged based on their order of presentation (first, second, or third) to investigate neural responses related to increasing WM load at encoding.", "Early N1 responses over occipital regions showed effects of stimulus order (F(1.55,135.04) = 12.61, p < .001, η2p = .13), where responses to both second (p < .001) and third (p < .001) stimuli were significantly greater (more negative) than those to first stimuli; similar patterns were observed across diagnostic groups.", "The LPP over central midline sites showed a significant interaction between presentation order and time window, F(12.38,1077.42) = 10.58, p < .001, η2p = .11.", "Examination of the group distributions of mean amplitude across time window suggested differential patterns of responses across groups (Fig. S2).", "Because PSZ showed notably deviant waveforms from CTRL, within group ANCOVAs of LPP responses were conducted and confirmed that presentation order by time window interactions were observed for CTRL, F(8.36,300.82) = 5.67, p < .001, η2p = .14, and REL, F(7.96,230.74) = 5.89, p < .001, η2p = .17, but not PSZ, F(8.07,177.52) = 1.59, p = .13, η2p = .07.", "Thus, LPP responses indexed WM load in CTRL and REL, but not in PSZ; early N1 responses, however, mirrored increases in WM load for all participants, regardless of diagnostic status.", "In order to investigate neural responses to probes at previously encoded locations, ERPs to probe stimuli were averaged based on whether they appeared in the position of a previous target, distractor, or elsewhere (“encoded type”).", "Early N1 responses over occipital brain regions showed an interaction between diagnosis and encoded type, F(4174) = 8.17, p < .001, η2p = .16 (Fig. 3).", "While a main effect of encoded type was observed for CTRL, F(2,72) = 4.09, p = .02, η2p = .10, and REL, F(2,58) = 24.56, p < .001, η2p = .46, PSZ showed no such effect, F(2,44) = 0.68, p = .51, η2p = .03.", "CTRL's N1 responses to probes at previous targets (p = .04, d = .26) as well as to probes at previous distractors (p = .04, d = .25) were larger than those to probes elsewhere; in REL, N1 responses to probes at distractors were greater than probes at targets (p = .003, d = .43), which in turn were greater than probes elsewhere (p = .002, d = .40).", "Thus, early N1 responses to probe stimuli for PSZ fail to be affected by prior encoding information, while such responses in REL and CTRL do differentiate previously encoded spatial locations.", "Late central midline LPP responses showed an interaction between encoded type and time window, F(12.58, 1094.03) = 2.31, p = .006, η2p = .03; Fig. 4a).", "Examination of the waveforms suggested possible differences in late responses across diagnostic groups (Fig. 4b–d); as such, within-group analyses were performed due to what appeared to be deviance in the PSZ and REL waveforms as compared to that of CTRL.", "Within-group ANCOVAs confirmed that significant encoded type-by-time-window interactions were observed for CTRL, F(9.85,354.53) = 2.77, p = .003, η2p = .07, but neither REL, F(8.33,241.48) = 1.39, p = .20, η2p = .05, nor PSZ, F(7.37,162.05) = 0.75, p = .64, η2p = .03.", "Fig. 4 illustrates that RELs' and PSZs' late responses to probe stimuli failed to differentiate between the previously encoded locations as compared to CTRL.", "Interestingly, LPP amplitudes to probes elsewhere were predictive of overall behavioral performance for PSZ alone between 400 ms and 450 ms post-probe (PSZ: r = −.43, p = .04; CTRL: r = .06, p = .74; REL: r = −.04, p = .83) as well as between 450 ms and 500 ms post-probe (PSZ: r = −.45, p = .03; CTRL: r = .01, p = .98; REL: r = −.04, p = .83).", "Thus, for PSZ, greater LPP responses to probes in task-irrelevant locations, perhaps reflecting errant recognition, predicted poorer performance in the 400 ms to 500 ms post-probe time window.", "In order to investigate neural responses to probe positions relative to the order of stimulus presentation, ERPs to probe stimuli were averaged based on whether they appeared in the position of a first, second, third stimulus, or elsewhere (“encoded order”).", "Early N1 responses recorded over occipital regions revealed a diagnosis-by-encoded order interaction, F(5.03,218.92) = 2.47, p = .03, η2p = .05.", "Post-hoc testing revealed that CTRL and REL showed greater N1 responses to probes in third stimulus positions than those in first positions and elsewhere (ps < .009, ds > .45), whereas PSZ demonstrated no effect of probe order (Fig. 5).", "Thus, CTRL and REL showed increasing N1 amplitudes to probes based on their position relative to the presentation order of the encoding stimuli, whereas PSZ failed to show such an effect.", "In addition, N1 responses to probes in the position of second stimuli predicted behavioral performance on trials featuring distractors in REL (r = −.43, p = .02) but neither CTRL (r = −.15, p = .39) nor PSZ (r = .02, p = .95).", "Thus, more robust N1 responses to probes in REL reflected more effective differentiation of previously encoded relevant and irrelevant locations.", "We administered a delayed response spatial WM task to PSZ, their first-degree biological relatives, and healthy controls during which we recorded EEG.", "We analyzed behavioral data and event-related neurophysiological responses in order to clarify the nature of spatial WM deficits in PSZ and their biological relatives (see Table 3 for a summary of findings).", "PSZ exhibited WM performance deficits and smaller neural responses as compared to controls.", "In particular, early posterior responses (N1) in PSZ recorded during retrieval failed to differentiate previously encoded locations while such responses in CTRL and REL varied depending on encoding status of the location.", "PSZ performed more poorly on the WM task than CTRL and REL across all task conditions.", "Such performance deficits are consistent with the widespread WM deficits typically observed in PSZ (Forbes et al., 2009; Lee and Park, 2005).", "Notably, all participant groups showed higher accuracy rates on trials that featured a distractor versus those that did not.", "Together with the observed reduction in reaction times to probes in the position of previous distractors, these findings suggest that participants benefitted from successfully encoding distractor stimuli.", "However, the interaction effects between group and trial type as well as group and reaction time indicate that PSZ were less able to use distractors to their advantage as compared to CTRL and REL.", "Hence, though PSZ are capable of successfully differentiating distracting stimuli when they are not overly salient (Erickson et al., 2015, 2014; Gold et al., 2006; Gray et al., 2014; Hahn et al., 2010), they do so less efficiently than healthy controls; such a result is consistent with fMRI studies demonstrating inefficiency of cognitive processing in PSZ (Cairo et al., 2006; Schlӧsser et al., 2008).", "Though PSZ gained some advantage from encoding distractors, their relative improvement was not as great as that observed in REL and CTRL.", "During retrieval, CTRL and REL demonstrated enhanced N1 responses to probe stimuli in the positions of previous stimuli, while PSZ showed no such response.", "N1 has been well established as an index of orientation to task-relevant locations (Luck et al., 1990); Dias et al. (2011) have reported similar modulations in PSZ and controls of N1 amplitude to probes based upon the prior cue.", "In the present study we found a failure in PSZ to modulate N1 responses to probes based on their spatial relation to encoding stimuli.", "It is possible that abnormalities observed during retrieval reflect failures to successfully encode stimuli in the first place; however, neural responses (namely, the P1 and P300) suggest some successful neural discrimination between differing stimuli during encoding.", "These findings are consistent with PSZs' behavioral performance and previous findings of preserved function of the parvocellular visual pathway in PSZ (e.g., Dias et al., 2011).", "Neural abnormalities at retrieval in PSZ may likewise reflect a failure to maintain encoded information in a manner that would affect early processing of stimuli at retrieval, consistent with previously reported maintenance deficits in PSZ (Fleming et al., 1997; Haenschel et al., 2007; Lee and Park, 2005).", "Furthermore, REL showed augmented N1 responses to probes at distractor locations, suggesting a potential sensitivity of biological relatives to distractor stimuli.", "Similarly, N1 responses to probes showed greater modulation by the presentation order of the stimuli in REL than CTRL, suggesting that the “traces” of these relevant positions are more clearly delineated.", "However, REL show LPP responses similar to PSZ for probe stimuli during retrieval.", "Functional MRI studies have shown similar activation deficits in PSZ and REL; however, REL also demonstrate increased activations as compared to CTRL in certain brain regions that have been posited to represent compensatory responses (reviewed in Zhang et al., 2016).", "The presently reported abnormalities in the modulation of early visual processes indexed by the N1 in REL for WM retrieval may similarly represent a compensatory neural mechanism that facilitates intact performance by biological relatives of PSZ on this spatial WM task featuring simple stimuli.", "Neural responses to probe stimuli proved most predictive of behavioral performance for PSZ and REL, but were not predictive for CTRL.", "PSZ performance related to LPP responses to probes that appeared in a location not previously occupied by an encoding stimulus, suggesting that PSZ who showed the strongest neural recognition of probes in a novel location exhibited errant probe location recognition.", "REL performance was predicted by N1 responses to probes in the second stimulus position.", "These associations were not significant for CTRL, suggesting that retrieval processes are particularly important to efficient WM ability in PSZ and REL, and that neural function during spatial WM retrieval may be the critical determinant of WM dysfunction in the disorder.", "In conclusion, the present study demonstrated neural deficits in PSZ during both encoding and retrieval.", "REL showed encoding responses comparable to CTRL, in addition to preserved and even heightened modulations of early responses to probes during retrieval despite late responses similar to PSZ.", "This notable difference between PSZ and REL during retrieval may suggest a compensatory mechanism for REL allowing them to perform at the level of CTRL.", "Consistent with this notion, both PSZ's and REL's task performance was predicted by neural responses during retrieval, emphasizing the importance of retrieval processes to WM function in PSZ and their unaffected relatives.", "The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.", "PAL programmed the behavioral task, collected much of the data, designed and performed data analyses and wrote the submitted manuscript.", "SSK wrote the base scripts used in processing the data and from which the analysis scripts were derived, reviewed the manuscript, and offered commentary.", "SRS designed the behavioral task and experiment, secured funding for the project, oversaw the recruitment and clinical assessment of participants, advised the data analyses and writing of the manuscript, and carried out editing of the submission.", "The authors have no conflict of interest." ]	[]
Forest harvest dataset for northern Colorado Rocky Mountains (1984–2015) generated from a Landsat time series and existing forest harvest records	This dataset provides a shapefile containing approximately 3500 polygons with the location, extent, size, and year of clearcut harvest events occurring between 1984 and 2015 in forested areas of the northern Colorado, Landsat WRS-2 scene Path 034, Row 032. Harvest events were modeled and mapped using a 32 year time series of Landsat imagery, the LandTrendr algorithm, and ancillary datasets. The dataset also conveys information on the elevation, aspect, ownership, distance to roads, and the watershed where each harvest event occurred.	[ "MULT" ]	[ "Forestry", "Harvests", "LandTrendr", "Landsat" ]	[ "1000" ]	[ "This forest harvest history dataset was derived from a 32-year time series of Landsat imagery using advanced image normalization and disturbance detection methods.", "Historical harvest spatial extents were modelled by integrating limited existing forest harvest records with time series data in a boosted regression trees classification algorithm.", "We acquired Landsat surface reflectance higher level data products for Landsat 4 and 5 Thematic Mapper (TM), Landsat 7 Enhanced Thematic Mapper plus (ETM+), and Landsat 8 Operational Land Imager (OLI) for the months of July, August, and September between 1984 and 2015 for WRS-2 Path 034, Row 032 via the USGS Earth Resources Observation and Science (EROS) Center Science Processing Architecture.", "All available images that contained less than 50% cloud cover were selected in our analysis, totaling 182 individual scenes across the 32-year study period.", "We processed these Landsat scenes using LandsatLinkr (LLR) [1], an R [2] package that creates annual, cloud-free, spectrally-consistent tasseled cap [3] composites for use in change detection analyses.", "The LLR package unpacks Landsat surface reflectance products, masks each scene for clouds, water, snow, and ice, and calculates tasseled cap brightness, greenness, wetness, and angle using standardized coefficients [3].", "In years where both Landsat 7 and 8 are available, spectral calibration is performed by the LLR tool using near date imagery to create an aggregate tasseled cap model that can be applied to all Landsat 8 OLI imagery to provide spectral consistency between TM/ETM+ and OLI sensors [4].", "The tasseled cap images produced for each scene are then composited using the mean pixel value of all available images for each year to create annual, cloud-free tasseled cap composites.", "We used LandTrendr [5] to delineate all spectrally-detectable disturbance events.", "We implemented a modified version of LandTrendr (LLR-LandTrendr) [6] that uses tasseled cap composites produced through LLR.", "LandTrendr is an advanced Landsat-based change detection algorithm that tracks changes in spectral trajectories at the pixel level to characterize disturbance events.", "We used tasseled cap wetness as our index for spectral segmentation and used default segmentation and trajectory-fitting parameters with no cover model.", "This resulted in multiple LandTrendr products including greatest disturbance magnitude, duration, and pre-disturbance event spectral vertex values which were used as predictors of harvest presence.", "Pixels identified as disturbed in LandTrendr outputs were used to limit the study area because the products represented spectrally-detectable disturbances of all magnitudes.", "Ancillary vector data containing forest management records were compiled from the 2014 LANDFIRE Public Events Geodatabase [7] and previously-validated harvest polygons (Woodward et al., unpublished data).", "Harvest polygons were visually inspected and validated using high-resolution (≤ 1 m2) aerial imagery.", "We buffered the validated harvest polygons (n=354) by 30 m to remove mixed pixels at the edge of harvests.", "Spatially-balanced points within the buffered harvest polygons (n=1510) and background points (n=4408) were generated to train the models described below.", "Modelling was performed using the Software for Assisted Habitat Modeling (SAHM) module package for VisTrails, an open-source provenance management and scientific workflow system [8].", "Predictor variables included greatest disturbance magnitude, duration, and pre-event vertex values, topography (elevation, slope, aspect, and compound topographic index) and distance to roads.", "We evaluated five classification models: random forests (RF), multivariate adaptive regression splines (MARS), generalized linear model (GLM), boosted regression trees (BRT), and maximum entropy (MaxEnt).", "We compared evaluation metrics from a ten-fold cross-validation to select the final model.", "Area Under Curve (AUC), Cohen's Kappa, True Skill Statistic (TSS), sensitivity, specificity, and percent correctly classified (PCC) metrics were used to assess model performance rather than relying on a single statistic to allow for a better overall model evaluation [9].", "We selected the BRT model (AUC=0.98, Cohen's Kappa=0.86, TSS=0.89, sensitivity=.93, specificity=0.95, PCC=94.8) to generate the final maps.", "Final outputs (Fig. 1) were refined by filtering out disturbances that were less than 11 contiguous pixels [5].", "Outputs were converted from raster to vector format and additional information was appended to each polygon: year of harvest derived from LandTrendr outputs, elevation, aspect, slope, area, perimeter, county, watershed, ownership, and distance from road.", "The final dataset contains 3531 polygons of clearcut harvest events occurring from 1984 to 2015." ]	[]
A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis	A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and up-regulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesis.	[ "BIOC" ]	[ "Endothelial to haematopoietic transition", "Gene trap", "Gfi1", "Haematopoiesis", "Haemogenic endothelium", "Zebrafish" ]	[ "1307", "1309", "1312" ]	[ "Haematopoietic stem cells (HSCs) are immature blood cells that can self-renew and give rise to mature cells of all blood lineages (Doulatov et al., 2012).", "HSCs first develop in the embryo.", "During embryogenesis, haematopoietic cells (HCs) arise in waves from mesodermal progenitors (reviewed in (Ciau-Uitz et al., 2014; Clements and Traver, 2013; Frame et al., 2013; Medvinsky et al., 2011)).", "In mammals, the first HCs are primitive red blood cells (prRBCs), macrophages and neutrophil granulocytes (Palis et al., 1999; Tober et al., 2007).", "They develop in the yolk sac from haemangioblasts, mesenchymal cells that are bipotent progenitors for blood and endothelial cells (ECs) (Huber et al., 2004).", "Haematopoietic progenitor cells (HPCs) and HSCs arise in subsequent waves.", "They form from haemogenic endothelial cells (HECs) (Chen et al., 2011; Chen et al., 2009; Frame et al., 2016; Yokomizo et al., 2001; Zovein et al., 2008) that undergo endothelial to haematopoietic transition (EHT) and form HC clusters inside the vessels.", "HPCs develop in yolk sac arteries and veins (Frame et al., 2016) while HSCs form from arterial HECs of the ventral wall of the dorsal aorta (vDA) and from other major arteries (de Bruijn et al., 2000; Gordon-Keylock et al., 2013; Medvinsky and Dzierzak, 1996; Taoudi and Medvinsky, 2007).", "Explant studies have visualized this process in the mouse vDA (Boisset et al., 2010).", "Once born, HSCs establish the definitive wave of haematopoiesis that maintains the blood system throughout life.", "In zebrafish, primitive blood cells also differentiate from mesenchymal haemangioblasts.", "The anterior and posterior lateral mesoderm (ALM, PLM) give rise to primitive myeloid and erythroid cells, respectively (Gering et al., 1998; Herbomel et al., 1999; Liao et al., 1998).", "PLM cells migrate to the midline to form the intermediate cell mass (ICM) where they differentiate into prRBCs, as well as ECs of the DA and the posterior cardinal vein (PCV) (Detrich et al., 1995; Kohli et al., 2013).", "Primitive erythroblasts first enter circulation between 24 and 25 h post fertilization (hpf).", "In circulation, they mature over the following days (Qian et al., 2007; Weinstein et al., 1996).", "After the onset of circulation, definitive HCs begin to arise from HECs in the zebrafish vDA.", "Unlike in mammals, zebrafish vDA HECs undergo basal epithelial to mesenchymal transition (bEMT) as they turn into HCs (Bertrand et al., 2010; Kissa and Herbomel, 2010; Lam et al., 2010; Zhen et al., 2013).", "These enter circulation through the vein (Kissa et al., 2008) and seed the caudal haematopoietic tissue (CHT) (Jin et al., 2007; Murayama et al., 2006).", "The CHT is a transient larval site of haematopoiesis in the tail mesenchyme, where haematopoietic stem and progenitor cells (HSPCs) occupy perivascular niches (Tamplin et al., 2015) before they leave for the kidney and the thymus (Traver et al., 2003).", "Gene expression analyses combined with gain- and loss-of-function studies in different vertebrate model organisms have defined sets of transcription factors and co-regulators expressed in developing HCs that tightly regulate gene expression during blood development and, thereby, control cell fate and identity (Orkin and Zon, 2008).", "The closely related transcriptional repressors Gfi1 and Gfi1b are two of these transcription factors.", "Gfi1 and Gfi1b play important overlapping roles in adult HSCs and fulfill non-redundant functions in cells of particular blood lineages (Möröy et al., 2015).", "Gfi1b knockout mice die during gestation with abnormal erythropoiesis and megakaryopoiesis (Saleque et al., 2002).", "By contrast, Gfi1 knockout mice are viable, but have inner ear defects and severe neutropenia (Hock et al., 2003; Karsunky et al., 2002; Wallis et al., 2003).", "Gfi1b expressed in place of Gfi1 can substitute for Gfi1 during haematopoiesis, but is not sufficient for normal inner ear development (Fiolka et al., 2006).", "The zebrafish genome harbors three Gfi1 paralogs (Cooney et al., 2013; Dufourcq et al., 2004; Wei et al., 2008).", "Two paralogs, gfi1aa and gfi1ab are orthologs of mammalian Gfi1, arisen during a genome duplication in the teleost lineage.", "The third paralog encodes a Gfi1b protein.", "Morpholino knockdown studies have indicated that zebrafish gfi1aa plays an important role in primitive erythropoiesis (Cooney et al., 2013; Wei et al., 2008) and that gfi1b alone is essential for the development of all definitive HC lineages in the embryo (Cooney et al., 2013).", "While observed defects in definitive erythrocyte and thrombocyte development are consistent with the phenotype of the mouse gfi1b knockout, the apparent deficiency in all definitive lineages, as well as the loss of primitive erythropoiesis in the gfi1aa morphant embryo were unexpected and suggested a remarkable reshuffling of responsibilities between Gfi1/1b proteins in the bony fish lineage since their divergence from the common teleost and tetrapod ancestor.", "Here, we report a zebrafish gfi1aa gene trap line that demonstrates that the loss of haematopoietic Gfi1aa expression is compatible with primitive erythropoiesis.", "Our data contradict the previous morpholino studies and suggest that the roles of the mammalian Gfi1 and Gfi1b proteins are conserved in the teleost lineage.", "A transposon-based gene trap approach was used in zebrafish to identify novel genes involved in embryonic haematopoiesis (Fig. 1A).", "F1 progeny of transposon-injected fish were analyzed for GFP expression at 26 hpf, once most prRBCs had entered circulation.", "One line, designated qmc551, displayed GFP in circulating blood cells and in spindle-shaped cells located between the DA and the PCV (Fig. 1B and C; Movie 1A).", "Sectioning of GFP-immunostained qmc551 embryos confirmed GFP expression in blood cells and suggested that the spindle-shaped cells are ECs located in the vDA (Fig. 1D-F).", "Confocal microscopy revealed additional mesenchymal GFP+ cells that appeared to be located just outside the blood vessels (Fig. 1G and H; red arrowheads; Movie 1B).", "Subsequent examination of double transgenic embryos at single cell resolution in 1.8–2.5 µm optical sections showed that both, the round GFP+ cells inside and the mesenchymal cells outside the vessels, co-expressed the prRBC marker gata1: dsRed (Traver et al., 2003), demonstrating that these cells were prRBCs and their precursors, respectively (Fig. 1I/S1A).", "The spindle-shaped GFP+ cells co-expressed the EC reporter flk1: tdTom and were indeed localized in the vDA (Fig. 1J/S1B).", "Co-expression of the Notch reporter gene csl: cer (Fig. 1K/S1C) confirmed these cells were arterial.", "Supplementary material related to this article can be found online at 10.1016/j.ydbio.2016.07.010.", "The following is the Supplementary material related to this article Movie 1.", ": In the gene trap line qmc551, GFP is expressed in primitive red blood cells and in spindle-shaped cells located between the dorsal aorta and the posterior cardinal vein at 26 hpf.", "This movie is related to Figure 1.", "(A) Live movie of the trunk and tail of a 26 hpf qmc551 transgenic.", "GFP is expressed in circulating and stationary blood cells and in elongated cells located between the dorsal aorta and the posterior cardinal vein.", "Anterior is to the left, dorsal is up.", "Pictures of this timelapse movie were taken every 100 ms with a Hamamatsu Orca-ER camera on a Nikon SMZ1500 dissection microscope with an epifluorescence attachment using a FITC filter set.", "The camera was controlled by IP lab software.", "Images were pseudo-colored in IP lab and saved as TIFF files.", "The series of TIFF files was imported into Imaris and turned into a video that was then annotated in iMovie.", "(B) Confocal analysis of the trunk of a fixed 26 hpf qmc551 transgenic embryo shows expression of GFP in blood cells and in spindle-shaped cells located between the lumen of the dorsal aorta and the lumen of the vein.", "An animation of a 3D maximum intensity projection of a 67.5 µm thick Z stack is shown.", "A still image of the maximum intensity projection of the Z stack highlights GFP+endothelial cells (green arrows), red blood cells in circulation (red arrow) and outside the vessels (red arrowheads).", "The embryo was first treated with an overdose of anesthetic and then fixed in 4% PFA for an hour.", "The Z-stack was taken on an inverted Zeiss Exciter confocal microscope and has the dimensions 161×161×67.5 µm, respectively.", "The movie starts with anterior to the left and dorsal up.", "The confocal images were acquired on a Zeiss Exciter microscope with an EC Plan-Neofluar 40x/1.30 Oil DIC M27 lens.", "HECs of the vDA are known to express the stem cell transcription factor Runx1 (Gering and Patient, 2005).", "Fluorescent whole-mount in situ hybridization (WISH) experiments showed that runx1 mRNA is localized in foci in flk1/kdrl+ ECs of the vDA (Fig. 1L/S2A).", "The same runx1 mRNA foci were also observed in GFP+ vDA ECs in qmc551 transgenic embryos (Fig. 1M/S2B).", "Moreover, a subset of the GFP+ ECs contained cmyb mRNA (Fig. 1N/S2C), which is known to be induced downstream of Runx1 (Burns et al., 2005; Kalev-Zylinska et al., 2002).", "To determine whether these GFP+ ECs could undergo EMT to give rise to HCs, confocal timelapse microscopy was performed on qmc551;flk1/kdrl: tdTom double transgenic embryos.", "These experiments revealed that individual ECs with elevated GFP slowly bent towards the mesenchyme, rounded up and eventually joined the mesenchyme as the endothelium closed up above them (Fig. 1O/S2D; Movie 2A).", "Throughout the process, the cells co-expressed the endothelial reporter transgene (Fig. 1P/S2E; Movie 2B).", "In addition to bEMT, we also observed that individual GFP+ cells left the endothelium apically to enter the DA directly, a process seen only up to 40 hpf (Fig. 1Q/S2F; Movie 3A).", "Cells, which had undergone bEMT, spent variable periods of time in the mesenchyme before they entered the vein to join the circulation (Fig. 1R/S2G; Movie 3B).", "These data strongly suggested that qmc551: GFP expression marks HECs prior to, during and after EHT.", "It is worth noting that the events observed in qmc551 transgenics were also seen in flk1/kdrl: gfp;csl:cer double transgenic embryos (Movies 4 and 5)).", "Altogether, these data confirmed the erythroid nature of the qmc551: GFP+ blood cells and showed that the spindle-shaped GFP+ cells were HECs of the vDA.", "Intrigued by the interesting expression pattern we wanted to know which gene was trapped by the transposon.", "Supplementary material related to this article can be found online at 10.1016/j.ydbio.2016.07.010.", "The following is the Supplementary material related to this article Movie 2, Movie 3, Movie 4, Movie 5.", ": qmc551: GFP+haemogenic endothelial cells undergo basal epithelial to mesenchymal transition.", "This movie is related to Figure 1.", "It shows image series and still frames of confocal timelapse microscopy experiments performed on qmc551: eGFP;flk1/kdrl: tdTom-double transgenic embryos.", "The embryo was immobilized in 1% low melting point agarose.", "The embryo faces to the left with dorsal up.", "The timelapse experiment started at 31 hpf.", "In (A) only GFP expression is shown, highlighting that qmc551: GFP marks HECs before, during and after epithelial to mesenchymal transition.", "In (B), both GFP and tdTom fluorescence are shown.", "tdTom expression confirms that spindle-shaped GFP+ cells undergoing EMT are vDA ECs.", "tdTom also highlights the outline of the vein.", "Please note that green horizontal lines in the DA and the PCV are caused by GFP+ prRBCs in circulation.", "The still frame shows the final image of the timelapse series shown in (B).", "It highlights the HEC-derived mesenchymal cell (yellow arrow) and the endothelium (white arrow) that has closed up above it.", "All images show single 2.1 µm optical sections.", "Images were processed in Imaris and annotated in iMovie.", ": qmc551: GFP+ haemogenic endothelial cells give rise to blood cells that join the circulation.", "This movie relates to Figure 1.", "(A) Timelapse movie of 1.5 µm thick optical slices through the trunk of uninjected qmc551;flk1/kdrl: tdTom double transgenic embryos from 33 hpf.", "The movie shows how a double-positive haemogenic endothelial cell leaves the endothelium apically to enter the DA.", "(B) Mesenchymal GFP single (green circle) and GFP/tdTom double-positive (yellow circle) cells migrate through the mesenchyme before they enter the vein to join the circulation.", "This movie is identical to the one shown in Movie 2, but different cells are highlighted.", "The cell marked with the yellow circle disappears in Z as it moves from below the vein to a position above the vein.", "It then re-appears (yellow arrow) before it enters the vein.", "All images show single 2.1 µm optical sections.", "Images were processed in Imaris and annotated in iMovie.", ": flk1/kdrl: GFP/csl: Cer-double positive haemogenic endothelial cells give rise to blood cells that join the circulation.", "This movie provides supporting evidence related to Figure 1.", "It shows data from timelapse confocal microscopy on flk1/kdrl: gfp/csl: cer-double transgenic embryos starting from 48 hpf.", "Images of single 1.5 µm optical sections are shown with anterior to the left and dorsal up.", "Images were taken every 3 min.", "At 48 hpf, endothelial cells of the dorsal aorta co-express both reporter transgenes, while vein endothelial cells are only GFP-positive.", "During the course of the timelapse, different events were observed that are all highlighted with colored circles.", "The red circle marks a haemogenic endothelial cell as it underwent basal endothelial to haematopoietic transition.", "In the mesenchyme, the cell divided once and the daughter cells remained more or less stationary.", "As time went on, haematopoietic cells in the mesenchyme lost their green fluorescence faster than their blue fluorescence.", "Cells that entered the vein to join the circulation were clearly less brightly green than cells that had only just undergone EHT.", "Based on the residual intensity of their green fluorescence, it was obvious that cells remained in the mesenchyme for different periods of time before they entered the vein.", "They also entered the vein by different routes.", "One cell was seen to migrate around the vein before entering it through its ventral wall (blue circle).", "Another cell entered the vein through its dorsal wall (green circle) while several other cells got trapped in an endothelial pocket formed by venous endothelial cells (yellow circle).", "The cells eventually left the pocket to join the circulation.", ": Some flk1/kdrl: GFP;csl:Cer double-positive haemogenic endothelial cells leave the endothelium apically.", "This movie supports findings reported in Figure 1.", "It shows data from timelapse confocal microscopy on flk1/kdrl: gfp/csl: cer-double transgenic embryos starting from 30 hpf.", "The arrow points at a haemogenic endothelial cell that leaves the endothelium apically to enter the dorsal aorta.", "The embryo was immobilized in 1% low melting point agarose.", "The images show single 1.5 µm thick optical sagittal sections of an embryo with anterior to the left and dorsal up.", "During the timelapse, images were taken every 3 min.", "To identify the gene, Southern blot experiments were performed using a probe embedded in the gfp gene on the transposon (Fig. 2A).", "These experiments detected seven copies of the transposon in the genomic DNA of qmc551 transgenics (Fig. 2B).", "To reduce the number of genomic integrations in the progeny, outcrosses with wild-type (wt) fish were performed.", "In all of these outcrosses, half of the progeny were GFP+ and displayed the full GFP expression pattern, demonstrating that the pattern was not a composite, but reflected GFP expression from a single transposon that was inherited in Mendelian fashion.", "After 4 generations, nested inverse polymerase chain reaction (PCR) was performed and identified a 134 bp sequence upstream of the transposon (Fig. 2C; for details see Fig. S3) that was identical to a sequence in intron 1 of the zebrafish gfi1aa gene (Fig. 2D).", "This integration site was validated in PCR experiments in which genomic DNA fragments were amplified across both intron 1-transposon boundaries (Fig. 2E).", "These experiments showed that the GFP expression strictly correlated with the presence of the transposon in gfi1aa.", "Furthermore, PCR experiments with two gfp-internal primers, confirmed the absence of silent transposon copies in the qmc551 line (Fig. 2F).", "Consistent with the transposon's position, reverse transcription PCR (RT-PCR) allowed successful amplification, cloning and sequencing of a cDNA in which exon 1 of gfi1aa was spliced to the splice acceptor on the transposon (Fig. 2G), demonstrating that GFP was transcribed under the control of the gfi1aa promoter.", "Consistent with its expression under the control of the gfi1aa promoter, embryonic GFP mRNA and protein expression patterns in qmc551 transgenic embryos reflected endogenous gfi1aa mRNA expression in non-transgenic embryos.", "Following early maternal expression (Fig. 3A), zygotic GFP and gfi1aa expression was first seen in prRBC progenitors of the PLM (Fig. 3B,C).", "While gfi1aa mRNA diminished in prRBC progenitors between 21 and 28 hpf (Fig. 3C,D), GFP mRNA and protein were still detected in prRBCs in circulation and in the posterior blood island, the posterior extension of the trunk ICM (Fig. 3D).", "This suggests that GFP mRNA and protein were more stable than endogenous gfi1aa mRNA.", "GFP and gfi1aa expression were also found in the inner ear and in ECs of the vDA (Fig. 3D).", "As GFP+ HSPCs left the AGM, they started to accumulate in the CHT where they displayed dynamic interactions with ECs (Fig. 3F, Movie 6A).", "At 2 and 3 dpf, GFP+ ECs expanded posteriorly into the ventral wall of the caudal artery (vCA)(Fig. 3F,G).", "In addition to the lateral line organ, the exocrine pancreas was seen to express GFP and gfi1aa mRNA (Fig. 3H,I).", "By contrast, only GFP could be detected in cells of the gut, which, based on their position and morphology, as well as in analogy to Gfi1 expression in the mouse (Bjerknes and Cheng, 2010), are likely to be mucus-filled goblet and crypt-base localized Paneth cells (Fig. 3I and K).", "While haematopoietic gfi1aa expression was undetectable by 3 dpf, GFP mRNA and protein could still be observed in the vDA, in the trunk mesenchyme and in the CHT at 3 and 5 dpf (Fig. 3H–K).", "In haematopoietic cells, gfi1aa mRNA may be present at lower levels that become increasingly difficult to detect by WISH as the larvae grow older.", "In the live qmc551 transgenics, GFP+ blood cells first seeded the thymus on day 3 (Fig. 3H,I,M) and the larval kidney at day 5 (Fig. 3L).", "On day 6, wandering GFP+ cells were visible throughout the head (Fig. 3O, Movie 6B).", "These were probably immature leukocytes, since they did not stain with the mature neutrophil marker Sudan black (Le Guyader et al., 2008) and did not co-express the macrophage reporter mpeg1: dsRed (Ellett et al., 2011) (Fig. S4).", "In adult kidney sections, individual GFP+ cells were found between the renal tubules.", "The sections displayed fewer GFP+ cells than kidney sections of the cd41: gfp fish, which are known to express GFP in HSPCs and in cells of the platelet/thrombocyte lineage (Ma et al., 2011) (Fig. 3P).", "Flow cytometry on kidney marrow (KM) cells of qmc551;gata1: dsRed transgenics demonstrated that (a) the GFP+ cells fall mainly into the progenitor and lymphoid gates of the forward and side scatter profile (Traver et al., 2003) and (b) do not co-express gata1: dsRed (Fig. 3Q).", "Thus, while qmc551: GFP was seen in embryonic prRBCs, there was no expression in definitive erythrocytes.", "Consistent with the flow cytometric data, cytospins of GFP+ KM cells identified very few neutrophils with multi-lobed nuclei (Fig. 3R) and macrophages with granules (Fig. 3S).", "Most of the stained cells were early progenitors with large nuclei and scant cytoplasm (Fig. 3T), and small lymphocytes (Fig. 3U).", "No erythrocytes were found among the GFP+ cells.", "Altogether, these data show that qmc551: GFP is not only expressed in HECs that initiate definitive haematopoiesis, but is also found in their HSPC progeny that seed subsequent sites of larval and adult haematopoiesis.", "Supplementary material related to this article can be found online at 10.1016/j.ydbio.2016.07.010.", "The following is the Supplementary material related to this article Movie 6.", ": qmc551: GFP-positive haematopoietic cells seed perivascular niches in the caudal haematopoietic tissue at 2 dpf and patrol the head tissues at 6 dpf.", "This movie is related to Figure 3.", "(A) Timelapse confocal microscopy on qmc551;flk1: tdTom double transgenic embryos starting from 48 hpf showing 2.0 µm thick optical slices through the caudal haematopoietic tissue.", "Here, GFP+ haematopoietic cells undergo dynamic interactions with tdTom+ ECs.", "One of the cells is highlighted with a yellow circle.", "Please note that ECs in the ventral wall of the caudal artery co-express the two transgenes.", "Anterior is to the left, dorsal is up.", "The embryo shown was immobilized in 1% low melting point agarose.", "(B) A 6 dpf qmc551 transgenic embryo was anesthetized and placed in a tiny depression in agarose under a fluorescent Nikon SMZ1500 dissection microscope using a Nikon DS-5Mc/DS-U1 camera setup.", "From minute 5 on, pictures of the head region of the embryo (facing right) were taken manually every 3 min.", "Images were imported into Photoshop.", "In Photoshop, the pictures were moved and rotated to correct for the drifting movement of the embryo under the microscope.", "Furthermore, annotations were added.", "All pictures were then imported into iMovie to generate the final video.", "Individual frames are also shown in Figure 3O.", "It had previously been shown that gfi1aa expression in prRBCs occurs downstream of cloche and scl/tal1, but is independent of gata1 and frs (Cooney et al., 2013).", "Here, we focused on the regulation of gfi1aa in HECs of the vDA.", "In HECs, runx1 expression is known to be induced downstream of a signaling cascade that includes Hedgehog, VegfA and Notch signaling (Burns et al., 2005; Gering and Patient, 2005; Rowlinson and Gering, 2010).", "We, therefore, tested whether qmc551: GFP and gfi1aa expression in HECs also required Vegf and Notch signaling.", "Treatment of double transgenic embryos with a VegfR inhibitor led to the pooling of qmc551: GFP+/gata1: dsRed+ prRBCs in the ICM and to a complete loss of GFP+ spindle-shaped ECs (Fig. 4A).", "Likewise, non-transgenic embryos lacked all gfi1aa and cmyb mRNA in the trunk HECs in the absence of Vegf signaling (Fig. 4B and C).", "To determine whether gfi1aa expression in the vDA required the Notch pathway, qmc551;gata1: dsRed embryos were either treated with the γ-secretase inhibitor DAPM (Geling et al., 2002) or injected with the Rbpja/b morpholino (Sieger et al., 2003).", "The qmc551 transgene was also crossed into the mindbomb (mibta52b) (Itoh et al., 2003) mutant background.", "All three types of Notch-depleted embryos specified GFP+ prRBCs, but lacked GFP+ HECs (Fig. 4D-G).", "A WISH experiment performed on non-transgenic mib mutants confirmed the loss of gfi1aa expression in the vDA (Fig. 4H).", "Increased expression of GFP and gfi1aa mRNA within the inner ear in these embryos (Fig. 4F and H) reflects an expected increase in the number of gfi1aa-expressing hair cells in Notch-depleted embryos (Haddon et al., 1998).", "Next, we examined whether qmc551: GFP induction in HECs was also dependent on Runx1.", "We found that the same runx1 morphant embryos expressed GFP in the vDA (Fig. 4I), but displayed a loss of cmyb expression after fixation and WISH staining (Fig. 4J).", "Likewise, non-transgenic runx1 morphants retained endogenous gfi1aa mRNA (Fig. 4K) while losing cmyb expression (data not shown).", "Midline gfi1aa expression had previously been shown to be independent of Runx1 and was, therefore, thought to be unrelated to definitive haematopoiesis (Cooney et al., 2013).", "Our transgenic line reveals that this Runx1-independent gfi1aa expression occurs in HECs.", "Interestingly, GFP expression in circulating prRBCs was reduced in runx1 morphants, suggesting that runx1 promotes gfi1aa expression in prRBCs (Fig. 4L).", "The location of the gene trap transposon (Fig. 5A) suggested that it might interfere with the expression of the gfi1aa gene.", "To address this issue, qmc551 heterozygotes and homozygotes were sorted based on the level of GFP fluorescence (Fig. 5B) and used in gfi1aa WISH.", "These experiments showed that gfi1aa expression was retained in the inner ear, but lost in the prRBCs of qmc551 homozygotes (Fig. 5C).", "At 16 hpf, quantitative RT-PCR (qRT-PCR) confirmed that gfi1aa mRNA was substantially reduced in qmc551 homozygotes (Fig. 5D).", "Despite this reduction, homozygous embryos did not appear to carry less prRBCs.", "Flow cytometric analyses revealed that the GFP+ qmc551 homozygous prRBCs had a twofold higher mean fluorescence (Fig. 5E and F), but that prRBC numbers were similar in heterozygous and homozygous carriers (Fig. 5E and G).", "The normal number of prRBCs was also reflected in normal gata1 and β-globin (hbee1) expression patterns in 22 hpf qmc551 homozygotes (Fig. 5H and I).", "By day 3, circulating prRBCs of qmc551 homozygotes stained normally with the hemoglobin peroxidase substrate diaminofluorene (DAF) (Fig. 5J) and displayed normal overall cell morphology (Fig. 5K), clearly demonstrating that prRBCs were not only specified, but also matured in the absence of Gfi1aa.", "Normal primitive erythropoiesis in qmc551 homozygous embryos could be sustained due to functional redundancy between the three zebrafish Gfi1 paralogs.", "While gfi1ab expression could not be detected in prRBCs in the presence or absence of Gfi1aa (Fig. 5L), gfi1b expression was present and unaltered in the homozygous qmc551 embryos (Fig. 5M).", "To test whether Gfi1b compensates for the loss of Gfi1aa expression during primitive erythropoiesis, Gfi1b morpholinos were injected into homozygous qmc551 embryos.", "The morpholinos were designed to target the splice junctions that flank exon 4 of the primary gfi1b transcript (Fig. 5N).", "RT-PCR showed that injected morphant embryos carried an alternatively spliced gfi1b mRNA (Fig. 5O).", "The sequence of its RT-PCR fragment revealed that exons 3 and 5 were spliced together, leading to a shift of the gfi1b reading frame (Fig. 5N and P).", "This frame shift is predicted to cause the production of a truncated Gfi1b protein that retains the N-terminal 20 amino acid-long SNAG (SNAIL/GFI1) domain and parts of the linker domain, but lacks all Zn-fingers of Gfi1b's DNA binding domain.", "Instead, a divergent sequence of 22 amino acids forms the C-terminus of the truncated product.", "The loss of the DNA binding domain is likely to interfere with the protein's function.", "While injected wt embryos displayed normal erythropoiesis at 3 dpf, morphant qmc551 homozygous embryos showed a dramatic reduction in DAF staining (Fig. 5Q).", "In comparison to the normal morphology of prRBCs in uninjected embryos, prRBCs of morphant qmc551 homozygotes appeared larger and their nuclei were less condense (Fig. 5R).", "Nuclear condensation and a reduction in cell size are hallmarks of RBC differentiation (Qian et al., 2007; Weinstein et al., 1996).", "Thus, in the absence of Gfi1aa and Gfi1b, prRBCs failed to mature.", "The apparently normal maturation of prRBCs in uninjected qmc551 homozygous embryos suggests that Gfi1b is sufficient to rescue primitive erythropoiesis in the absence of Gfi1aa.", "Next, we examined whether the transposon also interfered with gfi1aa expression at the onset of definitive haematopoiesis.", "WISH experiments on 26 hpf qmc551 homozygous embryos showed that gfi1aa expression was lost in the vDA, while expression was retained in the inner ear (Fig. 6A).", "QRT-PCR confirmed the substantial reduction in gfi1aa mRNA in the 26 hpf embryo (Fig. 6B).", "Despite this loss, cmyb expression in the vDA was normal (Fig. 6C) and GFP+ cells were observed to seed the CHT (Fig. 6D) and the thymus (Fig. 6E) of qmc551 homozygous embryos.", "Their green fluorescence was matched by the presence of cmyb-positive HSPCs of the CHT and rag1-expressing T cell progenitors of the thymus (Fig. 6F and G), demonstrating that definitive haematopoiesis commenced as normal.", "Within the vDA, we found no evidence for gfi1b mRNA in wt or qmc551 homozygous embryos (Fig. 6H and I).", "Gfi1b expression was clearly restricted to prRBCs in circulation over the yolk and in the PBI (Fig. 6H,I).", "By contrast, gfi1ab mRNA which had previously been shown to display a scattered expression pattern in the vDA (Dufourcq et al., 2004) was dramatically increased in the homozygous qmc551 embryos (Fig. 6J).", "A close-up of the trunk region shows that the gfi1ab expression pattern displayed an almost continuous line in the embryonic midline (Fig. 6K).", "Transverse sections demonstrated that the staining was associated with the vDA (Fig. 6L), suggesting that gfi1ab expression was upregulated and likely to substitute for Gfi1aa at the onset of definitive haematopoiesis.", "Upregulation of gfi1ab expression was first observed at 22 hpf (Fig. 6M).", "At this time, gfi1aa expression decreases in prRBCs and only individual cells located in the position of the future vDA display weak gfi1aa expression (Fig. 6N).", "Given that gfi1ab expression was not seen in prRBCs between 13 and 20 hpf in wt or qmc551 homozygous embryos (Fig. 5L), the gfi1ab-positive cells seen at 22 hpf are most likely definitive precursor cells (Fig. 6M).", "Interestingly, when gfi1ab expression was examined in qmc551 homozygotes that had been injected with a runx1 morpholino, de-repressed gfi1ab expression was completely lost at 26 hpf (Fig. 6O), suggesting that, unlike gfi1aa, gfi1ab expression at the onset of definitive haematopoiesis requires direct or indirect activation by Runx1 (Fig. 6P).", "Homozygous qmc551 larvae were successfully raised to adulthood.", "Adults were perfectly viable and fertile, and showed no phenotypic abnormalities.", "Flow cytometric analysis of adult KM cells showed that the relative numbers of GFP+ cells were identical in qmc551 heterozygous and homozygous adult fish (Fig. 6Q), despite a substantial reduction in gfi1aa mRNA in KM cells of qmc551 homozygotes (Fig. 6R).", "As Gfi1 knockout mice display neutropenia, KM cytospins were prepared to examine neutrophil granulocytes in qmc551 homozygous fish.", "KM cytospins stained with May-Grünwald and Giemsa revealed cells with bilobed and trilobed nuclei (Fig. 6S), which represented about 10% of the KM cells in wt and qmc551 homozygotes (Fig. 6T).", "These cells could successfully be stained with the neutrophil granulocyte stain Sudan Black (Fig. 6U).", "Thus, qmc551 homozygous fish displayed no obvious signs of neutropenia.", "Altogether, our data showed that although the gene trap transposon abrogates gfi1aa expression in haematopoietic cells, primitive and definitive haematopoiesis remain unaffected.", "The zebrafish line qmc551 carries a GFP gene trap transposon within intron 1 of the gene gfi1aa on chromosome 2.", "RT-PCR confirmed the presence of a spliced mRNA that fuses gfi1aa 5′UTR sequences encoded by exon 1 to the splice acceptor on the gene trap, showing that GFP transcription is under the control of the gfi1aa promoter.", "GFP translation is ensured by an ATG start codon at the beginning of the gfp open reading frame.", "The GFP reporter faithfully recapitulates early embryonic gfi1aa expression inside and outside the haematopoietic system.", "Furthermore, it provides a sensitive live read-out of gfi1aa promoter activity in cell types and at times when gfi1aa promoter activity cannot easily be detected by WISH.", "The expression pattern of qmc551: GFP is much wider than that of the previously published gfi1aa enhancer trap line gfi1.1: gfp whose GFP expression was limited to prRBCs (Wei et al., 2008).", "The gfi1.1: gfp line's enhancer trap was inserted 20 kb upstream of gfi1aa, and its GFP expression was probably dependent on a single local enhancer.", "By contrast, our gene trap's GFP reporter is likely to be under the control of all cis-regulatory elements that regulate the activity of the endogenous gfi1aa promoter upstream of exon 1.", "Our WISH and qRT-PCR data showed that the gene trap transposon interferes with gfi1aa transcription in pRBCs, in HECs of the vDA and in adult KM cells.", "The absence of exon 4/5-containing RNA sequences suggests that the primary transcript is terminated at the SV40 polyadenylation signal downstream of the gfp reading frame.", "Our WISH data also revealed that the transposon-mediated suppression of gfi1aa transcription is context-dependent.", "Hair cells in the inner ear express GFP, but also retain gfi1aa expression.", "The remaining expression may be due to inefficient transcript termination or the use of an alternative promoter downstream of the transposon.", "It is noteworthy that a transcript initiated in intron 1 would encode a full-length Gfi1aa protein.", "Interestingly, analysis of genome-wide data on histone 3 lysine 4 trimethylation, an epigenetic mark enriched at transcriptionally active promoters, in 24 hpf zebrafish embryos (Aday et al., 2011) shows that gfi1aa's intron 1 sequences are associated with this mark.", "The lack of inner ear defects in qmc551 homozygous embryos is likely due to the residual Gfi1aa expression and the co-expression of Gfi1ab in the sensory hair cells.", "This example highlights that one cannot simply assume that the transposon interferes with gfi1aa expression in all cell types and at all differentiation stages.", "A more detailed analysis into this context dependency was outside the scope of this study.", "Despite the loss of Gfi1aa expression in prRBCs, homozygous qmc551 embryos displayed normal primitive erythropoiesis.", "PrRBC progenitors were specified in normal numbers and DAF staining suggested that they matured normally.", "These finding contradict previous morpholino studies that proposed an essential role for Gfi1aa in primitive erythropoiesis (Cooney et al., 2013; Wei et al., 2008).", "The convincing reduction in gfi1aa mRNA in our mutants suggests that the reported morphant phenotype is possibly due to off-target effects (Lawson, 2016; Stainier et al., 2015).", "The normal development of prRBCs in our mutants is consistent with the phenotype of the mouse Gfi1 knockout.", "As in zebrafish, mouse prRBC progenitors co-express Gfi1 and Gfi1b (Moignard et al., 2015), and single Gfi1 and Gfi1b knockout mice display normal primitive erythropoiesis (Hock et al., 2003; Saleque et al., 2002).", "By contrast, loss of both proteins causes reduced embryonic βH1 globin expression in the murine yolk sac, suggesting a defect in primitive erythropoiesis (Lancrin et al., 2012).", "We show here that morpholino-mediated knockdown of Gfi1b in Gfi1aa-deficient embryos also interfered with primitive erythropoiesis in zebrafish.", "Initially, prRBCs were specified, but subsequently failed to mature.", "The lack of a maturation defect in either gfi1aa mutant or gfi1b morphant embryos demonstrated that Gfi1aa and Gfi1b could substitute for each other during primitive erythropoiesis in zebrafish.", "Homozygous qmc551 carriers were viable and fertile, and displayed none of the phenotypic abnormalities observed in the definitive blood system of Gfi1 knockout and Gfi1: GFP knock-in mice (Hock et al., 2003; Karsunky et al., 2002; Wallis et al., 2003; Yücel et al., 2004).", "In particular, the neutrophil granulocytes, which are severely reduced in Gfi1-depleted mice, were present in normal numbers.", "The normal blood phenotype was likely due to functional redundancy with Gfi1aa's paralogs (Fig. 7).", "At the onset of definitive haematopoiesis, loss of Gfi1aa expression lifted the repression of gfi1ab and led to the Runx1-dependent upregulation of Gfi1ab in HECs of the DA.", "Mammalian Gfi1 and Gfi1b proteins are known to auto- and cross-regulate their expression in a context-dependent manner (Doan et al., 2004; Montoya-Durango et al., 2008; Yücel et al., 2004).", "In homozygous qmc551 embryos, upregulation of gfi1ab was first detected in individual cells of the ICM.", "Based on their position within the ICM and the lack of gfi1ab upregulation in prRBCs at earlier time points, we suppose that these cells represent progenitors of HECs, i.e. haemogenic aortic angioblasts.", "These cells may be equivalent to the suspected HSC precursors recently reported to express gata2b (Butko et al., 2015), an issue that requires further attention.", "In the mouse, loss of Gfi1 alone does not abrogate EHT.", "Only Gfi1/Gfi1b double knockout mice display deficiencies in HECs of the YS and the vDA.", "In the YS, HEC-derived HCs cannot down-regulate EC genes and fail to enter circulation (Lancrin et al., 2012).", "In the vDA, HECs are specified, but fail to undergo EHT (Thambyrajah et al., 2015).", "In zebrafish, we did not see any convincing gfi1b expression in vDA HECs, but gfi1b expression was observed in definitive HCs of the CHT.", "Whether it plays an important role in EHT remains to be determined.", "In addition, the strong expression of gfi1aa in wt and gfi1ab in Gfi1aa-deficient embryos casts doubts on the previous morpholino-based assumption that Gfi1b alone is essential for the formation of all definitive haematopoietic lineages (Cooney et al., 2013).", "In the mouse, Gfi1 and Gfi1b are co-expressed not only during EHT, but also in HSCs, and only the loss of both genes completely abrogates HSC maintenance (Hock et al., 2004; Khandanpour et al., 2010; Zeng et al., 2004).", "Single Gfi1b knockout mice display severe defects only in cell types that do not co-express Gfi1, i.e. definitive erythrocytes and megakaryocytes (Saleque et al., 2002).", "The generation of gfi1ab and gfi1b single, as well as double and triple mutants in zebrafish is needed to shed more light on the redundant and non-redundant roles of these proteins during definitive haematopoiesis.", "The lines will also allow us to carefully examine dose-dependent requirements for Gfi1/1b proteins during tissue differentiation.", "Unlike the previous morpholino results, our data on the qmc551 line are consistent with findings in the mouse and strongly support the notion that the roles of Gfi1 and Gfi1b are at large conserved between teleosts and mammals.", "The high level of GFP expression in our qmc551 line and the transparency of the zebrafish embryo will be instrumental in unraveling the behavior of cells in gfi1aa/gfi1ab/gfi1b and other mutant backgrounds.", "This line will also be an excellent resource for HECs and HSPCs for use in transplantation and biochemical characterization.", "Information on zebrafish husbandry is provided in Supplementary data.", "Genetically altered zebrafish are listed in Table S1.", "Details on the transposon Tol2-embedded gene trap, the flk1: tdTom and the csl: cer constructs are available upon request.", "To generate transgenic lines, plasmid constructs were injected with Tol2 transposase mRNA into one-cell stage embryos (Kotani et al., 2006).", "Embryos that harbored cells with transient reporter expression that contributed normally to embryonic development were raised.", "Adults were crossed to wt fish to identify transgenic founders.", "Their progeny established the lines reported herein.", "Southern Blots followed standard procedures.", "The integration site in qmc551 was identified via inverse PCR as previously described (Kotani et al., 2006).", "Briefly, genomic DNA from qmc551 embryos was digested with MboI, self-ligated and used in a nested PCR.", "The amplification product was cloned and sequenced.", "A Blast search in ENSEMBL (Flicek et al., 2014) on zebrafish genome assembly zv8 allowed the localization of the transposon.", "Total RNA was isolated from qmc551 embryos using the RNeasy mini Kit (Qiagen) and was reverse transcribed using Superscript II reverse transcriptase (Life Technologies).", "Standard PCRs on cDNA and genomic DNA were performed using Taq polymerase (New England Biolabs).", "Quantitative TaqMan PCR on cDNA employed the qPCR mix plus Rox reference dye (Thermo Scientific).", "Oligo sequences are provided in Table S2.", "Statistical analyses were performed using Graph Pad Prism.", "Alkaline phosphatase and tyramide fluorescent WISH experiments were performed using published protocols (Broadbent and Read, 1999; Schoenebeck et al., 2007).", "Immunodetection followed standard protocols, using reagents summarized in Table S3.", "Some stained embryos were embedded in JB4 methacrylate (Agar Scientific, Cambridge) and sectioned on a Leica RM2265 microtome.", "Sudan Black staining of embryos followed (Le Guyader et al., 2008).", "Kidneys were isolated (Gerlach et al., 2011), fixed in 4% paraformaldehyde overnight, soaked in 30% sucrose overnight, frozen in OCT and sectioned on a Leica CM1850 cryostat.", "Morpholinos (see Table S2) were injected in a volume of 0.5 nl into 2–4 cell stage embryos.", "The two gfi1b morpholinos (0.5 ng each) were co-injected with 0.5 ng of p53 morpholino.", "The latter was used to block the frequently observed morpholino-induced upregulation of p53 and the p53-induced apoptosis (Robu et al., 2007).", "Inhibitors were added to the embryo medium and applied from tailbud stage.", "Control embryos were treated with the solvent DMSO.", "The Diaminofluorene staining followed (Weinstein et al., 1996).", "To block pigmentation and immobilize live embryos for confocal imaging, embryos were treated with MS222, and phenylthiourea, and embedded in 1% low melting point agarose as described in (Renaud et al., 2011).", "Details on chemicals are given in Table S3.", "Blood cells were collected from adult kidneys as described (Traver et al., 2003).", "Forward scatter, side scatter and GFP/dsRed fluorescence characteristics of KM cells were analyzed on a Beckman Coulter MoFlo Astrios cell sorter using the Kaluza software.", "Sytox was used to exclude dead cells.", "Using a Shandon Cytospin 4, all or just the GFP+ KM cells were cytocentrifuged for 3 min onto slides at 300 rpm and medium acceleration.", "RBCs were isolated from the sinus venosus of terminally anaesthetized 3 day-old embryos.", "The cells were subsequently stained with May-Grünwald, Giemsa or Sudan Black following manufacturer's instructions (see Table S3).", "Embryos were examined on a Nikon SMZ-1500 microscope.", "Sections and cytospins were analyzed on a Nikon Eclipse i80.", "Images were taken with a Nikon DS-5Mc/DS-U1 camera setup operated by the Nikon ACT-2U software or captured with a monochrome Hamamatsu Orca-ER camera via IP Lab software.", "Orca black and white images were pseudo-colored.", "Confocal microscopy was performed on Zeiss Exciter, LSM510 and LSM710 inverted confocal microscopes via ZEN software.", "All confocal images were analyzed in Imaris (Bitplane).", "Videos exported from Imaris were annotated in iMovie.", "Images were collated in Photoshop CS6.", "No competing interests declared.", "RT identified the qmc551 line and performed its initial characterization.", "DU revealed that haematopoietic gfi1aa expression is lost in homozygous carriers, and studied haematopoiesis in the absence of Gfi1aa.", "MJ performed all confocal timelapse analyses and immunohistochemistry on kidney sections.", "DU and YH performed the gfi1b morpholino experiments.", "RNW helped evaluate data and examined possible expression of qmc551: GFP in macrophages.", "KM and CM helped PhD students in experimental design and data analysis.", "MG supervised the project and wrote the manuscript.", "This work was supported by Medical Research Council grants [79780 and MR/J000841/1 to MG], a BBSRC PhD studentship to RT (student number: 4055674), an MRC studentship to YH (student number: 4254522), and University of Nottingham International PhD scholarships to DU (student number: 4154910) and MJ (student number: 4191879)." ]	[ "A zebrafish gene trap line expresses GFP in primitive and definitive blood cells.", "The gene trap transposon eliminates haematopoietic gfi1aa expression.", "Absence of Gfi1aa does not interfere with normal haematopoiesis.", "Gfi1aa and its paralogs Gfi1ab and Gfi1b play redundant roles in haematopoiesis.", "Our data suggest that their roles are conserved in all vertebrates." ]
Oxygen reduction reaction on TiO2 rutile (1 1 0) surface in the presence of bridging hydroxyl groups	The goal of this study is to provide insight into the mechanism of the oxygen reduction reaction on the TiO2 rutile (1 1 0) surface in the presence of bridging hydroxyl groups. Considering the Langmuir–Hinshelwood and Eley–Rideal mechanisms, each possible intermediate was identified using density functional theory and a cluster model along with the energy barriers of the reduction steps and the O[sbnd]O bond breaking. Our results show that the initial step, the O2 adsorption on the surface, is favored compared to the pure surface. At higher potentials, the oxygen reduction reaction was found to go through the formation of HO2, which can easily convert to two terminal hydroxyl groups. The rate-limiting step is the desorption of the first H2O with 0.58 eV energy requirement at zero applied potential, while at 1.23 V the reduction of the adsorbed OH to form H2O is the bottleneck with a barrier height of 1.71 eV.	[ "BIOC", "CHEM", "PHYS" ]	[ "Catalysis", "Oxygen reduction reaction", "Reaction mechanism", "Rutile" ]	[ "1303", "1606", "3104" ]	[ "Polymer electrolyte fuel cells (PEFC) are widely investigated, because they are promising alternatives to petroleum-based energy sources due to their high efficiency, high power density, low emission, and low operating temperature [1].", "However, there are still many challenges to be tackled before PEFCs can be commercialized.", "One of them is to find a sufficiently active and cost-effective catalyst for the oxygen reduction reaction (ORR).", "Nowadays Pt is used as a cathode catalyst, but the insufficient activity and high cost prevent its large-scale applications.", "TiO2 is a possible candidate owing to its high stability in acidic media.", "It has been used in several experiments as ORR catalyst itself [2–11] and also combined with other materials [12–14].", "These results show that the activity of pure TiO2 rutile is moderate compared to Pt, but it can be increased with doping [13,14] or introducing defects on the surface [15,16].", "Among the different crystal planes, the highest activity was attributed to the (1 1 0) plane [10], thus, in this work, we will focus on the TiO2 rutile (1 1 0) surface.", "However, it should be mentioned that stability superior to the Pt/C catalyst and increased activity were also found introducing defects on the TiO2 anatase (001) surface [15].", "On the TiO2 rutile (1 1 0) surface, the Ti atoms are either six- or fivefold coordinated.", "Furthermore, the O atoms are either threefold coordinated in the plane of the surface or twofold coordinated bridging atoms prominent from the surface.", "Several studies deal with the surface properties of rutile (1 1 0) [17], especially with the adsorption of H2O and with the adsorption of small molecules on it such as water or oxygen [18–28].", "The water adsorption follows an associative mechanism according to the experiments on the pure surface, while it follows a dissociative mechanism at bridging oxygen vacancies, the most common defect on this surface, leading to the formation of bridging hydroxyl groups [21,22,25].", "The first step of the ORR, the oxygen adsorption on the TiO2 rutile (1 1 0) surface, has been investigated both theoretically and experimentally in the presence of bridging hydroxyl groups.", "In a molecular dynamics study [29], the adsorbed OOH and hydrogen peroxide were found to be the most stable intermediates due to the stabilization by the hydrogen bonds formed with the neighboring bridging hydroxyl groups.", "Less stable intermediates, such as oxygen adatoms plus adsorbed water or bridging hydroxyls were not supported by the calculations.", "In fact, adsorbed OOH species were found to spontaneously form from the reaction of an oxygen molecule and a bridging hydroxyl group according to STM imaging [30].", "The adsorbed OOH species were observed to be quite stable and immobile especially in the presence of a second bridging hydroxyl group in the vicinity, and its dissociation to an oxygen adatom and a terminal hydroxyl occurred only in few instances.", "As a secondary reaction, the OOH species and a neighboring bridging hydroxyl group were found to produce a water molecule leaving an oxygen adatom on the surface.", "The resulting oxygen adatoms turned out to be able to accept a hydrogen atom from a nearby bridging hydroxyl or from the adsorbed OOH species leading to a stable and immobile terminal hydroxyl, which can further reduce to water accepting a hydrogen atom from an adjacent bridging hydroxyl group or another terminal hydroxyl.", "Adsorbed hydrogen peroxide intermediate was not observed probably because of its short lifetime.", "On partially hydroxylated surfaces, it was also revealed that water molecules can catalyze the reaction of oxygen adatoms and bridging hydroxyls, the reaction of terminal and bridging hydroxyls, and the delivery of oxygen adatoms to bridging oxygen vacancies, which finally leads to their mutual annihilation [31].", "Water molecules also resulted in shorter lifetime for the oxygen adatoms, terminal hydroxyls, and OOH species on fully hydroxylated surfaces due to the mediation of the diffusion of bridging hydroxyls to these reactants.", "Despite the interest, we still lack comprehensive studies about the mechanism of the ORR on the rutile (1 1 0) surface in the presence of defects, which would facilitate the understanding of the processes and the design of better ORR catalysts.", "The study of defective rutile surfaces is also justified as the increased catalytic activity of the related anatase has been observed previously.", "In this paper, DFT methods and a cluster model are applied to examine the reduction of the oxygen molecule on the TiO2 rutile (1 1 0) surface in the presence of bridging hydroxyl groups, taking into account the effect of the solvent and the electric potential.", "All the possible oxygen reduction reaction pathways are investigated, and the barriers for the OO bond breaking and the reduction steps are determined supposing the Langmuir–Hinshelwood (LH) or the Eley–Rideal (ER) mechanisms.", "For the geometry optimizations and frequency calculations, the cost-efficient but still reasonably accurate BP86 semi-local generalized gradient approximation functional [32,33] was selected with the smaller 6-31G(d) basis set [34].", "For the subsequent single-point energy calculations, the more accurate TPSSh functional with the larger def2-TZVP basis set [35] was utilized combined with the DFT-D3 dispersion correction [36] with Becke-Johnson (BJ) damping [37].", "The density fitting approximation was invoked in all the above calculations.", "Open-shell systems were calculated using spin-unrestricted DFT.", "If it is not specified otherwise, the SMD solvation model [38] was used during the calculations.", "The transition states of the OO bond breaking and the H-transfer on the surface to the reactant in the LH mechanism were confirmed by the imaginary frequency of the corresponding vibrational mode.", "In each reduction step, which is a coupled proton and electron transfer (CPET), the proton has to surpass the double layer from the bulk solution to the electrode.", "The modeling of this process is out of the scope of this study, and its effect is taken into account by taking the barrier to be 0.3 eV according to Jacob et al. [46].", "Therefore, the barriers of the ER mechanism are estimated to be 0.3 eV plus the reaction Gibbs free energy, if it is positive, while in the case of the barriers of the LH mechanism, the transition states of the H-transfer were also considered.", "The calculations for the bigger model systems (see later) were carried out with the Turbomole 7.1 package [47].", "For all the other calculations the Gaussian 09 quantum chemistry program (Revision E.01) [48] was utilized.", "We chose a cluster model for the description of the phenomenon studied.", "We note that we do not employ a periodic model, because periodic DFT calculations are still limited, compared to general DFT, regarding the available functionals (especially hybrid meta-GGA) and solvent models.", "Evaluation of the solvent effects are important to provide appropriate Gibbs free energy data for our processes.", "Also, for a periodic model a large enough cell should be used to avoid the overlap of the effect of bridging hydroxyl groups, which would be computationally demanding.", "First, a pure TiO2 rutile (1 1 0) surface model was built by cutting two trilayers from the bulk experimental rutile geometry (with TiO bond lengths of 1.948 Å and 1.981 Å) and closed by hydrogen atoms (with optimized positions) forming terminal hydroxyl groups, which led to a Ti21O54H24 cluster (Fig. 1).", "Two-trilayer thickness were used to have fivefold coordinated Ti atoms on the surface.", "Towards the other two directions, the size of the model was chosen to have a central fivefold coordinated Ti atom on the surface with two neighboring fivefold coordinated Ti atoms, and with two neighboring bridging oxygen atoms.", "For the optimization of the HO bonds in the terminal hydroxyl groups the calculations were performed with singlet spin-multiplicity in gas phase.", "To examine the convergence of the results with respect to the cluster size, we considered the effect of the next layer (in each direction) on the oxygen adsorption energies, on a Ti82O194H60 cluster.", "Due to the size of the system, frequency calculations were not possible, only the TPSSh-D3BJ/def2-TZVP adsorption energies were determined and compared to the results from the original model.", "The adsorption energies on the clusters of different size only differ by 0.10 and 0.09 eV, respectively, in the two orientations.", "These results support that the size of the cluster is appropriate.", "To further validate our model, the gas-phase adsorption energies of H, O, OH, OOH, and H2O were compared to literature values.", "The results are compiled in Table 1.", "Our adsorption energies only differ by 0.02, 0.03, 0.00, and 0.06 eV for H, O, OH, and OOH, respectively, from those obtained in the periodic PBE calculations of Wu [49] and Mom [50].", "The conformity with the experimental data is less satisfactory: the adsorption enthalpy of H2O is −1.57 eV from our calculations, while the experimental data is −0.96 eV [51,52].", "Nevertheless, these results suggest that the applied model qualitatively correctly describes the energetics of this system, and its predictivity is similar to that of periodic DFT calculations.", "In the following, a pair of bridging hydroxyl groups was built on the TiO2 rutile (1 1 0) surface cluster model.", "The positions of all cluster atoms were fixed with the exception of the attached H atoms of the bridging hydroxyl groups.", "Using singlet and triplet spin-multiplicity for the calculation of this model with a pair of bridging hydroxyl groups it was found that the triplet state is more stable than the singlet by 0.51 eV.", "The resulting model system will be denoted by h-TiO2 hereafter.", "Also, if necessary, the multiplicity of the system is presented in the upper left corner of the formula, for example, as 3h-TiO2.", "Then, the oxygen adsorption on the surface was investigated in the presence of the bridging hydroxyl groups.", "Due to the hydroxyl groups the observed binding modes are different than in the case of the pure TiO2 surface (see Fig. 3).", "In the first two cases, h-TiO2–O2(A) and h-TiO2–O2(B), both oxygen atoms are connected to a Ti atom, and the difference is the number of neighboring bridging hydroxyl groups.", "In the third possible position the two oxygen atoms attach to the same Ti atom, and the adsorbed O2 is perpendicular to the line of the surface Ti atoms [h-TiO2–O2(C)].", "A position similar to TiO2–O2(B) also exists where the O2 molecule is stabilized by a bridging hydroxyl group [h-TiO2–O2(D)].", "With singlet multiplicity only (A) and (B) binding modes are available with −1.34 and −1.21 eV adsorption energies, respectively.", "For triplet multiplicity all listed arrangements are possible, however, these are less stable than the singlet ones.", "The most stable triplet form is 3h-TiO2–O2(D) with an adsorption energy of −0.92 eV, followed by 3h-TiO2–O2(A) (−0.86 eV) and 3h-TiO2–O2(B) (−0.85 eV), and 3h-TiO2–O2(C) (−0.67 eV) in the stability order.", "These results demonstrate that the first step of the ORR, the O2 adsorption, is energetically more favorable in the presence of bridging hydroxyl groups than on the pure surface of rutile.", "During the discussion of ORR mechanism, both multiplicities will be considered.", "Even though 3h-TiO2–O2(D) is the most stable triplet form, 3h-TiO2–O2(A) was used as a starting point of the ORR in the triplet case, because the possible processes (the OO bond breaking and the first step of the reduction) are more likely from this binding mode, which is anyway by only 0.06 eV higher in energy than the most favorable orientation.", "For comparison, several key features of our h-TiO2 model agree well with the published experimental or theoretical results.", "The triplet state of the model is more stable than the singlet by 0.51 eV, which is quantitatively not so far from the literature value of 0.6 eV [53].", "The excess electrons are localized partially on the Ti atoms between and beside the two bridging OH groups as well as partially on the fivefold coordinated surface Ti atoms near the bridging hydroxyl groups.", "This indicates that our model describes well the hydrated surface since the unpaired electrons tend to migrate to the surface in the presence of water according to the previous findings [54].", "Moreover, in our model, the oxygen molecule is adsorbed on the surface only in the presence of bridging hydroxyl groups, which is qualitatively correct according to the literature [55].", "All these findings support that our model correctly describes the energetics, electronic structure, geometry, and adhesive properties of the TiO2 rutile (1 1 0) surface in the presence of bridging hydroxyl groups.", "Fig. 4 shows the general scheme of ORR in acidic media.", "In the top line the 2-electron pathway can be seen, where, after two reduction steps, H2O2 is formed.", "The requirement for the H2O formation, i.e., the 4-electron pathway, is the cleavage of the OO bond before the desorption of H2O2.", "Thereafter several routes are possible for the formation and desorption of H2O.", "Surface reactions can occur with the LH mechanism, where all reactants are adsorbed on the surface, or with ER, where the surface intermediate reacts with a species from the solution.", "In each reduction step, which is a coupled proton and electron transfer (CPET), both mechanisms were considered.", "Since in the LH mechanism every reduction is a two-step process, H adsorption to the surface then moving from surface to the reactants, here the higher of the two barriers is used to represent the process in these cases.", "In Fig. 5 we present the free energy diagram of the ORR at 0 V potential on the TiO2 rutile (1 1 0) surface in the presence of bridging hydroxyl groups.", "After the adsorption of O2, the resulting 3h-TiO2–O2 can be reduced to 2h-TiO2–HO2, or the OO bond breaking can take place to form 3h-TiO2–(O)(O).", "The barrier for the reduction is 0.30 eV for both the LH and the ER mechanisms, while the OO bond breaking requires 1.11 eV.", "However, from the more stable 1h-TiO2–O2 only the reduction is possible because the OO bond breaking to form 3h-TiO2–(O)(O) is a spin-forbidden reaction, and 1h-TiO2–(O)(O) (not shown in the figure) is with 1.50 eV higher in energy than the corresponding triplet state.", "The barrier for the reduction is 0.31 and 0.30 eV for both the LH and the ER mechanisms, respectively, while the OO bond breaking requires 1.11 eV, therefore the direct reduction is favored at this point.", "The 2h-TiO2–HO2 can be further reduced to 3h-TiO2–H2O2 or can form 2h-TiO2–(O)(OH) by breaking the OO bond.", "Although the latter process has the lowest barrier, 0.48 eV, reduction can also occur either through the ER or through the LH mechanism because of the low barriers of 0.52 and 0.65 eV, respectively.", "If the reduction pathway is followed, then the H2O2 formed can desorb with an energy investment of only 0.33 eV, while the formation of 3h-TiO2–(OH)(OH) from H2O2 requires the defeat of a 0.26 eV barrier.", "Thus both directions are feasible, which also means that the 2-electron pathway is possible at zero potential, however, not the main route.", "The formation of 1h-TiO2–(OH)(OH) from 3h-TiO2–(OH)(OH) is spin-forbidden, even though it is by 2.19 eV more stable than the triplet form.", "There are again two options for the further reduction from 2h-TiO2–(O)(OH): the formation of 1h-TiO2–(O)(H2O) or 1h-TiO2–(OH)(OH).", "The latter is more stable with 0.94 eV than the former, but the barriers to them are similar, 0.30 eV for both the ER and LH mechanisms.", "The third reduction step produces the same intermediate, 2h-TiO2–(OH)(H2O), from all the previous intermediates: from 3h-TiO2–(OH)(OH) and from 1h-TiO2–(O)(H2O) with barriers of only 0.30 eV, while from 1h-TiO2–(OH)(OH) with 0.30 and 0.41 eV barrier via the ER and LH mechanisms, respectively.", "This is the most stable intermediate with −5.37 eV.", "The last reduction step to 3h-TiO2–(H2O)(H2O) needs 0.48 and 0.66 eV energy to overcome the barrier in the case of the ER and the LH mechanism, respectively.", "The desorption of the first H2O from 3h-TiO2–(H2O)(H2O) requires 0.58 eV, but the desorption of the second one is spontaneous and releases 0.01 eV energy.", "That means that the usual coverage effect, i.e., the adsorption energy of the first molecule is the largest, the following molecules have lower adsorption energies, is not true here.", "The reason is that the hydrated surface is not favourable for the first H2O adsorption, but the following H2O molecules are stabilized by H-bridges.", "The H2O desorption can also happen earlier in the ORR, at 1h-TiO2–(O)(H2O) or 2h-TiO2–(OH)(H2O).", "In the two cases 0.32 and 0.49 eV is needed for this process, respectively.", "In this route, the barrier of the third reduction step from 1h-TiO2–O to 2h-TiO2–OH is 0.30 eV for both mechanisms, while, to overcome the barrier of the fourth reduction step, 0.57 and 0.83 eV is needed for the ER and LH mechanism, respectively.", "If the ideal electrode potential of 1.23 V, the Nernst potential of the ORR, is applied to the system, a different energetics can be seen (see Fig. 6).", "The energy levels are shifted upwards with 1.23 eV after each reduction step, and the corresponding barriers are also affected.", "However, the OO bond breaking barriers and the adsorption/desorption energies are unchanged.", "At this potential the reduction from 3h-TiO2–O2 to 2h-TiO2–HO2 has a barrier of 0.67 eV in the ER mechanism, but it is still preferred over the formation of 3h-TiO2–(O)(O) with a 1.11 eV barrier.", "The LH barrier for this step is 1.06 eV, therefore it is not likely to occur compared to the ER mechanism.", "From 1h-TiO2–O2 to 2h-TiO2–HO2 the barriers are 1.15 and 1.54 eV for the ER and LH mechanisms, respectively.", "From 2h-TiO2–HO2 the formation of 3h-TiO2–H2O2 is practically no longer possible.", "Even the barrier with the more favorable ER mechanism is 1.75 eV, which is very high compared to the 0.48 eV barrier of the OO bond breaking.", "It means that, at higher potentials, the 2-electron pathway is not possible.", "It is also instructive to compare the mechanism of the ORR on rutile and platinum.", "The results of Keith and Jacob [56], who studied this process on Pt(1 1 1) surface, show that, at low potentials on Pt, the H2O2 formation is the preferred pathway, and a mixed 2- and 4-electron mechanism can be observed.", "In contrast, in our case the HO2 pathway is favorable, and just a small amount of H2O2 can be expected.", "For Pt the rate determining step is the desorption of H2O requiring an energy of around 0.3 eV.", "At 1.23 V on Pt the OO bond breaking happens after the adsorption of O2.", "The highest barrier is 0.99 eV for the last reduction step, which is almost half of the corresponding barrier for h-TiO2.", "However, the rate of the first three reduction steps is expected to be similar on Pt and on the modified rutile surface even at high potentials.", "All in all, our results suggest that the key steps of the 4-electron ORR, the O2 adsorption and the OO bond breaking can occur on the rutile surface in the presence of bridging hydroxyl groups, which makes this material a possible ORR catalyst.", "However, it needs to be combined with other substances to reduce the barrier of the last reduction step of the ORR.", "The reduction of the oxygen molecule was investigated on TiO2 rutile (1 1 0) surface in the presence of bridging hydroxyl groups relying on a cluster model, which was validated using experimental and theoretical adsorption energies of H, O, OH, OOH, and H2O.", "First, a pair of bridging hydroxyl groups was built on the surface locally optimizing the geometry of the cluster model.", "Then, using this model, the reduction reaction pathway of the oxygen molecule was investigated on the TiO2 rutile (1 1 0) surface in the presence of bridging hydroxyl groups.", "The effect of solvation and potential were considered, and both the LH and the ER mechanisms were studied.", "At both 0 V and 1.23 V the ORR goes through the formation of HO2 and its dissociation and termination on the surface.", "Additionally, at 0 V a small amount of H2O2 can be expected due to the comparable barrier of the OO bond breaking and the second reduction step.", "Generally, in each possible reduction step the ER mechanism is the preferred, except where the proton transfer through the double layer is the biggest obstacle.", "The rate-limiting step is the desorption of the first H2O with 0.58 eV energy requirement at zero applied potential, while at 1.23 V the reduction of the adsorbed OH to form H2O is the bottleneck with a barrier height of 1.71 eV.", "However, the key steps of the reaction, the O2 adsorption and the OO bond breaking, are possible on the surface of rutile, which makes the latter a possible mixing material for ORR catalysts.", "Our findings can contribute to the understanding of the oxygen reduction reaction in polymer electrolyte fuel cells using TiO2 rutile catalyst and to the design of more efficient electrode materials.", "The authors declared that there is no conflict of interest." ]	[ "O2 adsorption on the defected surface is favored compared to the pure surface.", "At higher potentials, the oxygen reduction reaction goes through the formation of HO2.", "At lower potentials, the formation of a small amount of H2O2 is also expected.", "The rate-limiting step is the final reduction of the adsorbed OH to form H2O." ]
Mice lacking fat storage-inducing transmembrane protein 2 show improved profiles upon pressure overload-induced heart failure	Fat storage-inducing transmembrane proteins 1 and 2 (FITM1 and FITM2, respectively) are transmembrane endoplasmic/sarcoplasmic reticulum proteins involved in lipid droplet formation. The physiological functions of FITM1 have only been reported in skeletal muscle, while those of FITM2 were analyzed using genetically engineered mice. However, their roles in the heart have not been characterized. To examine their cardiac functions, we analyzed Fitm1- or Fitm2-knockout mice. Neither constitutive Fitm1 (−/−) aged nor heart failure model mice showed significant differences in heart size or function. Fitm2 (−/−) mice exhibited embryonic death, and aged Fitm2 (+/−) mice had shortened left ventricular end-diastolic dimension, and shortened left ventricular end-systolic dimension. However, body weight and ejection fraction of Fitm2 (+/−) mice were similar to those of wild-type littermates. In the chronic heart failure models, Fitm2 (+/−) mice showed significant suppression of increased left ventricular end-diastolic dimension and reduced ejection fraction. These results suggest the involvement of Fitm2 in chronic heart failure, whereas Fitm1 have a minor effect in this context in mice.	[ "MULT" ]	[ "Cardiology", "Physiology" ]	[ "1000" ]	[ "Fat storage-inducing transmembrane proteins 1 and 2 (FITM1 and FITM2, respectively) are members of a conserved gene family that are important for lipid droplet accumulation [1, 2, 3].", "Both human FITM1 and FITM2 are highly expressed in oxidative tissues such as the heart and skeletal muscle; specifically, FITM1 is highly expressed in skeletal muscle, whereas FITM2 is primarily expressed in the heart.", "In mice, Fitm1 and Fitm2 exhibit mRNA expression patterns similar to those of humans, but Fitm2 protein is most highly expressed in adipose tissues [1].", "On the basis of the expression pattern of FITM1, its functions were analyzed during different developmental stages of pig skeletal muscle and cultured cells.", "Porcine Fitm1 gene expression changes during skeletal muscle development and is regulated by MyoD1 in C2C12 cells during differentiation [4].", "In addition, overexpression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) induces FITM1 expression in cultured human skeletal muscle cells with the accumulation of lipid droplets [5].", "Meanwhile, the physiological functions of FITM2 were analyzed using adipose-specific-knockout mice [6], postnatal whole-body-knockout mice [7], and skeletal muscle-specific-overexpressing mice (CKF2) [8].", "These studies indicate that FITM2 has essential roles in normal fat storage and intestinal health.", "In addition, Fitm2 overexpression leads to decreased fatty acid oxidation, increased utilization of branched-chain amino acids, enhanced glucose uptake, and profoundly decreased cellular ATP levels in the skeletal muscle of CKF2 mice.", "These findings also indicate that Fitm1 and Fitm2 have important roles in skeletal muscle.", "Considering that both skeletal muscle and the heart are oxidative tissues, and obtained findings on the patterns of expression of FITM1 and FITM2, previous results suggest that these two molecules may have roles in the heart.", "Recently, we found that these proteins were identified in mice and/or human heart samples by proteomic analysis, affected the cardiovascular system and body development in zebrafish, and that their modulated expression or function can change lipid droplet formation, ER function, and cellular metabolism in cells [9].", "These data suggested their potential to modulate heart failure pathogenesis.", "However, their physiological functions in heart have not been elucidated.", "Therefore, we here report the profiles of FITM1 and FITM2 in mouse models of heart failure.", "The mRNA expression of Fitm1 and Fitm2 was decreased in heart failure model mice, and Fitm2 hetero-knockout [Fitm2 (+/−)] mice exhibited resistant profiles to pressure-induced chronic heart failure.", "However, neither Fitm1 (−/−) nor Fitm2 (+/−) status negatively affected cardiac development or function in normal conditions.", "Our findings collectively suggest that downregulation of Fitm, especially Fitm2 expression or function may have a beneficial effect in patients with heart failure.", "Seven-week-old male C57Bl/6 mice were purchased from Japan SLC, Inc.", "Fitm1- or Fitm2-knockout mice were generated at the Institute of Immunology Co., Ltd. (formerly Phoenix Bio).", "Seven- or eight-week-old male Fitm1 (−/−), Fitm2 (+/−), and littermate mice were purchased from the Institute of Immunology Co., Ltd.", "The mice were kept in a room at 55% relative humidity (permissible range: 30%–70%) and 23 °C (permissible range: 20–26 °C) under a 12-h light/dark cycle.", "The animal experiments were carried out in accordance with the guidelines of the local Institutional Animal Care and Use Committee and were reviewed and approved by it.", "At nine weeks of age, mice were intubated and anesthetized with isoflurane gas.", "For the chronic heart failure model induced by pressure overload, the transverse aorta was exposed by median incision, and pressure overload was produced by transverse aortic constriction (TAC) using a 27-gauge needle.", "For the chronic heart failure model induced by myocardial infarction (MI), the chest cavity was exposed by cutting the intercostal muscle, and the left coronary artery was subsequently ligated with a 7–0 silk suture.", "To generate sham-operated control mice for the pressure overload and MI models, mice underwent the same surgical procedures but without aortic constriction or coronary artery ligation, respectively.", "Cardiac function was assessed by transthoracic echocardiography in conscious mice (Aplio SSA-770A, Toshiba Medical Systems Corporation) using a 15-MHz linear-array transducer 1, 4, and 8 weeks after TAC surgery.", "Seven- or eight-week-old male Fitm1 (−/−), Fitm2 (+/−), and littermate mice were reared as described above.", "Cardiac function was assessed by transthoracic echocardiography in conscious mice using a 15-MHz linear-array transducer at the end of approximately two years of observation.", "Mice were sacrificed and the left ventricle (LV) was excised.", "Total RNA was extracted using TRIzol reagent (Life Technologies) and subsequently purified with an RNeasy Mini Kit (Qiagen) following the manufacturer's instructions.", "mRNA was quantitated using an ND1000 nanodrop spectrophotometer.", "cDNA was synthesized from 100 ng of total RNA using a QuantiTect Reverse Transcription Kit (Qiagen), in accordance with the manufacturer's protocol.", "Quantitative polymerase chain reaction (qPCR) using the TaqMan system (Applied Biosystems) was used to analyze gene expression in TAC and MI samples.", "TATA-binding protein (Tbp) was used as an internal control.", "The primers are listed below.", "Relative mRNA levels were quantified using the comparative ΔΔCT method with Tbp as a reference gene.", "Differences were tested by two-tailed t-test.", "Values of p < 0.05 were considered statistically significant.", "Data analysis was performed using Microsoft Excel 2010 (Microsoft).", "To investigate the functions of Fitm1 and Fitm2 in the heart, we generated knockout mice using a gene-targeting system (Figs. 1a, 2a).", "Fitm1 (−/−) mice were viable, but Fitm2 (−/−) mice exhibited embryonic lethality (Figs. 1b, 2b), as reported previously [10].", "As Fitm2 (−/−) mice died from embryonic days 9–10 (Nishihama and Nagayama, unpublished data), we used Fitm1 (−/−) and Fitm2 (+/−) mice in our experiments.", "Assessment of cardiac function by echocardiography at 22 months of age showed no differences in body weight (BW), left ventricular end-diastolic dimension (LVDd), left ventricular end-systolic dimension (LVDs), or ejection fraction (EF) between Fitm1 (−/−) mice and their wild-type littermates [Fitm1 (+/+)] (Fig. 3a–d).", "There was no difference in BW, left ventricle (LV) weight, or right ventricle (RV) weight at sacrifice at 24 months of age (Fig. 3e–g).", "Meanwhile, there was no remarkable difference in BW or EF between Fitm2 (+/+) and Fitm2 (+/−) mice.", "However, LVDd and LVDs were significantly shorter in Fitm2 (+/−) mice at 22 months of age (Fig. 3a–d).", "Although Fitm2 (+/−) mice had lower RV weight than Fitm2 (+/+) mice, they had similar BW and LV weight (Fig. 3e–g).", "These results suggest that Fitm1 deletion does not significantly affect cardiac development or function under normal conditions.", "Meanwhile, Fitm2 (+/−) mice exhibited short LVDd and LVDs.", "However, their EF was normal (Fig. 3b–d) and their survival rate did not differ from that of the wild-type mice (T. Nagayama and N. Nishihama, unpublished data).", "Thus, under normal conditions, Fitm2 knockdown does not have a significant impact.", "However, Fitm2 (−/−) mice exhibit embryonic lethality, and postnatal whole-body knockout of Fitm2 leads to lethal enteropathy [7].", "These results indicate that Fitm2 has important roles during development as well as in small intestinal health and function after birth.", "Next, we investigated whether Fitm1 or Fitm2 deletion affects the pathogenesis of heart failure.", "We induced TAC in Fitm1 (−/−) and Fitm2 (+/−) mice and monitored their BW and cardiac function by echocardiography 1, 4, and 8 weeks after TAC surgery.", "We also monitored the survival rate of the mice subjected to TAC.", "Fitm1 (−/−) and Fitm2 (+/−) mice exhibited increased survival (Tables 1 and 2).", "The BW of wild-type and Fitm1 (−/−) mice that underwent TAC decreased from 1 to 8 weeks after surgery.", "However, the BW of Fitm2 (+/−) mice that underwent TAC did not differ from that of the sham-operated mice (Fig. 4a; Tables 1 and 2).", "Within 8 weeks after TAC surgery, wild-type, Fitm1 (−/−), and Fitm2 (+/−) mice that underwent TAC developed dilation of the cardiac chamber and contractile dysfunction as measured by LVDd and EF (Fig. 4b, c).", "There were no significant differences in LVDd or EF between wild-type and Fitm1 (−/−) mice (Fig. 4b, c).", "Compared with wild-type mice that underwent TAC, Fitm2 (+/−) mice exhibited attenuated expanding LV and reduction of EF from 4 weeks after surgery until 8 weeks (Fig. 4b, c).", "After measuring cardiac function, we dissected the mice and weighed their LV, RV, and lungs.", "The LVs from Fitm1 (+/+) TAC mice were approximately twice as heavy as those from the sham-operated mice (Fig. 4d, Table 1); these results are consistent with the cardiac dysfunction described above (Fig. 4b, c).", "Furthermore, the RV of Fitm1 (+/+) and Fitm1 (−/−) TAC mice was approximately 1.5 times as heavy as that of the sham-operated mice (Table 1).", "There were no significant differences in RV weight between Fitm1 (+/+) TAC mice and Fitm1 (−/−) TAC mice, and the LV of Fitm1 (−/−) TAC mice was heavier than that of Fitm1 (+/+) TAC mice (Fig. 4d, Table 1).", "The lungs from Fitm1 (+/+) TAC mice and Fitm1 (−/−) TAC mice were approximately 2.5 times heavier than those from the sham-operated controls (Table 1); these results are consistent with the results regarding pulmonary congestion described above.", "However, there was no significant difference in lung weight between Fitm1 (+/+) TAC mice and Fitm1 (−/−) TAC mice.", "The tissue weights of Fitm2 (+/+) and Fitm2 (+/−) mice were greater in mice that underwent TAC than in those that did not (Fig. 4d, Table 2), reflecting their condition.", "The LV weight of Fitm2 (+/−) TAC mice was increased compared with that of the sham-operated mice; however, it is a smaller extent than that of Fitm2 (+/+) TAC mice (Fig. 4d).", "These results might be consistent with the suppression of dilation (Fig. 4b, c).", "Although the LV from Fitm2 (+/−) mice that did not undergo TAC weighed less than that of wild-type mice, there were no differences between groups with respect to LVDd or EF (Fig. 4b, c).", "There was also no significant difference in lung weight between Fitm2 (+/+) TAC mice and Fitm2 (+/−) TAC mice (Table 2).", "Together with the cardiac function data, our findings suggest that deletion of Fitm1 improves mouse survival in the TAC model without altering the pathological condition.", "On the other hand, Fitm2 (+/−) positively affects cardiac function and survival after pressure overload-induced heart failure.", "The different responses of Fitm1 (−/−) and Fitm2 (+/−) to TAC are thought to reflect their tissue expression patterns.", "Fitm1 mRNA expression is higher in skeletal muscle than in the heart, whereas Fitm2 mRNA expression shows the opposite pattern [1].", "Our data suggest that deletion of FITM2 has protective effects against heart failure.", "Moreover, we found that the mRNA expression of both Fitm1 and Fitm2 was decreased in model mice with chronic heart failure induced by pressure overload (TAC model) and myocardial infarction (MI model) (Fig. 5), suggesting that Fitm1 and Fitm2 are common response genes in heart failure due to volume and pressure overload.", "It would be interesting to analyze the effects of their deletion in chronic heart failure induced by MI, and the mechanistic insights together with that in TAC model.", "Previously, we found that Fitm1 were identified by proteomic analysis of endoplasmic reticulum/sarcoplasmic rich fraction of mice heart, but there was no significant difference between TAC-operated and sham mice, probably because, the protein expression changed little as a result of TAC [9].", "Therefore, it also would be important to analyze the detailed protein expression pattern in both heart failure model.", "In this study, we mainly analyzed the effects of Fitm1 and Fitm2 deletion in healthy and diseased heart by gravimetric analysis and echocardiography.", "Therefore, further studies are needed to investigate the molecular mechanisms behind the observed protective effects against heart failure especially by Fitm2 deletion, such as, whether structural changes are occurred in heart and cardiac cells of knockout mice.", "Determination of functional changes of cardiac cells of knockout mice is also informative.", "Fitm1 and Fitm2 are involved in lipid droplet formation [1, 2, 3], though relationship between lipid droplet accumulation and pathological condition of heart failure is still unknown, it is known that lipid content in myocardiocytes is increased in failing hearts, especially in patients with diabetes mellitus and the metabolic syndrome [11].", "It also would be interesting to determine lipid droplet number and size in tissues of knockout mice and analyze further mechanistic insights.", "In the view of the energy metabolism, heart failure is accompanied by reduced fatty acid utilization and ATP level [12, 13, 14].", "Mice with skeletal muscle-specific overexpression of Fitm2 are reported to exhibit decreased fatty acid oxidation, increased utilization of branched-chain amino acids, enhanced glucose uptake, and decreased cellular ATP levels in skeletal muscle [8].", "These findings suggest that the overexpression of Fitm2 in mice might alter cellular metabolism in a manner similar to the changes observed in heart failure conditions.", "Nevertheless, in this study, we have not evaluated the products of energy metabolism of Fitm1 (−/−) or Fitm2 (+/-) mice in the context of healthy condition and heart failure.", "Whether metabolic changes are occurred in knockout mice is an important question to be addressed.", "Considering that Fitm1 and Fitm2 are family genes, it would be important to investigate whether compensate pathway to each other is occurred in knockout mice.", "Furthermore, analysis of Ftim1 and Fitm2 double knockout mice will also be informative to determine their functions in heart and heart failure.", "Pharmacological agents generally cannot completely inhibit protein expression or function.", "Thus, considering our results from knockout mice that underwent TAC, pharmacological downregulation or inhibition of FITM2 may contribute to the treatment of heart failure without having negative effects in otherwise healthy conditions.", "Natsumi Nishihama: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.", "Takahiro Nagayama: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.", "Shinji Makino: Analyzed and interpreted the data.", "Ryuta Koishi: Conceived and designed the experiments; Analyzed and interpreted the data.", "This work was supported by Daiichi Sankyo Co., Ltd.", "The authors declare the following conflict of interest: Natsumi Nishihama, Takahiro Nagayama, and Ryuta Koishi are employees of Daiichi Sankyo Co., Ltd.", "No additional information is available for this paper." ]	[]
Valuing conservation benefits of an offshore marine protected area	Increasing anthropogenic pressure in the offshore marine environment highlights the need for improved management and conservation of offshore ecosystems. This study scrutinises the applicability of a discrete choice experiment to value the expected benefits arising from the conservation of an offshore sandbank in UK waters. The valuation scenario refers to the UK part of the Dogger Bank, in the southern North Sea, and is based on real-world management options for fisheries, wind farms and marine protection currently under discussion for the site. It is assessed to what extent the general public perceive and value conservation benefits arising from an offshore marine protected area. The survey reveals support for marine conservation measures despite the general public's limited prior knowledge of current marine planning. Results further show significant values for an increase in species diversity, the protection of certain charismatic species and a restriction in the spread of invasive species across the site. Implications for policy and management with respect to commercial fishing, wind farm construction and nature conservation are discussed.	[ "ECON", "ENVI" ]	[ "Discrete choice experiment", "Dogger Bank", "Environmental valuation", "Marine ecosystem services", "North Sea", "Offshore MPA" ]	[ "2002", "2300" ]	[ "With recent technological developments and an expanding global economy, there is increasing pressure from human activities on the offshore environment (Stojanovic and Farmer, 2013).", "Offshore waters are typically considered to be those outside territorial waters (i.e. beyond 12 nautical miles from the coast, including international waters) and have traditionally been exploited by the commercial fishing, oil and gas, and aggregate sectors.", "However, the expanding blue economy, which is considered to offer “smart, sustainable and inclusive economic and employment growth from the oceans, seas and coasts” (EC, 2012), exemplifies a growing interest in the offshore marine environment.", "It reflects a wider range of activities including: marine energy extraction; aquaculture; maritime, coastal and cruise tourism; marine mineral resources; and blue biotechnology (DG MARE, 2012; EC, 2012).", "With the resulting pressures on offshore sites, the challenges for the management of remote parts of the marine environment become far greater.", "These developments require more informed planning and management to ensure sustainable use of offshore marine resources.", "While marine planning has been effective in reaching out to particular user groups, such planning is increasingly required by legislation to engage all stakeholders including non-user groups such as the general public (cf. EC, 2001; Jobstvogt et al., 2014).", "This demand for wider engagement recognises that the marine environment provides ecosystem services which are fundamental to human well-being (Beaumont et al., 2007; Atkins et al., 2011; Börger et al., 2014).", "While the economic benefits of offshore activities such as fisheries are widely discussed (e.g. Sumaila et al., 2007), evidence on the wider societal benefits of the marine environment, in which these offshore activities take place, is less well known (Pendleton et al., 2007; Armstrong et al., 2010).", "Few published studies have explored the benefits provided by the offshore marine environment (Glenn et al., 2010; McVittie and Moran, 2010; Ressurreição et al., 2011, 2012; Jobstvogt et al., 2014).", "Much greater attention has focused on coastal areas (e.g. Atkins et al., 2007; Eggert and Olsson, 2009; Taylor and Longo, 2010; Hynes et al., 2013).", "Other studies have used benefit transfer to value the benefits of offshore sites (Hussain et al., 2010; Londoño and Johnston, 2012), but are limited by the scarcity of primary valuation data.", "Environmental impact assessments associated with the creation of wind farms or the designation of marine protected areas (MPAs) are starting to increase the offshore evidence base (e.g. Talisman Energy, 2006; Defra, 2012), but valuation evidence still remains limited.", "This paper focuses on gaining greater insight into how the general public perceive and value the benefits of the offshore marine environment.", "For this purpose a case study assessment is made employing a discrete choice experiment (DCE) to value benefits provided by the conservation of an offshore sandbank under UK jurisdiction.", "The Dogger Bank is the largest offshore sandbank in the North Sea, covering a total area of approximately 17,600 km2 (Diesing et al., 2009) (Fig. 1).", "The waters surrounding the Dogger Bank are relatively shallow (Diesing et al., 2009), are highly productive (Kröncke, 2011), and support an important seabed community (Wieking and Kröncke, 2003; Kröncke, 2011) which provides a vital food resource for the diverse fish assemblage (Sell and Kröncke, 2013).", "The Dogger Bank is also an economically important area.", "Historically it has been used as a rich fishing ground primarily by Dutch, German, Danish and UK fleets, and competition for space is intense and increasing.", "This area of the southern North Sea has further been used for natural gas exploration with five platforms still present across the site (JNCC, 2011).", "There are also two small areas which are licensed for aggregate extraction (JNCC, 2011).", "More recently, plans have been submitted for the development of the largest wind farm in Europe on the UK sector of the Dogger Bank, covering an area of 8660 km2 (Forewind, 2010).", "Given the shallow depth of water found above the Dogger Bank, very little commercial shipping takes place (JNCC, 2011), however the wrecks found on and around the Bank provide for a limited amount of recreational angling and diving (Forewind, 2013).", "The Dogger Bank is located within the exclusive economic zones (EEZ)1 of four European Union (EU) Member States (Fig. 1).", "Three of these Member States (the UK, Germany and the Netherlands) are in the process of designating the sandbank under their jurisdiction as a Special Area of Conservation (SAC), a form of MPA, under the EU Habitats Directive (EC, 1992).", "Within the EU, the establishment of a network of MPAs is driven by both international obligations (e.g. the OSPAR Convention in the North East Atlantic) and the implementation of European Directives (e.g. Good Environmental Status under the Marine Strategy Framework Directive (2008/56/EC) and Favourable Conservation Status for habitats and species under the Habitats Directive (92/43/EEC) and Birds Directive (2009/147/EC) (Potts et al., 2014).", "In the UK, the Joint Nature Conservation Committee (JNCC) is responsible for the identification of SACs in offshore marine waters for designation by the Government.", "The JNCC is therefore responsible for establishing conservation objectives for offshore SACs.", "The Dogger Bank was recommended by JNCC to the UK Government as a candidate SAC (cSAC) for the protection of ‘sandbanks which are slightly covered by seawater all the time’ (EC, 1992).", "Following a baseline assessment, JNCC reported the Dogger Bank as being in unfavourable conservation status.", "The site is vulnerable to physical disturbance or abrasion (e.g. from bottom trawl fishing gear2 and pipeline burial), selective extraction of species (e.g. by bottom trawl fishing), and physical loss by obstruction (e.g. permanent construction of wind farms) (JNCC, 2011).", "Its vulnerability to the introduction of non-native species has not been possible to asses (JNCC, 2011).", "To restore the site and achieve favourable conservation status, the establishment of a management plan is required to provide the basis for determining what current or future activities may have an impact on the overall health of the site.", "To develop such a management plan, the UK, Germany and the Netherlands formed the transnational Dogger Bank Steering Group (DBSG), in which the European Commission and Denmark have an observer status.", "In 2011, the DBSG invited the North Sea Regional Advisory Council (NS RAC), which consists mainly of representatives from the fisheries and wind farm industries and environmental non-governmental organisations (ENGOs), to develop a proposal for a fisheries management plan for the Dogger Bank (NS RAC, 2012).", "This novel bottom-up decision-making process was welcomed by most stakeholders and policy-makers.", "The process eventually stalled, however, because fishing representatives and ENGOs were unable to adopt a compromise proposal regarding habitat protection (NS RAC, 2012).", "This situation results in part from the lack of evidence on species, habitats and fisheries impacts on the Dogger Bank, and on what percentage of the Dogger Bank would need to be protected to achieve the conservation objectives set for the respective cSACs.", "Based on the above considerations, the main purpose of this study is to test the applicability of the DCE methodology to the valuation of the expected benefits of an offshore site as a result of management options currently under discussion.", "More specifically, the research objectives are to:", "Assess the general public's level of concern regarding the environmental consequences of offshore marine activities and potential support for marine management plans.", "Assess the social benefits of proposed management plans to an offshore sandbank (specifically the Dogger Bank).", "Determine the value of the potential outcomes of management plans that target specific aspects of biodiversity (both positive and negative attributes, such as species diversity, charismatic species and invasive species).", "Identify determinants of respondents' preferences for different aspects of the proposed management plans.", "Consequently, this paper contributes to the literature on the valuation of the offshore marine environment and, in particular, ecosystem services associated with protected offshore sandbanks.", "The remainder of the paper is structured as follows: Section 2 describes the DCE approach and survey development; Section 3 presents the results; and Section 4 provides the discussion and policy implications.", "Besides the contingent valuation method (CVM), discrete choice experiments (DCEs) (Hanley et al., 1998; Louviere et al., 2000) are one of the most popular approaches to assess the total economic value (TEV) of non-market environmental goods.", "These methods use surveys to elicit the willingness-to-pay (WTP) of respondents for securing some future positive environmental change (or to prevent some negative change from happening).", "WTP estimates are interpreted as indicators of the change in utility that respondents expect from the specific environmental change.", "Aggregated over the representative sample of the affected population, WTP estimates provide quantification of the benefits of environmental changes for that population, e.g. for users and non-users of a particular area.3", "Few non-coastal marine valuation studies have used quantifiable attributes.", "Glenn et al. (2010) and Wattage et al. (2011) report on a DCE on MPAs around cold-water coral reefs in Irish waters but do not provide any monetary values for the protection of corals or the banning of fishing activities.", "The CVM study in Ressurreição et al. (2011, 2012) reports significantly positive WTP of respondents in the Azores for the preservation of species diversity in waters around the archipelago.", "McVittie and Moran (2010) conducted a DCE survey to value the provision of ecosystem services resulting from the network of UK marine conservation zones.", "While these authors base their valuation scenarios entirely on a real-world management measure (the UK Marine Bill), the attribute levels are described qualitatively.", "This makes it very difficult to link changes in the underlying ecological indicators to the elicited values.", "The only non-coastal DCE study reporting WTP for the protection of deep-sea biodiversity and using quantifiable attributes is Jobstvogt et al. (2014).", "Their survey elicits WTP of Scottish households for additional MPAs in the Scottish deep-sea.", "They focus on two value categories of biodiversity: the existence value for deep-sea species and the option value of deep-sea organisms as a source of future medicinal products.", "Given the scarcity of evidence in this field, the present study implements a DCE that is firmly linked to real-world management measures for offshore marine sites.", "It also uses quantifiable attributes, which can be directly linked to measurable changes resulting from the proposed management.", "The use of clearly quantifiable attributes makes resulting value estimates suitable for benefit transfer (Loomis and Rosenberger, 2006; Richardson et al., 2014).", "The development of valuation scenarios was based upon the discussions of the DBSG regarding the management of the Dogger Bank.", "The range of management proposals brought forward by the different parties leave considerable scope for the development of relevant choice attributes.", "At the same time attributes represented certain ecosystem service categories.", "The two sectors likely responsible for the greatest current and potential future impact on the Dogger Bank are commercial fishing and offshore wind farm development (JNCC, 2011).", "Fishing regulations could affect a multitude of ecosystem services associated with the conservation of biodiversity, in addition to food provision.", "For example, the Dogger Bank has historically been important for sandeels (Cefas, 2007).", "They are important prey for other commercial fish species (e.g. whiting, plaice, mackerel and cod), seabirds (e.g. fulmar and kittiwake) and cetaceans, especially the harbour porpoise (Diesing et al., 2009).", "Prohibiting bottom trawling activities on part of the Dogger Bank would conserve the sandeel populations, as well as improve the health and diversity of the wider marine community (Kaiser et al., 2006; Olsgard et al., 2008).", "Seabed communities are also known to play a role in a number of regulating ecosystem services (Snelgrove, 1999; Austen et al., 2011), such as carbon sequestration and storage and the regulation of waste products.", "They also provide supporting services including nursery grounds for commercially important fish species.", "The Dogger Bank acts as a nursery ground for plaice, providing suitable habitat for foraging and maturing fish (Diesing et al., 2009; Hufnagl et al., 2013).", "In addition, by excluding fishing activities on part of the Dogger Bank, porpoises, seals and seabirds, charismatic species with cultural significance, are less likely to be caught as by-catch in the area (Vinther and Larsen, 2004; Zydelis et al., 2009; Sonntag et al., 2012; Brown et al., 2013).", "Changes in species diversity and protection of porpoises, seals and seabirds were therefore selected as choice attributes (Table 1).", "Recognising that a species is rare or endangered may lead to respondents stating a higher WTP for their protection (Christie et al., 2006), the conservation status of porpoises, seals and seabirds was therefore not mentioned.", "The attribute levels for species diversity and protection of porpoises, seals and seabirds were chosen based on the areas for closure proposed by the different stakeholders for the Dogger Bank and scaled to the UK section.", "The ENGOs and Germany are seeking closure of 50% of the area to all fishing activities, while the fishing industry would prefer a maximum of 25% of the area to be closed.", "Taking these as attribute levels, under the conservation scenario porpoises, seals and seabirds would be protected on 50% of the Dogger Bank by preventing potential by-catch by the fishing industry (e.g. Brown et al., 2013), but only on 25% under the fishing industry proposal.", "The link between the cessation of a particular type of fishing gear, namely bottom trawling, and change in species diversity is less clear.", "Drawing from correlations in Kaiser et al. (2006) suggests, however, that it is reasonable to assume that removal of trawling on 25% of the area could lead to a 10% increase in species diversity, while removal from 50% of the area could lead to an increase in species diversity of approximately 25%.", "The third attribute selected was the spread of invasive species across the North Sea as a potential outcome of the installation of offshore wind turbines.", "Many species are extending their ranges northwards due to increasing sea temperatures caused by climate change (Perry et al., 2005; Tasker, 2008; Rijnsdorp et al., 2009; Edwards et al., 2013; Mieszkowska et al., 2013).", "For hard substrate dwelling species the introduction of permanent hard structures into what is a dynamic, mobile sandbank has the potential to provide new habitat for species that are currently unable to colonise the locality (Petersen and Malm, 2006; Glasby et al., 2007; Bulleri and Chapman, 2010).", "This increases their potential to survive and, with the turbines acting as stepping stones, spread throughout the North Sea.", "Once established invasive species may compete with native species for resources such as food and space (e.g. Arenas et al., 2006).", "Invasive species are already found in the North Sea, e.g. the sea walnut (Mnemiopsis leidyi), colonial sea squirts such as Botrylloides violaceus, Atlantic blue crab (Callinectes sapidus), Japanese skeleton shrimp (Caprella mutica) (Galil et al., 2014).", "Of particular relevance in this case are the spread of species such as the colonial sea squirt and the Japanese skeleton shrimp which are both known to colonise artificial (man-made) hard substrata in shallow waters (Arenas et al., 2006; Page et al., 2007; Turcotte and Sainte-Marie, 2009) as well as the potential for the proliferation of those invasive jellyfish that require hard substrate for part of their life cycle (Purcell, 2012).", "Consequently, the attribute levels chosen for the spread of invasive species were ‘restricted’ and ‘wide spread’ across the North Sea, which reflects the fact that invasive species are already present in the North Sea.", "The valuation scenarios further specify that the implementation, monitoring and enforcement of the Dogger Bank management plan will come at a cost.", "Marine management within the UK is the responsibility of the Marine Management Organisation (MMO), funded ultimately by the taxpayer.", "The payment vehicle used was therefore an increase in annual tax for UK households over the next five years.4", "This attribute was given seven levels (Table 1).", "To develop the survey questionnaire 29 semi-structured interviews were conducted by the project team (an interdisciplinary team comprising marine ecologists and economists) with members of the general public.", "Interviews were completed in Hull (Northeast England) and Plymouth and Exeter (Southwest England) to sample respondents living close to and far from the North Sea.", "Based on insights from these interviews a preliminary choice questionnaire was tested in 19 face-to-face interviews with members of the public following a workshop on management of the Dogger Bank (held in Newcastle, Northeast England).", "Respondents were asked to think aloud while they completed the choice tasks (Ryan et al., 2009).", "This helped to detect inconsistencies and unclear wording in the scenario and attribute description.", "The same questionnaire was tested online with respondents from across the UK (n = 60).", "Findings from both pilot surveys were used to modify the questionnaire wording and additional pictograms were included to describe the choice attributes (Fig. 2).", "The results from the online pilot survey also informed the experimental design of the main survey.", "Coefficients indicating the influence of the choice attributes on choices from a mixed logit model (cf. next section) were used as priors when generating a Bayesian D-efficient design (Scarpa and Rose, 2008) in the software package Ngene (ChoiceMetrics, 2012).", "Unrealistic survey scenarios were excluded, such as choice options which yield the status quo for each attribute at positive cost, since these options would be dominated by the status quo.", "The final set included 24 choice tasks separated into four blocks of six tasks.", "Respondents were randomly allocated to one block and complete the six choice tasks in that respective block.", "Each choice task contained two alternative management plans at different cost levels and a ‘no change’ or status quo option at zero cost (Fig. 2).", "The final questionnaire consisted of four parts: part one contains 19 questions regarding the respondent's general knowledge of and experience with the North Sea and the Dogger Bank.", "Part two introduced the valuation scenarios, the hypothetical ‘Dogger Bank Management Plan’, and included the description of the choice attributes and the payment method (Table 1).", "Part three contained the actual choice experiment.", "Following each choice task, respondents were asked to indicate how certain they were of their choice on a 5-point scale from 1 “Not certain at all” to 5 “100% certain”.", "Part four contained a series of attitudinal questions, some of which are used to identify protest respondents.", "These are respondents who chose the costless status quo option in all six choice tasks and agreed to the statements “Taxes and fees are already too high, so there should be no additional financial burden”, “I already pay enough for other things”, “It is my right to have a well preserved Dogger Bank and I should not have to pay extra for it” and “The government should cut public spending on other things instead of expecting a contribution from me”.", "Answers to these questions were given on a 5-point Likert scale from “Strongly disagree” to “Strongly agree”.", "In the regression models, dummy variables indicating agreement (i.e. “Agree” or “Strongly agree”) are used.", "The questionnaire concluded with a series of socio-demographic questions.", "Three-digit postcode data (e.g. HU6, PL1) were collected to generate distance variables from the Dogger Bank, the North Sea coast and the nearest coastline using geographical information systems (GIS).", "Additional dummy variables indicating respondents that live within 1, 2, 4, 6, 10, 25 or 50 miles from a coastline compared to respondents living further inland were computed from these data.", "These are used to test for distance effects of elicited values.", "The survey was conducted online by a market research company with respondents sampled from their existing respondent panel.", "While online surveys allow researchers to reach a broad survey sample they systematically exclude people without internet connection.5", "However, the problem of hard-to-reach groups also exists for alternative survey modes, such as mail or direct interviews.", "Regarding stated preference valuation techniques it has been shown that online surveys do not produce significantly different WTP estimates compared to traditional mail surveys (Olsen, 2009; Windle and Rolfe, 2011).", "Empirical comparisons of WTP estimates of online and face-to-face surveys show mixed results (Canavari et al., 2005; Marta-Pedroso et al., 2007; Nielsen, 2011), but coverage of a large sampling area at justifiable costs can best be achieved by an online survey.", "The survey aimed to elicit views from a representative sample of the resident population of the UK, in terms of age, gender, and income, to assess potential non-use values held by respondents living far away from the actual study site.", "Sample characteristics are presented in the Results section.", "Both conditional and mixed logit models produce estimates of the coefficient vector β.", "The elements of β can be interpreted as the average utility weights of the attributes included in the choice tasks (i.e. the influence of these attributes on stated choices).", "Both models allow for the analysis of preference variation across respondents.", "If different respondents have different preferences for a certain attribute, respondent-specific variables can be interacted with attribute-specific variables to account for these different preferences (Train, 2009).", "As the conditional logit cannot account for unobserved preference heterogeneity, the analysis presented here relies heavily on the mixed logit model.", "By specifying the distribution of the elements of the coefficient vector β, the latter model can also account for random (i.e. unexplained) preference heterogeneity.", "The coefficients of all but the cost attribute are assumed to be normally distributed, so both means and standard deviations of coefficients of the respective attributes are reported.6", "Respondent-specific variables are included in the models in interactions with the attribute variables or as a dummy variable indicating whether the status quo or one of the policy options was chosen.", "Both models produce coefficient estimates through maximum likelihood estimation.", "This study uses the asclogit command in Stata (version 12) to compute the conditional logit model.", "The mixed logit model is estimated using the user-written command developed by Hole (2007).", "The main survey was conducted in early December 2013.", "In total 1022 respondents completed the questionnaire.", "To check whether the sample reflects the structure of the UK population, sample characteristics are compared against population means.", "Shares of age groups reflect age distribution in the population (Table 2) except for a slight oversampling of respondents aged 65 and over at the expense of the 18–24 and over group.", "The level of education is somewhat higher in the sample than in the general population.", "As an education indicator the share of people with at least a university degree is 32.8% in the sample but only 27.0% in the general population.", "The distribution of occupations closely resembles that of the general UK population.", "Self-employed and retired respondents are slightly underrepresented compared to the 2011 census figures.", "Average household size in the sample is 2.64 persons per household compared to 2.37 for the whole country.", "The mean monthly household income of £2275 is lower than average household income for the whole of the UK.", "Based on these comparisons, the survey sample was considered to reflect the structure of the UK population.", "The survey findings therefore have significance for the resident population as a whole and can be used in cost-benefit analysis (CBA).", "In terms of spatial distribution of the sample across the UK, Fig. 3 shows that respondents from almost all postcode areas of the country were sampled (115 out of 125).", "70.6% of respondents stated that they had previously visited the North Sea coast.", "About half the respondents had taken a ferry (49.9%) or flight (63.0%) across the North Sea.", "The more specific topic of the survey, the Dogger Bank, was new to most respondents.", "Only 50.2% of the respondents stated that they had heard of the Dogger Bank before completing the questionnaire, with the vast majority of them (80.3%) having heard of it in the shipping forecast.7", "After further introduction to the site, only 3.9% of respondents stated that they were previously aware of all the information presented, and 37.0% stated they were aware of some of it.", "The remaining 59.1% stated that they were unaware of this information before undertaking the survey.", "Similarly, regarding MPAs, only 19.9% of respondents had heard of the UK government's plans to create a network of MPAs by 2016.", "Part one of the questionnaire also elicited the level of support for the two main components of the hypothetical Dogger Bank management plan: regulation of fisheries and future wind farm development.", "Despite the low level of existing knowledge, the majority of respondents supported regulations both of fisheries and future wind farm development in that area (Table 3).", "Only a small proportion of respondents directly rejected the plans to regulate fishing and wind farm development introduced in the survey scenarios (3.8% and 6.5% respectively).", "For the analysis of choice data, 49 respondents were identified as protestors based on responses to attitudinal questions and were subsequently removed from the survey sample.8", "The variables used in all subsequent models and their description are listed in Table 4.", "In both the conditional and mixed logit models reported in Tables 5a and 5b, coefficients of all attributes were significant and exhibit the expected signs.", "Changes in species diversity and the protection of charismatic species positively influenced choices (i.e. on average, choice options describing an increase of these attributes had a higher probability of being preferred than those indicating no change).", "In contrast, the coefficients for wide spread of invasive species (INVASIVE) and the cost attribute were significantly negative in both models.", "The higher the cost of an option in terms of the household tax the smaller the probability that this option was selected.", "Tables 5a and 5b report WTP estimates computed from the conditional and mixed logit models according to Eq. (3).", "There was no clear pattern that either model produces only higher or lower WTP estimates.", "The relative importance of the attributes as measured in WTP did not change across models.", "However, the mixed logit model's fit to the data as indicated by the adjusted ρ2 and the Bayesian Information Criterion (BIC) was much better compared to the conditional logit model.", "WTP for the larger change in one attribute (e.g. 25% increase in species diversity) always exceeded WTP for the smaller change (e.g. 10% increase in species diversity).", "While respondents were willing to pay £4.19 (£5.70 in the conditional logit) per year on average for a 10% increase in species diversity on the Dogger Bank, their average WTP for a 25% increase was £7.76 (£7.22 in the conditional logit).", "WTP did not appear to increase linearly with the level of this attribute, but indicated decreasing marginal utility of species diversity.", "Respondents seemed willing to pay for a certain base level of change but their WTP did not increase proportionately for further positive changes.", "This pattern could also be detected for the protection of porpoises, seals and seabirds.", "Respondents were willing to pay £24.02 (£26.24 in the conditional logit) on average per year to protect these species on 25% of the UK Dogger Bank area, whereas the WTP for the protection on 50% of the area was £30.32 (£33.07 in the conditional logit).", "Designing a future wind farm on the Dogger Bank that results in wide spread of invasive species yielded a negative WTP of £− 25.39 (£− 22.93 in the conditional logit) on average to respondents in this sample.", "These estimates can be interpreted as an indicator of a negative welfare effect resulting from a decrease in environmental quality as indicated by this attribute.", "Relating these annual monetary figures to reported monthly household income (after tax and benefits), they make up shares between 0.018% and 0.112%.", "As the mixed logit model relaxes the assumption that attribute coefficients are fixed across respondents, the reported coefficients were means of the estimated distribution.", "An estimate of the standard deviation for each coefficient is reported in Table 5b.", "While there was no evidence of random preference variation for the coefficient of the protection of charismatic species on 25% of the Dogger Bank (PROT25), all other attribute coefficients modelled had standard deviations significantly different from zero.", "These results provide evidence of substantial random preference heterogeneity, consequently only the mixed logit model was employed to identify individual specific choice determinants.", "To identify determinants of choices, and thus of WTP, and to validate the survey responses, interactions between the management option dummy (ASC_CHANGE) and/or certain choice attributes and a set of respondent-specific variables were included in the mixed logit model.", "The three models reported in Table 6 contain an increasing number of explanatory variables.", "Often the number of observations decreases the more explanatory variables are included in the model resulting from the exclusion of respondents with missing values.", "It should be noted here that the number of observations remained constant across models due to the very high item response rate in this survey.", "Model 1 in Table 6 included choice attributes and 13 socio-demographic variables.", "In Table 6 the top section of the column reports mean estimates of attribute coefficients modelled as random.", "All attribute coefficients were significant and point in the same direction as in the basic model in Table 5b.", "Among the respondent-specific variables, the interaction between the ASC_CHANGE and MALE was negative, indicating that male respondents were less likely to choose any of the costly management options.", "Respondents who were members of an environmental organisation (ENVORG) or had previously taken a ferry (FERRY) or flight across the North Sea (FLIGHT) had a higher WTP for costly management options as indicated by the positive coefficients.", "The significant effects of MALE, ENVORG and FLIGHT were robust throughout all models in Table 6.", "The median response for each of the six choice certainty questions was 4 on the scale from 1 “Not certain at all” to 5 “100% certain”.", "The majority of respondents appeared to be confident to state their preferences by means of the choice tasks presented to them despite the low level of prior knowledge of the topic reported earlier.", "Beyond that, the level of choice certainty summed up for all six choice tasks (CERTAIN) did not significantly affect choice probabilities in the mixed logit models 1 to 3.", "The level of education of the respondent (approximated by the dummy variable UNI) did not show any significant effect on choices in models 1 to 3.", "There were no effects of respondents' age (AGE) and household income (INCOME) on choices.", "Similarly, there was no influence of respondents' previous experience of sea angling (ANGLING) or scuba diving (SCUBA), professional experience in any marine sector (MARSEC) or their distance from the Dogger Bank (DIST_DB).", "Alternative distance variables to the North Sea and the nearest coast, as well as the natural logarithm of the three different distance variables were tested, however, none of them had an impact on choices when interacted with ASC_CHANGE.", "In an alternative model, dummy variables indicating respondents that live within 1, 2, 4, 6, 10, 25 and 50 miles from a coastline were interacted with ASC_CHANGE, but again there were no significant effects of these variables on choices.", "In model 2, a set of dummy variables drawn from attitudinal questions, some of which were also used to identify protest respondents,9 were added.", "All significantly influence choices.", "Coefficients of statements adverse to the Dogger Bank management plan (NOTNEC) and its financing (BURDEN, ENOUGH) were negative and remain so after including further variables in model 3.", "This means that respondents agreeing to the statements “I think a Dogger Bank management plan is not necessary” (NOTNEC), “Taxes and fees are already too high, so there should not be an additional financial burden” (BURDEN) and “I already pay enough for other things” (ENOUGH) had a lower probability of choosing one of the management options.", "The effect of a fourth statement critical of the financing of the management plan reading “The government should cut public spending on other things instead of expecting a contribution from me” (CUT) was significantly positive in models 2 and 3.", "In contrast, respondents agreeing to the statements “I enjoy contributing to a good cause no matter what it is” (ENJOY) or “I think it is better to ask experts whether or not to carry out this Dogger Bank management plan” (EXPERTS) were more likely to prefer a costly management option over the no-change (zero cost) option.", "Model 3 included further attitudinal variables, which are related to specific attributes and were therefore interacted with these attributes rather than with ASC_CHANGE.", "Results in Table 6 show that respondents agreeing with the statement “Without a management plan the diversity of species on the Dogger Bank will continue to decrease” (SPECDIV) had a stronger preference for species diversity.", "The coefficients of the main effects of SPEC10 and SPEC30 (Table 6) were not significant in model 3.", "While respondents who agreed with the statement (SPECDIV) preferred higher species diversity more strongly, choices of respondents who did not agree with this statement were not influenced by this attribute (i.e. indifferent to changes in species diversity).", "In a model not reported here, choices of respondents who did not agree to SPECDIV were completely indifferent to changes in species diversity.", "This supports the finding that agreement to this attitudinal statement is a valid predictor of preferences for the respective benefits.", "Respondents agreeing to “Porpoises, seals and seabirds need protecting through the management plan” (PROTECT) valued this particular feature of the plan significantly more than respondents not holding this view.10", "Preferences for a reduction of the spread of invasive species (INVASIVE) were not influenced by the inclusion of these interactions.", "In all of the reported models, the model fit to the data was very good.", "Louviere et al. (2000) state that adjusted McFadden's ρ2 between 0.2 and 0.4 indicate extremely good model fit for this type of logit model.", "Overall, model fit improved from model 1 through to model 3 as indicated by an increasing adjusted ρ2 and a decreasing BIC.", "The inclusion of attitudinal variables in models 2 and 3 further increased the predictive power of the model and thus better explained stated choices.", "The main focus of this study was to test the applicability of DCE for the valuation of benefits arising from the management of an offshore MPA.", "While there are a very limited number of stated preference studies relating to offshore sites, this is one of the few studies to explicitly link the valuation scenarios to current marine management discussions.", "This particular approach includes a set of largely quantifiable attribute specifications that can be directly linked to real-world management measures.", "Establishing these links and conveying them in a credible and readily understandable way in the valuation scenarios proved challenging with respect to an offshore marine site.", "Yet results from valuation studies of this kind can be most informative to marine management if the link between proposed management measures, ensuing environmental change, and the assessment of the resulting benefits is as clear and direct as possible (McVittie and Moran, 2010).", "The importance of the coastal and marine environment to an island nation such as the UK is reflected in the finding that a large majority of respondents have visited and/or travelled across the North Sea.", "As anticipated this study found low levels of self-rated knowledge about the case study site and its management, an observation also made for the case of the deep-sea by Jobstvogt et al. (2014).", "Nevertheless, following the provision of limited information on the implications of management of the main commercial activities on the Dogger Bank, support for their regulation was found.", "This provides evidence of the level of concern of the general public regarding the consequences of these offshore activities and supports the approach taken in the subsequent choice experiment.", "Nevertheless, these findings should be interpreted with caution due to the potential influence of the provided information on responses.", "Results further show that the UK public hold significant values for environmental benefits generated by conservation measures in an offshore location.", "The elicited values obtained through this study correspond to real-world management options that are currently under discussion for an offshore MPA in the UK.", "Regarding the specific choice attribute values, protection of charismatic species (porpoises, seals and seabirds) on 25% of the UK section of the Dogger Bank is proportionately greater than for protection on 50% of the area.", "This non-linear increase is important given the disagreement over such protection between the fishing industry and the ENGOs who advocate for these respective protection levels.", "Similarly, WTP for increasing species diversity by 25% resulting from the exclusion of bottom trawling from 50% of the UK section of the Dogger Bank is higher than for an increase of only 10%, if bottom trawling was excluded on just 25% of the area, although again there is no linear relationship.", "Comparing these two attributes, values for the protection of charismatic species exceed those for general species diversity.", "This supports findings elsewhere that suggest that respondents are capable of distinguishing between different (quantitative) levels of species protection but that they react more strongly to charismatic species compared to general diversity (Martín-López et al., 2007; Jacobsen et al., 2008).", "However, this might depend on the cultural background of respondents (Ressurreição et al., 2012).", "A confounding issue in this survey could result from the link suggested between the regulation of two commercial sectors on the Dogger Bank and the resulting environmental changes.", "While the choice tasks focus specifically on the different levels of environmental changes, it is conceivable that some respondents' choices were motivated by the underlying restrictions to fishing and wind farm development.", "This ambiguity often occurs when basing valuation scenarios on real-world management decisions where environmental improvements may come at the expense of economic or social factors.", "Despite respondents' low levels of prior knowledge of the study site and its characteristics there are several indicators supporting the validity of the elicited values.", "Self-reported choice certainty, as assessed after each choice task, was found to be high.", "This can be considered an indication that the information provided was sufficient for respondents to link the scenarios to their existing attitudes, and consequently to state choices based on individual preferences.", "Choice certainty, however, does not affect choices indicating no systematic difference between the preference structures of respondents who feel certain about their choices and those who are less certain.", "The validity of the elicited values is further supported because preferences for the proposed benefits are predicted by certain respondent characteristics in a plausible manner.", "The higher likelihood of respondents who have seen the North Sea (from a flight or ferry) to prefer one of the management options indicates that familiarity with the good or some feeling of connection (but not direct use) increases the benefits to be expected from the proposed measures.", "Similarly, members of environmental organisations value the proposed measures more than non-members.", "This effect is frequently found in studies valuing biodiversity (e.g. Jobstvogt et al., 2014; Yao et al., 2014).", "Indicators of direct use of the marine environment, such as recreational angling and scuba diving or professional experience in a marine sector do not systematically affect choices.", "This is in contrast to the results of Jobstvogt et al. (2014) with respect to deep-sea ecosystem services.11", "The findings of the present study therefore indicate that values for the proposed benefits are mostly non-use in nature.", "This interpretation is further supported by the lack of any distance effects.", "Attitudes towards the management plan and recognition of existing financial burdens for households influence choices and WTP mostly as expected.", "In addition to preference heterogeneity explained by these attitudinal variables, there is evidence of further random heterogeneity in preferences for the proposed benefits.", "This heterogeneity is particularly strong for changes in species diversity and the spread of invasive species, the two attributes which are likely to be more difficult to understand.", "Consistent with this, the results indicate rather uniform preferences for the protection of some charismatic species on 25% of the UK sector of the Dogger Bank — a benefit potentially more easily understood by respondents.", "The failure to detect any decreasing or increasing distance effect in the choice data is interesting against the backdrop of ongoing discussions relating to distance decay of values elicited in stated preference surveys.", "Previous studies have found evidence for distance decay for use and non-use values in terrestrial (Bateman et al., 2006) and coastal settings (Luisetti et al., 2011).", "Schaafsma et al. (2012) have further shown that distance decay with respect to a terrestrial environmental good also depends on the availability of substitutes, which might differ between different geographical directions from the good to be valued.", "The present study, however, does not find evidence for any of those spatial patterns in values for Dogger Bank ecosystem services.", "Nor are there differences between respondents living near the coast (and potentially having substitutes for the proposed benefits available) and further inland.", "In an island nation such as the UK it is conceivable that respondents living at another part of the coast value the benefits from conservation of the Dogger Bank differently because (some of) these benefits are provided by the marine environment on their doorstep.", "Yet such differences could not be found.", "These findings reflect those reported by Rolfe and Windle (2012) and Choi (2013).", "They indicate that considerations about distance effects found for terrestrial and coastal environmental goods do not translate to an offshore site, such as the Dogger Bank.", "Non-use values, which constitute the major motivation for the benefits assessed in this study, might not exhibit linear distance effects but instead could show other spatial patterns (Johnston et al., 2011).", "It is possible that respondents living close to each other exhibit local clusters of relatively high or low WTP.", "Further research in this area is needed to clarify these preference patterns for marine environmental goods and services.", "Finally two further caveats require mention.", "The first concerns values for species protection.", "There is still debate about how to include the conservation status of species (Jacobsen et al., 2008).", "This survey avoided explicit mentioning of conservation status of the harbour porpoise because it is not one of the features that led to the designation of the UK part of the Dogger Bank as a cSAC.", "Nevertheless conservation value may be an important component of non-use value.", "Secondly, standard survey practice requires the description of the payment mode to be as plausible as possible.", "The present survey did not further specify the household tax.", "Reasons for this are mentioned earlier, however, it cannot be said with certainty that this did not diminish the perceived realism of the valuation scenarios.", "The results should therefore be interpreted against this background.", "Despite the mentioned limitations, an online survey allowed respondents from across the UK to be sampled.", "The resulting sample reflects the general structure of the UK population in terms of certain demographic characteristics.", "This is crucial when valuation estimates are to be used in an environmental CBA to support decision-making.", "It has also been shown that WTP estimates from postal and online surveys are comparable (Olsen, 2009; Rolfe and Windle, 2011).", "However, online surveys offer limited control on how respondents complete the questionnaire and take in the information.", "Christie and Rayment (2012) have shown that in workshop settings complex valuation topics can successfully be conveyed to respondents, it is not clear whether this is also true for online surveys.", "Most variables in the regression models affect choices in a plausible way, but it remains unclear to what extent respondents really grasp the concepts of species diversity and invasive species.", "Proper testing of questionnaires such as the use of “think-aloud” interviews proved to be crucial in this respect.", "It should also be stressed that the use of pictograms to increase understanding of choice attributes is not undisputed.", "While such pictograms ease the cognitive burden for respondents, they may cause emotional reactions in the respondent that might bias choices but are unobservable to the analyst.", "In terms of marine management, the Dogger Bank provides a case study of a transnationally governed offshore MPA.", "Although there are likely to be additional cross-border benefits of consistent management of the MPAs by respective Member States, these were not assessed in the present survey but should be incorporated into future studies.", "Nevertheless results of studies such as this one can inform bio-economic models used to identify optimal locations for offshore wind farms or MPAs.", "For example, the model developed by Punt et al. (2009) determines the optimal location of a wind farm in the Dutch EEZ given local physical conditions and impact on ecosystem services but does not take into account the changes in values resulting from the ecological changes.", "Börger et al. (2014) have argued that valuation is indispensable for the incorporation of ecosystem services into such marine planning efforts as required by recent legislation, such as the EU Marine Strategy Framework Directive (Bertram et al., 2014) or in a less explicit manner by the US National Ocean Policy (NOC, 2013).", "The survey clearly identifies the conservation benefits from an offshore MPA for the general public.", "Through the use of a DCE, the present study has significance beyond this particular case.", "It demonstrates the applicability of stated preference methods to inform detailed policy-making for offshore sites.", "Despite limited prior knowledge about the study site, survey respondents are able to develop and express preferences on specific attributes based on the information provided in the survey.", "Studies of this kind can be employed to assess the attainment of potential benefits from marine management by the general public, a group beyond the immediate stakeholders and direct marine user communities.", "In negotiations about marine management plans, valuations can inform negotiations by providing the wider societal perspective and, thus, help to establish priorities among conflicting management goals." ]	[]
Clustering of commercial fish sauce products based on an e-panel technique	Fish sauce is a brownish liquid seasoning with a characteristic flavor that is produced in Asian countries and limited areas of Europe. The types of fish and shellfish and fermentation process used in its production depend on the region from which it derives. Variations in ingredients and fermentation procedures yield end products with different smells, tastes, and colors. For this data article, we employed an electronic panel (e-panel) technique including an electronic nose (e-nose), electronic tongue (e-tongue), and electronic eye (e-eye), in which smell, taste, and color are evaluated by sensors instead of the human nose, tongue, and eye to avoid subjective error. The presented data comprise clustering of 46 commercially available fish sauce products based separate e-nose, e-tongue, and e-eye test results. Sensory intensity data from the e-nose, e-tongue, and e-eye were separately classified by cluster analysis and are shown in dendrograms. The hierarchical cluster analysis indicates major three groups on e-nose and e-tongue data, and major four groups on e-eye data.	[ "MULT" ]	[]	[ "1000" ]	[ "Fish sauce is produced from seafood of various types.", "Table 1 displays the geographic origins and ingredients of the commercially available fish sauce products analyzed.", "Anchovy and sardine appear to be widely used in fish sauce production in Thailand and featured in fish sauce products from Vietnam, China, and Italy.", "A fish sauce product from the Philippines contained mackerel.", "Flying fish, which is often used as dashi, a broth employed in various dishes in Japan (especially in the Kyushu area), was found in several Japanese fish sauce products.", "Bonito is also employed for dashi preparation throughout Japan, and was also present in many of the fish sauce products tested from this country.", "In addition, Japanese sandfish, tuna, cod, sea bream, cutlassfish, deep-sea smelt, sea urchin, oyster, shrimp, and squid were found to be used in the manufacture of fish sauce products in Japan.", "Commercial fish sauce products from Japan, Thailand, Vietnam, China, the Philippines, and Italy clustered into three major groups when the e-nose data were analyzed (Fig. 1).", "Cluster analysis of the e-tongue data also identified three groups (Fig. 2), whereas that of the e-eye data yielded four major groups (Fig. 3).", "Fish sauce possesses distinctive smells, tastes and colors depending on the ingredient fish, fermentation process, geographic origins.", "Here we analyzed these features of fish sauces from Asian countries as well as Italian products.", "To avoid the effects of subjective assessment, we used an e-nose, e-tongue, and e-eye to precisely analyze the smell, taste, and color of fish sauce products from several countries.", "The e-nose and e-tongue consist of arrays of non-selective gas and liquid sensors with broad and partially overlapping selectivity towards the compounds present in a sample [1].", "In addition, an e-eye was used to distinguish color components using camera-equipped apparatus and computer-assisted analysis.", "Of the commercial fish sauce products selected, 30 were produced in Japan, 11 were from Thailand, two derived from Vietnam, and one was from each of China, the Philippines, and Italy (Table 1).", "The list of numerical data indicating signal intensities on e-nose, e-tongue and e-eye sensors was brought to hierarchical cluster analysis to classify the fish sauces based on their smell, taste and color.", "All of the 46 fish sauce products were purchased at local markets in Tokyo, Fukuoka, and Abashiri (Japan).", "The ingredients of each product as listed on their labels are summarized in Table 1.", "For analysis, the products were assigned product IDs as follows: J1–J30 for the Japanese products; T1–T11 for the Thai products; V1 and V2 for the Vietnamese products; and P1, C1, and I1 for the Filipino, Chinese, and Italian products, respectively.", "E-nose analysis was performed with the αFOX 4000 smell analysis system (Alpha M.O.S., Toulouse, France), which has 18 metal oxide gas sensors for different selectivity patterns.", "Fish sauce product samples (0.5 g) were collected in 10-ml vials, placed in an autosampler, and analyzed under the following conditions: syringe temperature, 50 °C; oven temperature, 40 °C; injection speed, 2 ml/s.", "The analyzed data was obtained as numerical values of signal intensities.", "E-tongue analysis was performed with the αASTREE taste analysis system (Alpha M.O.S.), which has seven liquid potentiometric sensors (SRS, GPS, STS, UMS, SPS, SWS, and BRS) and a reference electrode (Ag/AgCl).", "Fish sauce products diluted 21-fold were collected in a beaker and placed in an autosampler.", "Each sensor was immersed in the samples for 120 s at 20–25 °C with agitation to elicit a sensor response.", "A stable response value was then recorded at 120 s.", "The analyzed data was obtained as numerical values of signal intensities.", "E-eye analysis of fish sauce products was carried out using an IRIS VA300 visual analyzer (Alpha M.O.S.) with a charge-coupled device camera.", "Five milliliters of fish sauce product was collected in a transparent plastic dish and placed in the measurement chamber.", "The color of the surface of each sample was measured three times.", "The collected color data were represented by IRIS color codes, which encompass 4096 colors.", "The analyzed data was obtained as numerical values of signal intensities.", "Hierarchical cluster analysis was used to classify the fish sauces based on the smells, tastes and colors analyzed by e-nose, e-tongue and e-eye sensor sets.", "The datasets, comprising a series of sensor values from the e-nose, e-tongue, and e-eye tests, were subjected to cluster analysis by Ward's method using the program R-3.4.2 (http://www.R-project.org) [2]." ]	[]
Bounce regime of droplet collisions: A molecular dynamics study	Droplet collisions have complex dynamics, which can lead to many different regimes of outcomes. The head-on collision and bounce back regime has been observed in previous experiments but numerical simulations using macro- or mesoscale approaches have difficulties reproducing the phenomena, because the interfacial regions are not well resolved. Previous molecular dynamics (MD) simulations have not reproduced the bounce regime either but have reported the coalescence and/or shattering regimes. To scrutinize the dynamics and mechanisms of binary collisions especially the interfacial regions, head-on collision processes of two identical nano-droplets with various impact velocities both in vacuum and in an ambient of nitrogen gas are investigated by MD simulations. With the right combination of the impact velocity and ambient pressure, the head-on collision and bounce back phenomenon is successfully reproduced. The bounce phenomena are mainly attributed to the “cushion effect” of the in-between nitrogen molecules and evaporated water molecules from the two nano-droplets. The analysis has verified and also extended the current gas film theory for the bounce regime through including the effects of evaporated water molecules (vapour). Some similarities and some dissimilarities between nanoscale and macro-/meso-/microscale droplet collisions have been observed. The study provides unprecedented insight into the interfacial regions between two colliding droplets.	[ "COMP", "MATH" ]	[ "Binary droplet collision", "Droplet bounce", "Molecular dynamics", "Multiphase flow", "Nanoscale" ]	[ "1700", "2611", "2614" ]	[ "Droplet collisions are encountered in both natural and industrial processes, such as, in the formation of clouds and rain drops [6], in the operation of nuclear reactors and in the process of spraying [5].", "In recent decades, efforts have been made to unveil the fundamental mechanisms associated with coalescence and bounce of two identical droplets.", "Experimental studies have identified that under different atmospheric conditions most of head-on liquid-gas droplet collisions can be described using three distinct regimes: coalescence, bounce and reflexive separation [1,2,9,30].", "Other studies using experimental and numerical methods such as the level set, volume of fluid, immersed boundary method, lattice Boltzmann method have already presented various phenomena of droplet collisions [4,7,14,17–19].", "Even so, details of the collision process are difficult to obtain through experiments and continuum simulation methods, especially when the approaching droplets are within a distance comparable to the molecular mean free path.", "It should be noted too that the lattice Boltzmann method has been successful in reproducing all the collisions regimes except the head-on collision and bouncing regime [18].", "Thus, computer simulations considering atomic effects become the only method to understand droplet dynamics when the separation distance comes within nanoscale.", "Yet up to the present time, the published simulations have only reproduced coalescence and shattering regimes [8,11,12,16,22,26,31] but not the bounce regime.", "Such an absence is intriguing as it suggests the possibility that the bounce regime may not exist in nano-droplet collisions while it was observed in micro- and macro-droplet collisions.", "Our study has, however, successfully observed the bounce behaviours with two nanoscale water droplets, thereby completing the regime map of the nano-droplet collision phenomena.", "In this research, molecular dynamics (MD) simulations of droplet collisions in vacuum and in ambient gas have been conducted to reproduce the bounce behaviours of nano-droplets.", "Our research will demonstrate that bounce frequently occurs in nanoscale droplet collisions.", "Such occurrences are influenced by parameters including pressure and impact velocity.", "Moreover, a “cushion effect” will be proposed to reveal the impact of the ambient gas and vaporized water molecules on droplet behaviours during the bounce process.", "Fig. 1 illustrates the initial setup of the simulation system with two nano-droplets submerged in the nitrogen gas.", "Equilibrium simulations of droplets and ambient gases were conducted before the collision simulations.", "For droplets, the equilibrium was undertaken in vacuum at the temperature of 300 K, and the canonical ensemble (NVT) was employed.", "After equilibrium of 2 ns, a single droplet can be generated by removing all the vaporized molecules around.", "The second droplet was obtained by duplicating the first one.", "Meanwhile, ambient nitrogen molecules were also equilibrated at 300 K in a parallel equilibrium simulation.", "After equilibrium, the two droplets were first placed in the required positions, and then equilibrated ambient gas molecules were added to fill the remaining space of the box.", "A further simulation was conducted for 2 ns to equilibrate the final system consisting of both droplets in ambient gas molecules.", "In the formal collision simulations, each droplet was assigned the same impact velocity along the x direction.", "Droplet collisions were first simulated in vacuum with impact velocities varying in a broad range from 5 to 500 m/s.", "Results have shown that the binary droplet collisions in vacuum follow either coalescence regime or shattering regime regardless of how the impact velocities vary, which is consistent with previous studies [13,20].", "However, a bounce regime could never be found under the vacuum condition.", "Binary collisions were then simulated with the existence of the ambient gas.", "With the impact velocity 8 m/s (v = 8 m/s) and the ambient pressure 2.7 atm (p = 2.7 atm), we found that the binary collision fell within the bounce regime (Fig. 2b and c).", "The whole process consists of three key stages.", "At the very beginning (t = 0–2.8 ns), the two droplets approach each other with very small deformation, whilst a small number of water molecules escape from the two droplets.", "The second stage (t = 2.8–4.2 ns) is reached when the distance between the two droplets reaches the minimum, where the droplets appear to be stationary.", "At this stage, the inter-droplet distance remains almost constant, whilst water molecules continue to escape from both droplets.", "Moreover, some of the water molecules escaping from one droplet are absorbed by the other droplet.", "The last stage (t = 4.2–5.6 ns) – the bouncing stage – occurs when an increase in the inter-droplet distance is clearly observed.", "Interestingly, the droplets are found to separate from each other along routes that deviate from the trajectories of the approaching droplets.", "Moreover, the separation velocity of the two droplets is detected to be smaller than the impact speed (v = 8 m/s).", "The deviating routes are attributed to the interactions between the water and nitrogen molecules, which are not entirely deterministic.", "The smaller separation velocity (as compared with the impact velocity) is due to the momentum loss through the escaping molecules as well as interactions with the ambient gas molecules.", "As for the cause for bouncing only in the presence of ambient gases, the theory of “gas film” is a popular explanation for droplet dynamics at microscale.", "Qian and Law [25] stated that the ambient gases form a gas film between the two droplets as the inter-droplet distance becomes tiny in the approaching stage.", "As the gap between the droplets decreases, the gas film pressure increases dramatically, leading the droplets to bounce back.", "Our results appear to support this physical picture, but at nanoscale.", "Furthermore, we have extended the “gas film” theory, which will be shown in detail in the following discussion.", "The first step is to scrutinize the composition of the gas film.", "To distinguish whether a water molecule belongs to the parent droplet or has evaporated, the surface of the droplet should be defined.", "However, at the molecular scale, a nano-scale droplet has no well-defined surface as its macro-scale counterpart usually does.", "Thus, a criterion has been proposed to definite the surface.", "For two random molecules in the simulation box, if their distance (rij) is smaller than a critical value (Rcl), the two molecules are regarded in the same droplet.", "The droplet is defined as a set of molecules, each of which is within the Rcl from one or more molecules in the droplet Powles [23] had applied this method and suggested Rcl = 1.9σ.", "Any water molecule can be regarded exactly as an evaporated molecule or an intra-molecule of the droplet with this method.", "Using the artificial surface method, the instantaneous numbers of nitrogen and evaporated water molecules in the gas film between the two droplets have been counted and shown in Fig. 3.", "The gap which is the rectangular region between the two droplets can be defined by the position of molecules in the surface in all the directions.", "For example, at time = 0 ns, the two droplets with diameters of 10 nm were placed along the x direction at the central part of the box with x coordinates being 200 Å and 600 Å.", "The 300 Å × 100 Å × 100 Å rectangular region between the two droplets is where the 225 nitrogen molecules in.", "In Fig. 3, when the two droplets are approaching each other (t = 0–2.8 ns), the number of nitrogen molecules between the two droplets decreases, which means that the nitrogen molecules are squeezed out of the space between the droplets.", "Meanwhile, an increasing number of evaporated water molecules occupies the in-between space.", "Interjacent molecules between the droplets therefore constitute both the nitrogen molecules and the evaporated water molecules.", "In the second stage (from 2.8 ns onwards), the two droplets have slowed down significantly, and the number of interjacent nitrogen molecules stops decreasing, but more water molecules continue to evaporate to fill the in-between space.", "As evaporating water molecules further reduce the momentum of the droplets, the droplets are not able to overcome the gas film and consequently come to an almost standstill (t = 2.8–4.2 ns).", "The process resembles droplets hitting a cushion, which can be called the “cushion effect” of the interjacent molecules.", "Afterwards, momentum exchange between the two droplets through the interjacent gases leads to the bounce back of the two droplets, which is aided by the continuing evaporation of water molecules from the droplet surfaces.", "In our research, ambient pressure and impact velocity of droplets are two key parameters which may influence the droplet behaviours.", "To scrutinize their effects on the bounce regime, simulations with different ambient pressure and impact velocity have been conducted.", "As mentioned previously, coalescence happens (Fig. 2a) when two droplets collide in vacuum which is an extreme case of decreasing ambient pressure.", "When the ambient pressure increases to 8 atm, the bounce regime occurs as expected.", "Compared with the lower pressure case (p = 2.7 atm) in which the minimum distance between the two droplet frontiers is 141 Å, the higher pressure causes a larger droplet frontier distance (155 Å), as shown in Fig. 4.", "The number of water molecules in each droplet at pressures of 2.7 atm and 8 atm are also recorded and shown in Fig. 5.", "Obviously, a higher pressure inhibits the evaporation of water molecules.", "In fact, higher pressure contributes to a more compact layout of molecules and shorter inter-molecules distances, and thus stronger atomic or molecular interactions.", "In the meantime, when pressure is higher, nitrogen molecules are more densely distributed and more nitrogen molecules stay in the in-between space.", "The gas film would feel more “stiff” less “elastic”.", "Consequently, droplets would find it more difficult to approach each other and the minimum distance between the two droplet frontiers increases.", "When the impact velocity increases to 15 m/s, coalescence, instead of bounce, of the two droplets can be observed (Fig. 6a).", "Analogous to the vacuum low-impact-velocity collision simulation (Fig. 2a) and experimental observation [28,29], the two droplets rapidly approach each other before they finally merge into a large droplet (t = 1.6 ns).", "During the approaching process, the two droplets dramatically decrease the in-between space for accommodating nitrogen molecules and evaporated water molecules.", "Thus, “cushion effect” of the gas film is not strong enough and the two droplets combine into a large one.", "These results indicate that the bounce regime occurs as a balance between the competing factors of impact velocity and pressure, leading to different levels of the “cushion effect”.", "Meanwhile, another case in which two droplets in an ambient of nitrogen gas are placed at the original positions without impact velocities (that is, v = 0) has been simulated (Fig. 6b).", "Several cases have been considered for ambient pressures p = 0, 1 atm, 2.7 atm and 8 atm, respectively (with the corresponding nitrogen densities of 0, 1.138 kg/m3, 3.074 kg/m3, and 9.116 kg/m3).", "The two droplets oscillate in all three directions with a maximum offset of 12 Å.", "At this separation distance, the van der Waals attractive forces are not effective while there are no bulk velocities to move the droplets in any specific directions.", "Instead, the oscillating positions of the nano-droplets reflect the random interactions between the in-between nitrogen molecules and the water vapour.", "These results have unveiled different behaviours of nano droplets from their macro or micro counterparts which tend to remain stationary without an impact velocity.", "Both the higher ambient pressure and the lower impact velocity have been seen to promote bounce back, which can be attributed to the stronger interactions between interjacent molecules and two moving droplets.", "In vacuum, interjacent molecules only comprise water molecules evaporated from the droplets.", "The number of evaporated water molecules is too limited to push the droplets back, which allows further approaching, contacting and coalescence of the droplets.", "This kind of coalescence in vacuum at low impact velocities has been corroborated by previous studies [28,29].", "When nitrogen molecules are introduced to provide an ambient pressure, interjacent molecules include both nitrogen and water molecules.", "The interactions between interjacent molecules and moving or stationary droplets become large enough to prevent coalescence, thereby enabling bounce to occur.", "When the impact velocity exceeds a certain threshold, however, the large momentum of the two moving droplets would overcome the “cushion effect” of the interjacent molecules, get into contact and coalesce.", "The above zero impact velocity simulation indicates that nanoscale collisions have different behaviours from their macro- or micro-counterparts.", "The macroscale collision regimes successively contain stationary (v = 0), coalescence, bounce, coalescence and separation as the impact velocity increases [27].", "The nanoscale collision regimes include approaching (v = 0), bounce, coalescence, shattering (in our follow-on paper) and possibly other regimes as the impact velocity increases.", "More detailed analysis of these issues are beyond the present paper and will be examined in future studies.", "MD simulations of droplet collisions in vacuum and in ambient gas have been conducted to scrutinize the dynamics and mechanisms of binary collisions at molecular scale.", "The head-on collision and bounce phenomenon is successfully reproduced for the first time.", "The bounce phenomena are mainly attributed to the “cushion effect” of the in-between nitrogen molecules and evaporating water molecules from the two nano-droplets.", "The study has verified and extended the current gas film theory for the bounce regime by including the additional effects of evaporating water droplets.", "Moreover, the MD results show that low impact velocity and high ambient pressure promote bouncing.", "The behaviours of nanoscale droplet collisions have both similarities and dissimilarities to macro-/micro-droplet collisions.", "Finally, a set of regimes for nano-scale droplet collisions has been identified, but further details such as regime boundaries require extensive further investigations, which will be pursued in our future studies." ]	[ "Reproduced the binary collision bounce regime in nanoscale by MD simulation.", "Extended the gas film theory for bounce regime.", "Considered impact velocity and ambient pressure as two key parameters affecting the appearance of the bounce regime.", "Discussed the differences between nanoscale and microscale droplet dynamics.", "Completed the regime map of nanoscale binary droplet collisions." ]
Seed storage and germination in Kumara plicatilis, a tree aloe endemic to mountain fynbos in the Boland, south-western Cape, South Africa	Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at - 80. °C, 4. °C, 25. °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at - 80. °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0-90.4%). The highest percentage germination overall was for seeds stored at - 80. °C for four months (90.4%) and the lowest was for seeds kept at 4. °C and - 80. °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation. © 2014 South African Association of Botanists.	[ "AGRI" ]	[ "Aloe", "Dormancy", "Ex situ conservation", "Fynbos", "Germination", "Seeds", "Storage", "Temperature", "Viability" ]	[ "1110" ]	[ "Seed storage is vital for the long-term preservation of germplasm, and the maintenance of genetic diversity for the conservation of threatened and commercially important plant species, which has become a global concern over recent decades (Bonner, 1990; Vertucci and Roos, 1990).", "During storage, all seeds undergo deterioration, the rate of which is dependent on storage temperature, seed moisture content and the species concerned (Tang et al., 1999).", "Hence, germination tests are important for determining the viability of a seed lot, the efficacy of a particular pre-sowing treatment (e.g. storage), or the number of seedlings that may be expected from sowing a certain number of seeds in the field or nursery (Czabator, 1962).", "Seed germination may be immediate, but usually there is a delay, either as a result of quiescence, or dormancy, which may be morphological, physical, or physiological (Fenner and Thompson, 2006).", "Quiescent seeds will germinate as soon as conditions conducive to germination are present, while dormant seeds preclude germination when conditions are favourable, but the probability of seedling survival and growth is low (Fenner and Thompson, 2006; Finch-Savage and Leubner-Metzger, 2006).", "Hence, germination of dormant seeds occurs only when conditions for establishing a new plant generation are likely to be suitable (Fenner and Thompson, 2006; Finch-Savage and Leubner-Metzger, 2006).", "During dry storage, seeds of certain species undergo physiological changes in a process known as dry after-ripening, which is often reflected in a decline in the level of innate dormancy, and decreased specificity of germination requirements (Probert, 2000).", "Importantly, for species adapted to regions of seasonal drought and dry soils, physiological changes recorded during dry storage reflect a natural mechanism, which governs the timing of germination in the wild (Probert, 2000).", "Thus, studying a species' dormancy and germination characteristics following seed storage, may provide insight into its regeneration strategies in situ.", "The Alooideae is the largest subfamily in the Asphodelaceae, a family of succulent-leaved, petaloid monocots, primarily found on the African continent (Smith and Van Wyk, 2009).", "The subfamily comprises the genus Aloe (± 350 species), the newly established genera Aloidendron (five species) and Aloeampelos (six species) and the reinstated monotypic genus Kumara (Grace et al., 2013), collectively termed ‘aloes’ herein.", "With approximately 140 aloe taxa, South Africa has the highest aloe species richness in Africa (Klopper et al., 2009).", "Aloes flower primarily during winter and produce large quantities of small, double-winged, air-borne seeds (Smith and Van Wyk, 2009).", "Although there has been a recent increase in research on aloe seed biology and ecology (see Bairu et al., 2009; Mukonyi et al., 2011; Symes, 2012; Arena et al., 2013; Cousins et al., 2013; Kulkarni et al., 2013), knowledge on the subject, including dispersal, germination, seed bank dynamics, and especially dormancy, longevity and storage, is still limited (Cousins and Witkowski, 2012).", "Hence, in an effort to build on our currently narrow understanding of aloe seed biology, this study investigates the effects of various seed storage treatments on the viability and germination behaviour of the seeds of Kumara plicatilis, a charismatic tree aloe endemic to mountain fynbos in the Boland, south-western Cape.", "K. plicatilis (L.) G.D. Rowley (Asphodelaceae: Alooideae), recently segregated from Aloe s.s. (Daru et al., 2013; Grace et al., 2013; Rowley, 2013), is one of six tree aloes indigenous to South Africa, and the only one that occurs in the Cape fynbos (Van Wyk and Smith, 2008).", "K. plicatilis has a restricted distribution in mountainous parts of the Boland in the south-western Cape.", "It occurs from the Groot Winterhoek Mountains near Tulbagh in the north to the Franschhoek Mountains in the south.", "From east to west it occurs in the Du Toit's Kloof Mountains near Rawsonville/Worcester to the Paardeberg between Malmesbury and Wellington.", "K. plicatilis grows in well-drained, acidic soils on steep rocky slopes and outcrops in winter rainfall areas (400–2000 mm/year) (Cousins et al., 2014).", "The species is relatively long-lived and slow-growing, reaching a maximum height of 5 m, with dichotomously branching stems, each ending in a set of 12–16 strap-shaped succulent leaves.", "(Fig. 1) (Van Wyk and Smith, 2008).", "Its flowering season is from August to early November, with dispersal of the small, double-winged, wind-dispersed seeds commencing in December/January (Van Wyk and Smith, 2008).", "Fresh seeds were collected from 2 to 3 inflorescences on 40 K. plicatilis individuals at a population near Rawsonville/Worcester in December 2010.", "The seeds were initially kept under ambient conditions in a laboratory in brown paper bags for three months.", "All the seeds were sorted by hand to separate intact from empty seeds.", "Only the intact and therefore potentially viable seeds were used for the experiment.", "Initial germinability of the seeds was determined using 300 seeds, the results of which are presented in Cousins et al. (2013).", "Two-thousand-four-hundred seeds from the original batch were then separated into eight sets of 300.", "The seeds were placed into 20 ml centrifuge vials, 20 seeds/vial, giving a total of 15 vials for each set.", "The vials were placed in sealed brown paper bags to maintain dark conditions for the duration of the storage period.", "The eight seed sets were spilt into two groups of four: one group to be stored for four months (4-month stored seed henceforth) and the other for nine months (9-month stored seed).", "A set from each of the two groups of four was then stored using the following four temperature treatments: (a) in a freezer at − 80 °C, (b) in a cold room at 4 °C, (c) in an incubator at 25 °C and (d) under ambient conditions (typical December to February (summer) temperatures and humidity) in a laboratory at the University of the Witwatersrand, Johannesburg.", "Seed water content was calculated on a fresh mass basis pre-storage using 100 fresh seeds, and again after four and nine months of storage using 50 seeds/treatment.", "The samples of 50 seeds were taken from each set of 300 seeds, leaving 250 seeds/treatment for the germination trials.", "Seeds were weighed on a Precisa 92SM-202A scale correct to 0.0001 g, then oven-dried at 80 °C for 24 h and reweighed to determine their water content.", "Seeds were germinated in a phytotron chamber with a 12 hour day/night cycle and light provided by fluorescent bulbs at ~ 650 nm.", "Temperatures were set at 25 °C during the day and 15 °C at night and relative humidity at a constant 50%.", "For each treatment, the 250 seeds were divided equally among ten 90 mm Petri dishes (25 seeds/dish), with two layers of sterilized filter paper under them and one on top.", "They were supplied with sterile distilled water until the filter paper was saturated.", "The germination trials were conducted for 18 weeks for all four treatments and for both storage durations.", "Monitoring for germination took place once a week, and watering twice a week.", "Germinated seeds were removed from the dishes and planted in seedling trays to be grown for ex situ conservation.", "Seeds that had not germinated by the end of the trials were tested for viability using standard tetrazolium tests, with a 1% solution of 2,3,5-Triphenyl-tetrazolium chloride salt under ambient, dark conditions for 24 h (Savonen, 1999).", "The peak value (PV) and germination value (GV) were calculated according to the methods described in Czabator (1962).", "To obtain the PV, which is an index of seed vigour, the T value (percentage germination) / (number of weeks) is calculated for each week, and the highest T value is equal to the PV.", "The GV is then calculated as (PV) × (mean weekly germination), and gives a composite index of percentage germination and germination rate (germination index).", "Mean weeks to germination was calculated as another indicator of seed vigour.", "A one-way ANOVA tested for the difference between the water content of stored seeds and initial water content, while two-way ANOVAs tested for an interaction between storage duration and temperature treatments for (a) seed water content and (b) mean weeks to germination.", "Chi-square contingency tables were used for 4-month and 9-month stored seeds to test for associations for (a) percentage germination and (b) post-storage viability (germinated plus ungerminated viable seeds).", "There were no differences between the water content of stored seeds and the initial water content of 6.91 ± 0.18% (mean ± S.E.) except for 9-month stored seeds kept at 25 °C, which were significantly lower (4.63 ± 0.60%) (F4, 294 = 4.51, p = 0.0014) (Table 1).", "There was no interaction between storage temperature and duration for seed water content (F3, 392 = 0.537; p = 0.657), however, the water content of 9-month stored seed at 25 °C was significantly lower than that of 4-month stored seed at 4 °C (p = 0.015) (Table 1).", "Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks), while the slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks).", "For percentage germination, there was an association between length of seed storage (4-months versus 9-months) and storage treatment, with decreased germination for the − 80 °C and 4 °C treatments after 9 months compared with those stored for only 4 months (χ23 = 60.56; p < 0.001).", "Percentage germination was comparable across the four treatments for 4-month stored seed (78.0–90.4%) (Fig. 2a).", "Within the four-month stored seed lot, seeds kept at 4 °C had the lowest GV due to a slow germination rate (Table 2, Fig. 2a).", "Those kept at ambient and 25 °C had similar GVs and shared the same PV, while the − 80 °C seeds had the highest GV, since they had the highest germination rate and percentage germination of all the 4-month stored seeds (Table 2, Fig. 2a).", "The GV and PV of the 9-month stored seeds at ambient and 25 °C were substantially higher than those at 4 °C and − 80 °C owing to a greater germination rate and percentage (Table 2, Fig. 2b).", "The highest GV and PV overall were for 9-month stored seeds under ambient conditions (97 and 19.6 respectively) (Table 2).", "Percentage germination of 4-month stored seeds at − 80 °C was the highest overall (90.4%) — in sharp contrast to seeds stored at − 80 °C for nine months.", "This treatment shared the lowest overall percentage germination (39%) with 9-month stored seeds at 4 °C (Fig. 2).", "Despite the low percentage germination of 9-month stored seeds kept at 4 °C and − 80 °C, 82% and 87% of the ungerminated seeds respectively, were still viable as demonstrated by tetrazolium salt staining (Fig. 3).", "Overall, there was no difference in post-storage total viability (as shown by germination and tetrazolium tests combined) of 4-month and 9-month stored seeds (χ23 = 0.507, p = 0.917), which ranged from 89% to 98% across the treatments (Fig. 3).", "The most important factors that influence extended seed longevity during ex situ storage are seed moisture content and storage temperature (Hartmann et al., 1997).", "The low water content of K. plicatilis seeds (6.91%) indicates that they are orthodox seeds, and can therefore probably be stored for long periods at sub-zero temperatures (Bonner, 1990).", "The initial germinability of the seed lot as determined by Cousins et al. (2013) indicated that an after-ripening period is required before K. plicatilis seeds will germinate, since after six weeks of germination trials, only 28% of 3-month-old seeds stored under ambient conditions germinated, while ~ 80% of the remaining ungerminated seeds were still viable.", "By seven months of storage under ambient conditions (the initial three months, plus the four months reported here) 81.6% of seeds germinated.", "This after-ripening period (between three and seven months) likely corresponds with the months of summer drought that seeds in the wild must endure directly post-dispersal (December–April).", "The end of the after-ripening period therefore coincides with the onset of the winter rains, when conditions for germination and establishment are more favourable.", "Short-term storage of K. plicatilis seeds at all four temperature treatments appeared to have very little impact on seed vigour and viability, as germination rate and total germination were high for all four, and almost all the ungerminated seeds were viable.", "A striking result was that seeds stored at the coldest temperature (− 80 °C) for four months showed the highest germination rate and percentage overall, but those stored at the same temperature for a further five months displayed the exact opposite trend.", "Storage under cold conditions (both 4 °C and − 80 °C) for nine months had similar effects on germination rate and percentage, and appeared to induce dormancy in the majority of seeds (~ 60%).", "It is most likely that the K. plicatilis seeds entered physiological dormancy, whereby an inhibiting mechanism of the embryos prevented radicle emergence (Baskin and Baskin, 1998).", "While it is widely known that chilling can be used to break dormancy, particularly among species adapted for spring germination, there is considerable evidence that low temperatures can also result in the induction of dormancy (Probert, 2000).", "Furthermore, within a single seed population, genetic differences in the depth of dormancy between individual seeds can result in differential effects of chilling (Probert, 2000).", "The germination of a small proportion of K. plicatilis seeds after extended cold storage may have been a result of such genetic differences.", "Extended cold storage of A. plicatilis seeds may have acted as an environmental stressor, and triggered a response to a reduction in the probability of the arrival of conditions favourable to germination and establishment.", "Since reduced temperature invariably lengthens the storage life of seeds (Hartmann et al., 1997), by entering dormancy, the aloe seeds would extend their longevity, and thereby improve their chances of survival after release from cold storage.", "Kulkarni et al. (2013) showed that treatments of 10-month-old Aloe arborescens seeds with smoke water (1:500 v/v) and smoke-isolated KAR1 (10− 8 and 10− 9 M) significantly increased percentage germination in comparison with the untreated control seeds when incubated under a 16 h photoperiod at 25 °C.", "Bairu et al. (2009) obtained similar results for Aloe ferox using smoke-water-treated seeds germinated under the same conditions.", "Although the effect of smoke water treatment on the germination of K. plicatilis seeds was not tested, the high percentage germination (76–92%) in its absence suggests that smoke is unlikely to act as a germination cue in the species.", "This hypothesis ties in with a total absence of recruitment at a population 22 months post-fire reported by Cousins et al. (2013), suggesting that mass post-fire recruitment (which is common in many other fynbos plant species) as a general rule in K. plicatilis is unlikely.", "A more probable recruitment strategy involves the germination and establishment of small numbers of seedlings over time in the absence of major disturbances as suggested by Cousins et al. (2014).", "The high percentage germination achieved using seed stored under ambient conditions (80%) shows that K. plicatilis seeds germinate readily without any special treatment such as cold stratification, varying light conditions, pH modification and smoke water treatment.", "However, the higher germination rate and percentage after storage at − 80 °C suggests that chilling enhances germination in the species.", "Further studies on long-term storage, germination cues and dormancy breaking mechanisms in K. plicatilis seeds, and those of other taxa in the Alooideae, would be beneficial for developing long-term seed storage protocols for the family Alooideae for ex situ conservation." ]	[ "We studied the viability & germination of stored Kumara plicatilis seeds.", "Seed viability was not reduced after storage at 25 °C, 4 °C, − 80 °C & ambient.", "All seeds showed similar percentage germination after four months of storage (78–90%).", "Lowest percentage germination was for seeds kept at 4 °C & − 80 °C for nine months.", "High viability of ungerminated seeds kept at 4 °C & − 80 °C indicates dormancy induction." ]
Personal name in Igbo Culture: A dataset on randomly selected personal names and their statistical analysis	This data article contains the statistical analysis of Igbo personal names and a sample of randomly selected of such names. This was presented as the following: 1). A simple random sampling of some Igbo personal names and their respective gender associated with each name. 2). The distribution of the vowels, consonants and letters of alphabets of the personal names. 3). The distribution of name length. 4). The distribution of initial and terminal letters of Igbo personal names. The significance of the data was discussed.	[ "MULT" ]	[ "Distribution", "Igbo name", "Linguistics", "Onomastics", "Personal name", "Statistics" ]	[ "1000" ]	[ "The data contained in this article are listed as follows.", "The dataset of randomly selected Igbo personal names and their respective gender associated with each name, and the distribution of the name length.", "This data can be assessed as Supplementary data 1.", "Secondly, the distribution of the vowels, consonants and alphabets of the Igbo personal names was included in this data article.", "This data can be assessed as Supplementary data 2. Lastly, this data article contains the distribution of initial and terminal letters of Igbo personal names.", "In addition, tables showing the statistical analysis of the above listed datasets were also included.", "Personal names can be classified as given name, first name, middle name, forename, Christian name, local name or adopted name.", "These are opposite of last name, surname, family name or clan name.", "Igbo is one of the major tribes in Nigeria and the language is spoken by over 25 million and characterized by dialects.", "The Igbo people are originally from the eastern part of Nigeria but can be found in virtually every country of the world.", "Similar to any other ethnic groups in Africa, naming in Igbo is premeditated venture that is designed to speak to the future of the newly born child.", "Igbo people are not careless in naming because of their belief that names are tied to destinies and as such have religious, philosophical, psychological, historical, social and linguistic interpretations.", "Personal names in Igbo land are characterized by the following: 1).", "Names are clustered along the lines of dialects, largely because of geographical proximity, migration and historical ties.", "2).", "Sentential names are heavily been replaced with Pentecostal names.", "3).", "The influence of God called “Chi” or “Chukwu” is very strong in Igbo personal names.", "4).", "Superstitious beliefs also influence the naming system.", "5).", "Sociological effects such as procreation and the importance of children over barrenness, wealth, status, riches for example “Nwako”, caste system like “Osu”, “Umeh”, traditional post or monarchial lineage, for example; “Adaeze”, “Ezedinobi”, innuendo or response to mockery, childlessness or taunting for example “Iroahushi”, superiority of their siblings, clan or kingship or kinsmen over others, or their wealth, beauty, riches, sexual or intellectual abilities for example “Akubuilo”, “Ofunneka”.", "6).", "Igbo personal names are gender sensitive because of the patriarchal nature of Igbo people.", "The males are often named based on issues such as: gods or deities, physical and spiritual objects, intellectual prowess and dexterity in trade or agriculture, natural or mysterious phenomena, sportsmanship and craftsmanship, animals and so on.", "On the other hand, female names are often associated with good lineage, fruitfulness, beauty and intelligent, moral responsibility, favor, good luck and tidings, joy, happiness, wealth, purity and so on.", "7).", "Maternal lineage or descent can influence the personal names given to Igbo people.", "8).", "Historical or geographical events for example waterfall, market days, birth of prince or princess, disease outbreak, war, famine, draught, great harvest, fruitful period and so on.", "Numerous investigators have worked on various aspects of personal names and naming in Igbo but the actual distribution and frequency of the alphabets that made up each name have not been reported to the best of the knowledge of the authors.", "This research was as a result of rigorous research gaps observed from the works of numerous authors.", "Few of which are listed [3–48].", "The limitations of accessing the target population is compensated with a well-defined sample which must be a true representative of the studied population.", "See [49–67] for some selected survey research done to study some observed population attributes.", "Simple random sampling of some selected Igbo personal names yielded 965 names which are subsets of larger population.", "The samples were collected in such a way as to reflect the dialectal classification of Igbo people.", "The data was obtained from freely available textbooks, online baby name websites, oral interview, published articles and online discussion forums.", "Statistical analysis of the personal name (word) length of Igbo people are summarized in Table 1.", "This was done using simple statistical tools.", "On the average, a randomly selected Igbo personal name will have a word length of eight.", "The description can be done using histogram as shown in Fig. 1.", "It is most likely that the word length will be greater than eight as seen in the histogram (skewness).", "Igbo language is made up of 36 letters of the alphabets comprising of 8 vowels and 28 consonants.", "This is shown in Table 2.", "The research was restricted to 25 letters of the alphabets of the written form of Igbo language (Anglo-Igbo) version which is currently used in child registry, school registration, international passport and national identification and so on.", "The form is shown in Table 3.", "Excel command was used to determine the frequency of letters of alphabets of Igbo personal names.", "The command is: =SUMPRODUCT(LEN(A2) – LEN(SUBSTITUTE(A2, \"letter\", \"\"))).", "The result was presented along with their corresponding comparative ranks.", "This is shown in Table 4.", "The rank is from the most frequent to the least.", "Igbo language comprises of nine double letter consonants.", "These are shown in Table 5.", "Excel command was used to determine the frequency of double letter consonants of alphabets of Igbo personal names.", "The command is: =SUMPRODUCT(LEN(A2) – LEN(SUBSTITUTE(A2, \"letters\", \"\")))/2.", "The result was presented along with their corresponding comparative ranks.", "This is shown in Table 6.", "The high frequency of occurrence of “ch” is a pointer to the influence of God in the naming systems of Igbo people.", "This is because of the presence of “Chi”, Chukwu, Chuku in almost 50% of Igbo personal names.", "It should observed from Table 4 that the 5 vowels are rank first to fifth and the consonants are ranked after that.", "However this can be clearly seen in the histogram.", "The histogram of the distributions of consonants and vowels of 965 randomly selected Igbo personal names are shown in Figs. 2 and 3.", "The total number of vowels and consonant and their respective percentages were shown in Table 7.", "There are a total of 8049 letters.", "On the average any random selection of Igbo personal name would likely comprised of 48% vowel and 52% consonant.", "Initial and terminal letters constitute a major component of the study of words, nouns, proper nouns and personal names.", "Excel command was used to determine the frequency of initial and terminal letters of Igbo personal names.", "The command for the initial letter is: =COUNTIF(A2: A966, “letter”).", "The command for the terminal letter is: =COUNTIF(A2: A966, “letter”).", "The result was presented along with their corresponding comparative ranks.", "This is shown in Table 8.", "Areas of similarity and differences and relationship of the initial and terminal letters can be obtained by further analysis and use of statistical methods like correlation and chi-square." ]	[]
Ionic liquid-based water treatment technologies for organic pollutants: Current status and future prospects of ionic liquid mediated technologies	Water scarcity motivated the scientific researcher to develop efficient technologies for the wastewater treatment for its reuse. Ionic liquids have been applied to many industrial and analytical separation processes, but their applications in the wastewater treatment, especially in the removal of organic pollutants, are still not well explored. Potential applications of ionic liquids include solvent extraction, solvent membrane technologies and ionic liquid-modified materials that are mainly used as adsorbents. Aforementioned technologies have been examined for the abatement of phenol, chloro- and nitrophenols, toluene, bisphenol A, phthalates, pesticides, dyes, and pharmaceuticals etc. Present review enlightens the application of different ionic liquids in wastewater treatment and suggests the versatility of ionic liquids in the development of rapid, effective and selective removal processes for the variety of organic pollutants. Implementation of ionic liquid based technologies for wastewater treatment have lots of challenges including the selection of non-hazardous ionic liquids, technological applications, high testing requirements for individual uses and scaling-up of the entire pollutant removal, disposal, and ionic liquid regeneration process. Toxicity assessment of water soluble ionic liquids (ILs) is the major issue due to the widespread application of ILs and hence more exposure of environment by ILs. The development of effective technologies for the recovery/treatment of wastewater contaminated with ILs is necessary from the environmental point of view. Furthermore, the cost factor is the major challenge associated with ionic liquid-based technologies.	[ "ENVI" ]	[ "Ionic liquid", "Liquid membrane", "Organic pollutant", "Solvent extraction", "Wastewater treatment" ]	[ "2304", "2305", "2310", "2311" ]	[ "Ionic liquids (ILs) have been applied to a huge range of industrial processes ever since the first IL was synthesized, 100 years ago (Walden, 1914).", "Generally, ionic liquids consist of organic cations (such as pyrrolidinium, imidazolium, and pyridinium and ammonium) and inorganic anions (for e.g. Chloride, bromide, tetrafluoroborate, and hexafluorophosphate) (Costa et al., 2017; Hashemi et al., 2018; Wang et al., 2016).", "At temperature below 100 °C, ILs behave as non-volatile liquids.", "Due to non-volatile nature, thermal stability, and null flammability, ILs are known as environmentally friendly solvents and considered as “designer solvents” due to limitless freedom to tailor their properties for specific applications (Sas et al., 2018; Yan et al., 2019; Zhu et al., 2019).", "ILs can be used for the selective recovery many organic pollutants such as dyes, amines, herbicide, toluene, PAHs, phenolic compounds (Zhu et al., 2019; Liu et al., 2005).", "By several combinations of anions and cations, the physicochemical properties of ILs can be easily tuned for task specific requirements.", "The miscibility of ionic liquid depends on the particular ions that constitute it.", "Anion plays a crucial role in the hydrophobicity of any ILs (Sulaiman et al., 2019).", "The hydrophobicity of ILs is highly dependent on the magnitude of the charge on ions and also the size of ions.", "Hydrophobic ILs can be used for the treatment of extracting pollutants from aqueous solutions.", "ILs can be classified according to their anionic or cationic counterparts.", "Fig. 1 represents the structure of some common anions and cations for ILs preparation.", "The first-generation hygroscopic ILs consisted of Cl− and Br− anions and organic cations such as 1-alkyl imidazolium (CnMIm], n = 4, 6, or 8) (Mallakpour and Dinari, 2012).", "With progress in ionic liquids study with time, halides have been replaced with more stable tetra- and hexafluoroborate (BF4−, BF6), hexafluorophosphate (PF6−), nitrate (NO3−), or sulfate (SO4−2).", "Further, ILs have been synthesized by using trifluoro methanesulfonate [CF3SO3]− and bis(trifluoro methanesulfonyl)imide [(CF3SO2)2N−] (or [NTf2]−) for having more hydrophobic anions.", "Based on the head group in the cation, ILs can be classified as organic salts with an aromatic (imidazolium, pyridinium, e.g.) or alicyclic (pyrrolidinium, e.g.) head group (Petkovic et al., 2011).", "Other important ILs are based on quaternary ammonium salts, such as cholinium (2-hydroxy ethyltrimethylammonium) and choline (cholinium chloride), and quaternary phosphonium salts.", "Due to high thermal stability, immiscibility in organic solvents and negligible volatility, ILs have been actively investigated in separation processes as a substitute of traditional organic solvents (Ho et al., 2011; Mallakpour and Dinari, 2012; Poole and Poole, 2010).", "Treatment of industrial wastewater is one of the most prospective fields of application for ILs.", "In general, separation and concentration of dissolved pollutants into a smaller liquid volume via extractive or adsorptive techniques offers specific advantages when compared with the direct decomposition of pollutants in the effluent (Sekar et al., 2012).", "Biological treatment systems need time and care for microbial growth and adaptation, they are sensitive to seasonal variations and toxicity of wastewater, and their capability for the treatment of recalcitrant organics and metals is limited.", "Chemical treatments, such as Advanced Oxidation Processes (AOPs), can be too non-specific and ineffective for some pollutants and wastewater types, leaving toxic residuals or intermediate products in the treated effluent, which could further lead secondary pollution due to degraded product and sometimes degraded product show more toxicity than the parent compound.", "In contrast, a rapid extractive process leaves several options for subsequent treatment: decomposition of pollutants in a concentrated liquid volume, recovery of valuable substances, or disposal of the reject.", "Feasibility of an extractive water treatment process depends on the technical design of the extraction system as well as the selection of the solvent.", "The first process stage, contact between solvent and solute, is influenced by the physical properties of the solvent, such as density, viscosity, vapor pressure, and miscibility with water, in combination with operational conditions (time, mixing, temperature, e.g.) and properties of the contactor device (size of the contact surface, solvent flow, e.g.).", "Partitioning of the pollutant into the extraction solvent is determined by physical and chemical solvent-solute interactions, thermodynamic feasibility of partitioning, and diffusivity of the solute.", "In the last process stages, such physical properties of the solvent that promote convenient phase separation and regeneration of the solvent, are valuable.", "In this review paper, a number of recent studies on IL-based extraction of organic pollutants from aqueous solutions have been compiled.", "Our aim is to assess the current status, prospects, limitations, and further R&D needs of ionic liquid- based water treatment technologies that could be used for the removal of organic pollutants from industrial process waters and effluents.", "IL-based technologies available for the industrial-scale extraction of aqueous feeds comprise two major types of processes.", "In liquid-liquid extraction (LLE), hydrophobic ILs are used similarly as conventional organic solvents.", "The hydrophobic IL phase settles below the less dense aqueous phase and it can be easily separated and recovered (Mallakpour and Dinari, 2012).", "If hydrophilic ILs are used, phase-separation promoters must be added to split the water-IL mixture into two immiscible aqueous phases.", "Several room-temperature ILs have been found to be capable of producing aqueous two-phase systems (ATPSs) (also referred to as aqueous biphasic systems, ABSs) that had earlier been established for different polymer–polymer combinations, polymer–salt combinations, or salt–salt combinations (Freire et al., 2012; Li et al., 2010).", "Aqueous IL-rich and salt-rich phases are formed above certain concentrations and phase volume ratios of water, IL, and salt such as K2HPO4/KH2PO4.", "An increase in salt concentration strengthens the water-salt interactions and an increase in IL concentration improves the contact and interaction between solute and IL, and higher partitioning of the solutes into the IL phase is observed (Sheikhian et al., 2014).", "One more alternative could be amphibilic IL-based surfactants.", "Variety of ILs has been applied for the separation of organic compounds from water and sediment samples for analytical purposes (Delgado et al., 2012; Pino et al., 2012, 2008).", "However, the dissolution of halide anions makes them impractical for water treatment purposes.", "Full-scale LLE systems based on conventional solvents are in industrial use.", "For example, in biotechnological processes, the solvent extraction method has been tested for the treatment/recovery of organic acids, amino acids, penicillin, and alcohols (Haan and Bosch, 2013).", "The extraction process may consist of several contacting stages for the feed and solvent.", "Feed/solvent contact can be improved by using different flow patterns and agitation in the LLE columns.", "Conventional organic solvents, such as toluene and dichloromethane have also been used for wastewater treatment.", "Treated effluents include wastewater streams from coal gasification (Yang et al., 2006), chemical manufacturing (Jiang et al., 2003; Chen et al., 2007), and dyeing processes (Elumalai et al., 2014; Muthuraman, 2011; Muthuraman et al., 2012, 2009).", "Solvent extraction suits for the removal of organic pollutants especially when the pollutant concentrations are high, as in the manufacturing of organic chemicals, and/or the pollutant has enough reuse value to offset the treatment costs (Scovazzo et al., 2006).", "The application of full-scale LLE systems based on ILs has not been well explored.", "This would require the adoption of the unit processes from conventional LLE systems and incorporation of IL-and feed-specific modifications due to different solvent-to-feed ratios, viscosities, volatilities, and solvent recovery systems.", "IL extraction studies on organic pollutants have mostly been carried out at miniature scale, using as little as a few microliters of IL on a few-milliliter water sample, which is appropriate for chemical analytics (Han and Row, 2010; Rezaee et al., 2010; Sarafraz-Yazdi and Amiri, 2010; Zaijun et al., 2011).", "Several reviews have documented impressive lists of environmental pollutants that have been successfully extracted by IL-based dispersed liquid-liquid microextraction (Han et al., 2012; Johansson and Johansson, 2013; Saraji and Boroujeni, 2014; Vičkačkaite and Padarauskas, 2012) The solvent-feed contact stage of microextraction cannot be scaled up to more realistic systems for water treatment, and full-scale applications is not available.", "Hence, we have summarized recent experiments that have been classified as solvent extractions (not as microextractions) with a focus on the removal of ubiquitous organic wastewater pollutants in Table 1 (McFarlane et al., 2005; J. Fan et al., 2008; Brinda Lakshmi et al., 2013; Khachatryan et al., 2005; Bhosale et al., 2015; Egorov et al., 2008; Zhang et al., 2010) and organic dyes in Table 2 (Chen et al., 2013; Ferreira et al., 2014; Lin et al., 2014; Pei et al., 2007a; Sheikhian et al., 2014; Talbi et al., 2014; Li et al., 2007; Kermanioryani et al., 2016).", "Optimization of the LLE process has obviously not been targeted by most of the researchers, but they have rather reported on comparative tests for different organics and ILs.", "The most promising technology seems to be the magnetic extraction system based on trihexyl tetradecyl phosphonium tetrachloroferrate(III) ([3C6PC14][FeCl4]) that can be substantially scaled up from the 6-mL lab tests (Deng et al., 2011).", "An increase in treat ratio had no substantial impact on the partitioning of chlorophenols until a sharp drop at treat ratios exceeding 120:1.", "Furthermore, a successful extraction of pentachlorophenol from contaminated soil suspension indicated that the method could have a potential for the treatment of real wastewater.", "The LLE system that worked well for the removal of phenol, nitrophenols, naphthols, and aromatic amines utilized quaternary ammonium–based ILs, tetrahexyl ammonium dihexyl sulfosuccinate (THADHSS) and trioctylmethyl ammonium salicylate (TOMAS) (Egorov et al., 2008).", "Generally, the D values were higher with THADHSS than TOMAS, and both ILs were very efficient extraction solvents for the investigated compounds than [C6MIm][Tf2N] and [C10MIm][Tf2N], or [C4MIm][PF6] in the study of (Khachatryan et al., 2005).", "Furthermore, the treat ratio that could be used for the quantitative extraction of 2,4-dichlorophenol was as high as 50:1, whereas the capacity of the imidazolium-based ILs was limited to a treat ratio of 3:1.", "In some studies, ILs were blended with conventional solvents or surfactants.", "With the addition of as little as 0.5% [BMIm][BF4] to tributyl phosphate, higher extraction efficiency for chlorophenols was obtained at a treat ratio of 7:1 than by using pure tributyl phosphate at a treat ratio of 1:1 (Brinda Lakshmi et al., 2013).", "At higher treat ratios, a saturation of the solvent became a limiting factor.", "A drop in the extraction percentage from 96 to around 60% was also noted with all the phenols as their concentrations in the feed solution increased from 10 to 50 mg/l.", "In another study, [C4MIm][PF6] was mixed with a surfactant Triton X-114 to extract methylene blue (Talbi et al., 2014).", "An association between methylene blue cations and IL anions neutralizes the ionic charge and allows better access of the dye-IL complex into the surfactant micelles.", "On the other hand, at alkaline pH, the neutral form of methylene blue predominates and becomes more easily solubilized by a non-ionic surfactant or a hydrophobic IL than at acidic pH. The extraction was accomplished above the cloud point, i.e., the temperature at which the surfactant forms two phases in aqueous solutions.", "Methylene blue was separated with the surfactant-rich phase, but a drawback would be residuals of the surfactant in the purified water phase.", "It was demonstrated that the complete phase separation can't be achieved in an ATPS.", "It is reported that the salt concentration range was observed around 1.5–3.5 mol/kg in the salt-rich phase.", "The molar fraction of water was >0.8 in the two phases in ATPSs (Li et al., 2010).", "Further, in another study, Sulaiman et al. investigated the solubility of different 3-chlorophenol, 2,5-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol in six hydrophobic bis(trifluoromethylsulfonyl)imide based ILs at different temperatures was investigated in hydrophobic ionic liquids and the found that solubility is highly dependent on ILs structure and it was demonstrated that dichlorophenol (DCP) has the highest solubility in pyridinium-based ILs and the lowest solubility in sulfonium- based ILs (Sulaiman et al., 2019).", "In another study, Sas et al. reported the efficiency of the hydrophobic ionic liquid, 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide, [C2mim]FSI, to extract o-cresol, 2-chlorophenol, resorcinol and phenol from water.", "They reported that ILs could be easily recovered by using NaOH solution (Sas et al., 2018).", "It was demonstrated that during interaction with Il and phenolic compound, the hydroxyl group of phenolic compound make a hydrogen bond with the fluorine of ([C8mim][BF4] (Ma and Hong, 2012).", "From the point of view of a wastewater treatment technology, the high salt content and residuals of IL in the effluent are of great concerns, and the water content in the extract infers regeneration of the solvent.", "A liquid membrane system hosts all the separation steps in a single device: nondispersive extraction of solutes (typically) from an aqueous feed phase, selective separation of the solutes according to their solubility and diffusion through the membrane, and recovery of the solutes in an aqueous stripping (or receiving) phase (Vladimir, 2010; Lozano et al., 2011).", "A liquid membrane may consist of an organic solvent, IL, or a mixture of liquids and dissolved solids with different functionalities.", "Their role is to act as selective extractants and carriers for the solutes.", "Three major classes of liquid membranes can be considered for the water treatment applications: a supported liquid membrane (SLM) system is preferable because immobilization of the liquid phase reduces leaking of the solvent.", "In bulk liquid membrane systems, the use of hydrophobic liquid and semipermeable film helps to prevent leakage to some extent, too.", "A third major class of liquid membranes is an emulsion liquid membrane system.", "A solvent-surfactant emulsion serves as a semipermeable liquid membrane in globules filled with a stripping solution.", "The extraction step is carried out by stirring the emulsion globules with the feed phase (Dżygiel and Wieczorek, 2010).", "Several industrial and laboratory scale applications of SLMs (Venkateswaran and Palanivelu, 2006) and emulsion liquid membranes have been developed for wastewater treatment (Correia and de Carvalho, 2003).", "Liquid membrane-based separation techniques with conventional solvents, especially SLMs, have gained interest as selective, relatively simple, and easily scalable alternatives to classic absorption, liquid-liquid extraction, and distillation techniques to separate the metals and organic compounds (Parhi, 2013; Petra, 2011).", "The high extraction and concentration degrees that can be achieved in a single treatment which makes them attractive for the removal of highly toxic substances or the recovery of valuable substances from dilute solutions (Yurtov and Koroleva, 2014).", "Water treatment in closed-circle processes, e.g., in the nuclear industry, could be an attractive field of application (Shen et al., 2015).", "The incorporation of ILs to SLM systems has been investigated in laboratory and pilot plant studies (Özevci et al., 2018; Petra and Katalin, 2009; Sun et al., 2017).", "SLMs have been frequently used for the selective separation of variety of organic compounds (de los Ríos et al., 2012).", "It is believed that the replacement of organic solvents with ILs would help to overcome the obstacles of instability and short lifetime of SLMs encountered with conventional liquids that are currently limiting the industrial use of SLM systems (Lozano et al., 2011).", "ILs have many advantages over traditional organic solvents such as the selectivity, functionality, relatively high viscosity, and low volatility of ILs that can minimizes the liquid consumption, especially with SLM configuration (de los Ríos et al., 2012; Fehrmann and Riisager, 2014; Lozano et al., 2011).", "Improved mechanical, chemical, and thermal stability extend the life-time of the SLM.", "However, the selectivity of the extraction may disrupt if any water migrates into the IL phase (Fortunato et al., 2004, 2005).", "Few studies have been published on IL-based membrane extraction of organic pollutants from aqueous effluents (Table 3) (Balasubramanian and Venkatesan, 2012; Chaouchi and Hamdaoui, 2014; Ng et al., 2011; Panigrahi et al., 2013; Pilli et al., 2014).", "The reviewed experiments have been conducted in laboratory-scale with synthetic wastewater volumes typically around 50–300 mL (Chaouchi and Hamdaoui, 2014; Ng et al., 2011).", "The studies made so far have brought out some benefits and limitations of the technology that should be taken into account in the planning of waste-water treatment systems.", "The partitioning of organic compounds between aqueous and IL phase follows the same principles in solvent extraction and membrane extraction systems.", "Extraction conditions, such as solution pH and hydrophobicity of the IL, can be adjusted to improve affinity and selectivity for a specific solute.", "The quality of the support material in SLM systems may have a decisive impact on the extraction efficiency: in the study of (Kouki et al., 2014), 68% of acetaminophen was recovered by immobilizing a hydrophilic IL Aliquat 336 (trioctylmethyl ammonium chloride) in propan-2-ol on porous polypropylene, whereas only 29% was recovered with a polyvinylidene difluoride support.", "One important issue with membrane extraction is the sensitivity of membranes to chemical and physical stress.", "For example, too high stirring speed or the acidity of highly chlorinated phenols may destroy the membrane (Balasubramanian and Venkatesan, 2012).", "To prevent the liquid membrane from leaking, the membrane should be chemically and physically stable.", "Hydrophobicity of IL reduces dissolution of the membrane into the water phase.", "The high viscosity of IL and low solute concentration in the feed have a negative impact on membrane permeation and stripping efficiency (Ng et al., 2011; Panigrahi et al., 2013).", "IL consumption and treat ratios for different membrane systems cannot be calculated in the same manner as for LLE.", "The consumption of ILs is minimized in membrane systems by preparing emulsions of ILs, organic solvents, and surfactants, or by immobilizing ILs on solid support materials.", "The extraction capacity of IL is constantly renewed by stripping of the extracted solute into the receiving phase.", "The extraction time in different studies has varied enormously.", "It should be checked whether extraction or equilibration times of up to 24–30 h (Panigrahi et al., 2013; Pilli et al., 2014) are necessary and feasible, as the shortest reported treatment time was as short as 6 min (Balasubramanian and Venkatesan, 2012).", "The extraction of organic pollutants has been carried out by both bulk, emulsion, and supported membrane systems.", "As mentioned before, leaking of hazardous solvents may pose a severe limitation to the use of systems other than SLMs.", "The bulk membrane was able to extract up to 96% of phenol into 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [C4MIm][NTf2], and strip up to 98% of the phenol into the receiving phase (NaOH solution).", "However, 7,4% of [C4MIm][NTf2] was found in the aqueous phase (Ng et al., 2011).", "In emulsion liquid membrane systems, ILs have typically been used as co-extractants and co-carriers in the liquid membrane phase.", "Balasubramanian and Venkatesan mixed [C4MIm][PF6] and tributyl phosphate, diluted the mixture with kerosene, and emulsified it with a surfactant.", "The IL enhanced the transport rate of phenol into an internal phase (NaOH) of the emulsion globules, and >99% of phenol was removed from an aqueous feed solution (pH 6.5) with 0.02% IL content in the membrane and 6 min stirring time (Balasubramanian and Venkatesan, 2012).", "The IL had a stabilizing effect on the emulsion membrane, but still, around 0.5–1.3% leakages were found in different experiments.", "This could lead to an IL release of approximately 0.3–0.9 mL/m3 in treated water.", "In another emulsion liquid membrane study, a hydrophilic IL Aliquat 336 (trioctylmethylammonium chloride) was emulsified with organic solvents (Chaouchi and Hamdaoui, 2014).", "Nearly 100% of acetaminophen [paracetamol or N-(4-hydroxyphenyl) acetamide)] was recovered from distilled water.", "However, treatment of spiked water samples was more challenging: the extraction efficiencies were 50.5%, 63.7% and 78.9% in sewage water effluent, sea water, and in natural mineral water respectively.", "SLMs have been tested for the removal of pharmaceuticals with Aliquat 336 (Kouki et al., 2014), pesticides with 1-butyl-2,3-dimethylimidazolium hexafluorophosphate [C4DMIm][PF6] (Pilli et al., 2014) and phenol with 1-butyl-3-methylimidazolium hydrogensulfate [C4MIm][HSO4] (Nosrati et al., 2011) from aqueous effluents.", "The membrane permeability of bisphenol A from tap water (62% in 24 h) was higher with tetrabutylphosphonium hexafluorophosphate, [TBP][PF6], than with other phosphonium-, ammonium- and imidazolium-based ILs investigated (Panigrahi et al., 2013).", "The [TBP][PF6] was the most stable IL, with 0.35% loss from the SLM during the experiment.", "The highest losses, 2–3%, were attributed to ILs with chloride anions.", "The effect of Tributyl phosphate (TBP) was used as a carrier in the membrane while for stripping, NaOH solution was selected for the IL supported membrane for levulinic removal (Baylan and Çehreli, 2018).", "It was demonstrated that higher removal of levulinic can be achieved with the higher concentration of TBP and NaOH.", "ILs have been frequently used for the modification and enhancement of adsorption efficiency of adsorbent materials (Yang et al., 2019a, 2019b).", "1-butyl-3-methylimidazolium ionic liquid was used in the modification of chitosan and used for the removal of malachite green (Naseeruteen et al., 2018).", "Kinetic of malachite removal followed the pseudo-second order as the best-fitted model.", "A bi-functional smart film using Poly(styrene-butyl acrylate-ionic liquids) and TiO2 was fabricated for the adsorption and photodegradation of methylene blue, brilliant red, phenol, and metallic ions and found very efficient for the treatment of selected pollutants (Dang et al., 2018).", "Further, magnetic hydroxyethyl cellulose/ionic liquid (MHEC/IL) adsorbent material was synthesized for the selective adsorption of hydroquinone (Ding et al., 2018).", "The MHEC/IL adsorbent showed very high adsorption capacity (335.68 mg g−1) for hydroquinone at pH 5.0.", "Magnetic properties make its separation easy by using an external magnet and IL impregnation enhanced the adsorption efficiency.", "Zeolite has been proved as a good adsorbent material for the variety of pollutants.", "Li et al., synthesized an acidic ionic liquid viz.1-butyl-3-(triethoxysilylpropyl) imidazolium hydrogen sulfate [BTPIm][HSO4]).", "Synthesized IL was grafted on MCM-36 zeolite to enhance the adsorption property for isobutane and 1-butene (Li et al., 2016).", "The reduced surface area was noted in grafted MCM-36 while surface density was increased for the acid.", "Due to IL impregnation, increased interaction was observed in the case of isobutane over MCM-36-IL, while for 1-butene, interaction was bit reduced.", "Application of C8mimBF4, C10mimBF4 and C12mimBF4 imidazolium based ionic liquid was investigated for the oil recovery (Pillai et al., 2018).", "It was demonstrated that the surface activity can increase while the interfacial tension (IFT) can reduce with the increase in alkyl chain length of ILs.", "The capability of ILs for trapped oil was assessed by the emulsification tests.", "Further, IL modified activated carbon was used for the adsorption of congo red and reactive blue dye (Lawal et al., 2017).", "Activated carbon was derived from peanut shell and then Peanut shell and AC was modified with 1-methyl-3-decahexyl imidazolium ionic liquid (IL) and named as (ILNS) and ILAC for nut shell and AC respectively.", "Adsorption of dyes was highly dependent on pH and enhanced adsorption was observed in IL modified AC for both dyes.", "For CR, the adsorption capacity was 136.4 and 150.0 mg g−1 for ILNS and ILAC respectively.", "In the case of RB, the adsorption capacity 290.0 and 364.4 mg g−1 for ILNS and ILAC respectively.", "In another study, 1-benzyl, 3-hexyl imidazolium, and 1-benzyl, 3-decahexyl imidazolium ionic liquids were tailored for the functionalization of CNT for sulfamethoxazole and ketoprofen adsorption (Lawal et al., 2018).", "Increased adsorption efficiency was reported by IL functionalized CNT for both pollutants compared to the unmodified ones.", "Belbel et al. demonstrated the adsorption performance of 1-butyl-3-methylimidazolium chloride modified homoionic montmorillonite for the dye (Belbel et al., 2018).", "In another study, IL modified Montmorillonite (Mt) clay and used as an adsorbent for diclofenac sodium (DS) (Wu et al., 2018).", "It was reported that DS adsorption was enhanced by the increasing IL dosage on montmorillonite clay.", "IL modification affects the surface charge of Mt. IL-modified Mt. could be an inexpensive adsorbent material for water treatment.", "Golmohammadi et al. used the dimethyl octadecyl[3-(trimethoxysilylpropyl)] ammonium chloride for immobilization on the magnetite nanoparticles coated (Fe3O4@SiO2@IL) for reactive yellow 15 (anionic dye) removal by ultrasound assisted adsorption (Golmohammadi et al., 2019).", "The Langmuir capacity of Fe3O4@SiO2@IL was determined to be 63.69 mg g−1.", "Exothermic nature was observed for dye adsorption on Fe3O4@SiO2@IL.", "Further, silica-ionic liquid composite (SiO2-ClPrNTf2) was applied for the selective adsorption of 4-chlorophenol and it was reported that efficiency of SiO2 was enhanced by 96.86% when IL-based silica composite was used for the adsorption (Marwani and Bakhsh, 2017).", "The adsorption capacity of IL based silica composite was found to be 626.25 mgg−1 at pH 1.0.", "A classical approach for the selection of an extraction solvent focuses on reaching a feasible compromise between solute solubility and selectivity, with full respect to the safe and practical use of the solvent (Haan and Bosch, 2013).", "Feasibility of an extractive water treatment process depends on the technical design of the process as well as solvent properties.", "Octanol/water partitioning coefficient (Kow or LogKow) of a solute does not directly correlate with IL/water partitioning because of the influence of other solute-IL interactions than hydrophobicity.", "In particular, the distribution of acidic solutes in IL tends to be less than in octanol (Han and Row, 2010).", "On the other hand, it is often possible to enrich more polar compounds into a water-immiscible IL phase than by using conventional organic solvents.", "Hydrophobicity of an IL reduces its dissolution and mixing with water but improves its capability of solvating non-polar organics.", "ILs used for extraction processes range from hydrophilic to hydrophobic.", "The anion has a great influence on the hydrophobicity of an IL.", "Changing the anion of an imidazole-based IL from PF6− to BF4− or Cl− may transform the corresponding IL from hydrophobic to hydrophilic (Freire et al., 2007; Mallakpour and Dinari, 2012).", "Because of the mutual solubility of water and IL, water is also dissolved into the IL phase in an extraction process.", "The mutual solubility of water and [C4Mim][PF6] is around 2 weight-% (Poole and Poole, 2010).", "Polarity-based classification is often used to screen the solubilizing power for specific solutes.", "In essence, a polar solvent is one that dissolves and stabilizes dipolar or charged solutes (Welton, 1999).", "It comprises all intermolecular interactions between the solvent and solute that do not result in chemical interactions (Poole and Poole, 2010).", "Polarity values have been reported for some ILs.", "Polarities of typical imidazolium-based ILs, [C4MIm][BF4] and [C4MIm][PF4], are in the range of medium chain alcohols, such as 1-hexanol or 1-octanol (Mallakpour and Dinari, 2012).", "However, the polarity parameter is considered deficient for understanding the behaviour of complex solvents such as IL, which possess multiple mechanisms for interacting with solutes (Cláudio et al., 2014).", "Besides direct solvent-solute interactions, anion-cation interactions inside the solvent are important for ILs (Mallakpour and Dinari, 2012).", "Ions with strong, pH-dependent electrostatic interactions make ILs highly different from classical organic solvents (Berthod et al., 2008).", "Systematic empirical testing or modelling is recommended to select an appropriate IL for a specific solute (Pilli et al., 2013; Poole and Poole, 2010; Welton, 2014).", "Solvation models developed to predict the ability of an IL to solvate another substance (solute) have focused on dipolarity/polarizability, hydrogen bond (HB) basicity, and HB acidity as solvent-dependent parameters (Ho et al., 2011; Poole and Poole, 2010).", "In particular, Conductor-like Screening Model for Real Solvents (COSMO-RS) is based on calculating the HB basicity (ability to act as HB acceptor) of an IL to predict its capability of solvating HB donors (Cláudio et al., 2014).", "The model has been used to screen ILs for their selectivity for several solutes.", "Fundamental studies on IL/water partitioning of organic compounds have been conducted by (Berthod and Carda-Broch, 2003).", "The chemistry of an organic pollutant and an IL have a significant influence on the partitioning of the pollutant between water and IL phase.", "Other wastewater chemistry, especially pH, plays a major role.", "Researchers attempting to treat industrial effluents have faced the fact that in there's no IL which could be applied for the removal of a large range of organic compounds (McFarlane et al., 2005).", "Highly polar organics, such as acetic acid and chlorinated ethanes, tend to be too hydrophilic to be effectively extracted with hydrophobic ILs (Berthod and Carda-Broch, 2003; Kujawski et al., 2004).", "Other compounds that are usually poorly extractable with ILs include such hydrophobic aromatic and aliphatic organics, which don't possess electron-withdrawing groups and don't preferably act as hydrogen donors, for example, benzene and nonane (Bhosale et al., 2015).", "When the pH is less than the logarithmic acid dissociation constant (pKa) of the acid, the longer chain aliphatic acids partition into the IL phase in a protonated form (Berthod and Carda-Broch, 2003; McFarlane et al., 2005).", "Phenolic compounds are common pollutants in industrial process waters and their IL/water partitioning has been investigated by several research groups.", "Phenol (hydroxybenzene) is a weak acid with pKa close to 10, and hence, it exists almost exclusively in the undissociated, unprotonated form at acidic to neutral pH. The unprotonated hydroxyl group has a strong hydrogen bond donor (HBD) strength, and it interacts readily with a hydrogen bond acceptor (HBA).", "Typical HBAs in ILs are anions with unshared electron pairs and a partially negative charge.", "Hydrogen bonding is the primary extraction mechanism for phenols for example with imidazole-based ILs, such as [C4MIm][BF4] and [C4MIm][PF6] (Egorov et al., 2008; Y. C. Fan et al., 2008; Nosrati et al., 2011).", "Chlorine substituent has an electron-withdrawing impact that enhances the HBD character of phenol.", "The HB mechanism explains the higher affinity of ILs for monochlorophenols than phenol (Khachatryan et al., 2005).", "With a higher number of chlorine substituents, the importance of hydrophobic interactions increases (Deng et al., 2011).", "Higher HBD strength (smaller pKa) and higher lipophilicity (higher LogKow) of pentachlorophenol than phenol led to higher partitioning of pentachlorophenol into a relatively lipophilic/hydrophobic IL, [C8mim][PF6] (J. Fan et al., 2008).", "Nitro group (NO2) in the phenol molecule is also an electron-withdrawing group and it improves the HBD capability of a phenolic compound in the same manner as chloride.", "Since nitrophenols formed HBs with [C4MIm][PF6] in undissociated form, the optimal extraction range for a weak acid 4-nitrophenol was wider, pH 1–7, than for stronger acids 2,4-dinitrophenol and 2,6-dinitrophenol, for which a drop was noted at pH > 4 (Khachatryan et al., 2005).", "Bhosale et al. (2015) studied the influence of IL cations [CnMIm] and anions [NTf2] and [PF6] on the partitioning of three nitrogen compounds, 2,4,6-trinitrotoluene (TNT), 2,4,6-trinitrophenyl-n-methylnitramine (tetryl), and 2,4,6-trinitrophenol.", "They concluded that the strength of hydrogen bonding between the CH at position 2 of the N-alkyl imidazolium cation and pi bonds/electrons of the aromatic ring explained the highest D value, which was obtained for TNT.", "Formation of a hydrogen bond with ILs is not similarly preferred for bases as for (weak) acids, and the D values for bases are generally lower.", "Weak bases (e.g., aromatic amines: aniline) may act as HBAs for imidazolium cations, and they are preferentially extracted in molecular form, at pH > pKa (Egorov et al., 2008; Zhang et al., 2010).", "In addition to hydrogen bonding mechanism, hydrophobic interactions often contribute to the affinity of ILs for organic solutes.", "Some partitioning of phenol still occurs at higher pH. The partitioning mechanism has been attributed to unspecific hydrophobic interactions (Nosrati et al., 2011).", "Increase in hydrophobicity of a phenolic compound has denoted higher IL-to-water distribution ratios (D values) for alkylphenols and napththols relative to a simple phenol at alkaline pH (J. Fan et al., 2008; Khachatryan et al., 2005; Ng et al., 2011).", "On the other hand, the addition of more hydroxyl groups to the benzene ring, as in catechol and resorcinol, makes the pollutant too hydrophilic to be separated from the aqueous phase (Khachatryan et al., 2005).", "When HB mechanism is not effective, ion exchange or electrostatic interactions may involve in the extraction process showed that the partitioning of 2,4-dinitrophenol into TOMAS phase at pH > 7 was driven by anion exchange mechanism (Egorov et al., 2008).", "The more hydrophobic the phenolic compound, the more likely it was assumed to partition via anion exchange at basic pH. The distribution of picrate in different IL/water systems was demonstrated (Watanabe and Katsuta, 2014) and it was revealed that both ion pair extraction with IL cation are included in the extraction mechanism, which was enhanced when the hydrophobicity of the IL cation was increased.", "Dying wastewater from textile and other industries may consist of a mixture of organic dyes with different functional groups, thus representing different affinities and extraction mechanisms.", "Pei et al. (2007b) examined the partitioning different dyes viz. methyl orange, eosin yellow and orange G between conventional hydrophobic imidazolium-based ILs and deionized water (Pei et al., 2007b).", "Partitioning of all dyes in the IL phase increased with increasing hydrophobicity of the IL cation: [C8MIm] > [C6MIm] > [C4MIm].", "Methyl orange was preferably extracted in an anionic form at pH > pKa (3.46), presumably due to hydrogen bonding between the dissociated sulfonate (-SO3−) or/and azo group (-N=N-) of methyl orange (an HBA) and the acidic 2H of the imidazolium cation (HBD).", "Eosin yellow displayed the highest D values among the three dyes.", "In molecular form (pH < pK1, 3.25), the two –OH groups of eosin yellow act as potential HBDs for hydrogen bonding with the PF6 anion.", "The extraction of orange G was not sensitive to the changes in acidity at pH 1–11, but the D value was low.", "The orange G molecule contains one –OH group and two sulfonate groups, thus being capable of acting either as a HBD or HBA with the investigated IL.", "Not only the hydrophobicity of the ILs, but the pH of the aqueous phase/feed solution also affected the IL/water partitioning of the dyes.", "In another study, [C4MIm][PF6]/water partitioning was determined for weak acid, acid blue 6B (Lin et al., 2014).", "No change in the structure of acid blue 6B was observed after the extraction.", "Electrostatic interaction between the –SO3 or C=N+ groups of the dye and an ionised IL cation ([C4MIm]+ or anion [PF6]− was assumed.", "Partitioning of thirteen different kinds of textile dyes was also tested visually, showing that all the other dyes but direct blue 5R and reactive B-3BD were extracted by [C4MIm][PF6].", "In addition to wastewater treatment, IL also showed a promising substitute for inorganic salts as retarding agents in the actual silk dying process.", "Li et al. investigated the IL/water partitioning of 15 dyes.", "They found that acid dyes were more effectively extracted by [C4MIm][PF6] than weak acids and reactive dyes.", "Changes in the 1H NMR spectra pointed out to involvement of an ion exchange mechanism, which implies that the transfer of an anionic dye molecule into the IL phase was accompanied by the transfer to dissolved IL anions into the aqueous phase (Li et al., 2007).", "The extraction efficiency for a reactive dye, reactive black KN-G2RC, was increased from approximately 31% to 82% on addition of a crown ether (dicyclohexyl-18-crown-6), a complexing agent that formed hydrogen bonds between the dye and IL.", "Instead of using [C4MIm][PF6] as a hydrophobic IL, quaternary ammonium–based IL, tricapryl methylammonium thiocyanate [N1888][SCN] was used for the extraction of dyes (Chen et al., 2013) The removal efficiency, D value, and recovery of the solute with a dye-specific stripping solution was higher for an anionic azo dye methyl orange (sodium 4-[(4-dimethylamino) phenyldiazenyl] benzenesulfonate) than for a cationic dye methylene blue (3,7-bis(dimethylamino)-phenothiazin-5-ium chloride).", "Hydrogen bonding with SCN−, a strong HBA, was suggested as an extraction mechanism for the dyes.", "A negative influence of salt addition on the [N1888][SCN]/water partitioning of methyl orange and positive influence of pH increase was observed.", "The removal of acid azo dyes from synthetic solutions and real tanning effluent was carried out by using a hydrophobic IL, N-butyl N-methylpyrrolidinium bis(trifluoromethanesulfonyl) amide [p14][Tf2N] has been used for (Vijayaraghavan et al., 2006).", "The extraction efficiency for acid blue Navy 5RE was 56% at pH 7 and 3, suggesting that the IL had the affinity for the azo groups (-N=N-) or sulfonate groups, irrespective of the dissociated/undissociated form of the sulfonate.", "The extraction efficiency for the acid red, which contains –OH groups, was higher at pH 7.", "This could indicate that electrostatic interactions between charged ions played a role in the extraction.", "In order to obtain removal efficiencies ≥95%, synthetic samples were extracted 3 times and real tanning effluent 4 times with fresh IL.", "Further, IL-based aqueous two-phase systems of hydrophilic imidazolium-based and phosphonium-based ILs (45 wt-%), water (45 wt-%) and inorganic aluminium sulfate [Al2(SO4)3] or organic potassium citrate [K3C6H5O7] salts was composed for the dye removal from synthetic textile wastewater (Ferreira et al., 2014).", "The most hydrophobic dye Sudan III partitioned completely into the IL phase by using any IL and either organic or inorganic salt.", "Moderately hydrophobic dye indigo blue partitioned completely into phosphonium-based ILs but precipitated at the IL/water interphase of imidazolium-based ILs.", "Hydrophilic dye chloranilic acid was completely extracted with phosphonium-based ILs, but with imidazolium-based ILs the partitioning coefficients were low.", "Both evaporation and cooling processes were found to induce precipitation of the extracted dyes, which could be used for the regeneration of ILs.", "The authors estimated that a filtrated IL-rich phase could be recovered and reused up to 800 times.", "Partitioning of dyes in IL-based aqueous two-phase systems was also evaluated in the study of (Sheikhian et al., 2014).", "The ABS consisted of [C4MIm][Br], potassium phosphate salts, and an azo dyes methyl orange, PAR [4-(2-pyridylazo)-resorcinol], or reactive red 120 dissolved in water.", "Phase diagrams were established for neutral to basic pH range, corresponding to the dissociation of the phosphate buffer.", "Increase in dye concentration first enhanced partitioning, but the extraction capacity of the ILs appeared to be limited.", "Partitioning of methyl orange and reactive red into the IL-rich phase was found to be greater at basic than neutral pH, which was explained by the better salting-out capability of phosphate salts in the case of methyl orange and by the more hydrophobic nature of the dye in the case of reactive red.", "Extraction with a hydrophobic IL was used to recover the spent IL.", "The investigated azo dyes, reactive red-120, 4-(2-pyridylazo)-resorcinol and methyl orange, exhibited high partitioning coefficients in the two-phase system of [C4MIm][Br] and potassium phosphate salts.", "It was demonstrated that in functionalized ILs, dye removal was enhanced and recovery and reuse of ILs were easier.", "An IL-based extraction process transfers wastewater pollutants into an IL phase that can be separated from the system either by salting out a hydrophilic IL, settling or centrifuging a water-immiscible phase or by mechanically removing IL-containing membranes or resins from the system (Mai et al., 2014).", "Direct decomposition in the IL phase can be a practical treatment method for the extracted pollutants, ILs provide an ideal medium for photolytic decomposition of chlorinated pollutants.", "Subramanian et al. (2007) examined the photodegradation of pentachloro phenol (PCP) dissolved in [C4MIm][PF6] in pure solutions and soil suspensions.", "They found that 90% of PCP (1.57 mM) in pure IL solution was degraded in 6 h under 253.7 nm UV-radiation.", "Natural soil components and additions of Fe(II)-activated Oxone (KHSO5) at low concentrations accelerated photolysis reactions.", "It was also shown that the high stability of ILs towards oxidation and reduction reactions makes it possible to reuse the IL in the soil cleanup process.", "In an earlier study, the photodegradation rate of PCP in [C4MIm][PF6] was found to be approximately 0.009 mM/min (Yang and Dionysios, 2004).", "The researchers found an increase in transformation rate with increasing concentration with 2-chlorophenol, decreasing the number of chlorine substituents and the number of chlorines at ortho-positions.", "It was proposed that the investigated IL acts as a hydrogen donor in the photodegradation process.", "Furthermore, IL additions appear to have beneficial impacts on some chemical destruction methods for chlorinated hydrocarbons.", "In another report, the contribution of ILs to alkaline polyethylene glycol (KPEG) dehalogenation was investigated (Kastanek et al., 2011).", "The role of 1-ethyl-3-methyl imidazolium ethyl sulfate ionic liquid with Pseudomonas stutzeri strains was assessed for the Acid Black 48 (AB48) and Reactive Black 5 (RB5) degradation (Álvarez et al., 2016).", "Major environmental concerns in the use of ILs are related to the use of volatile solvents and non-renewable raw materials in the synthesis of ILs, and secondly, to the degradation and toxicity of the disposed ILs (Amde et al., 2015; Cvjetko Bubalo et al., 2014).", "Depending on the composition, the ionic liquid may pose the toxic effect and can show the poor biodegradability, hence the development of an effective chemical treatment is of great importance from the environmental point of view (Neves et al., 2014; Zhou et al., 2018).", "The proliferation and dissemination of antibiotic resistance genes (ARGs) were found to be in environmental bacteria by 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6]) (Luo et al., 2014).", "It is reported that most imidazolium-based ILs have a potential toxic effect on bacteria, algae, and zebrafish.", "Furthermore, 1-alkyl-3-methyl-imidazolium salts are poorly degradable by microbial communities of activated sludge.", "Many ubiquitous ILs contains ions such as quaternary ammonium cations and fluoride, bromide and chloride anions, and the use of hydrophilic ILs produces an effluent with high salinity.", "Hydrolysis of [BF4]− and [PF6]−based ILs in water releases toxic and corrosive hydrofluoric acid, HF (Freire et al., 2012).", "It was reported that the toxicity of ILs with alkyl side chains decreases with increasing length of the side chain, but recent studies also suggest that dicationic ILs could be less toxic than monocationic ILs (Steudte et al., 2014) However, with the increase in the relative number of oxygen atoms leads a reduction in toxicity (Zhao et al., 2014).", "Though ionic liquid has been designated as the green solvent but recently, some studies reported that ILs could show phytotoxicity (Yu et al., 2018).", "Phytotoxicity of ([Bmim]OAc [Bmim]C7H5O2 [Bmim]BF4 [Bmim]Br, [Domim]Br [BPy]Br [Omim]Br [Bmim]Br [Emim]Br.)", "ionic liquids were studied and it was demonstrated that anion species, cation chain length, and IL's concentration can affect seeds growth.", "This effect was more prominent when the alkyl chain length with cation was longer and fluoride anion was present in ILs (Yu et al., 2018).", "It is important to recover and regenerate the spent IL phase economically, but it is also important that IL residuals in the treated effluents are eliminated for ecological and human health reasons, too.", "Many IL-based techniques incorporate a procedure for regenerating the spent IL for reuse, such as stripping off or back-extracting the pollutants with an alkaline solution or vacuum.", "For example, when phenolic pollutants were back-extracted or stripped of with NaOH solution after LLE process, recovery rates of >97% were obtained (Brinda Lakshmi et al., 2013; J. Fan et al., 2008).", "Several successfully applied chemical and electrochemical methods are available for the destruction of residual ILs (Cvjetko Bubalo et al., 2014).", "Different AOPs based on hydroxyl radicals have also been investigated for the degradation of organic pollutants from wastewater.", "Recently, processes such photodegradation, heterogeneous Fenton, electrolysis, photoelectrolysis, photocatalysis, electro-Fenton, heterogeneous electro-Fenton, and adsorption technologies have been successfully used for the treatment of ionic liquids from water (Bocos et al., 2017).", "Advanced oxidation processes (AOPs) have been proved very efficient for in the treatment of emerging pollutants (Siedlecka et al., 2012).", "Microbial degradation methods have not been effective for ILs treatment (Álvarez et al., 2016; Liwarska-Bizukojc et al., 2015).", "Due to toxic and recalcitrant properties of ILs may severely inhibit their removal in activated sludge processes.The Fenton based degradation of the ionic liquids of different families viz. imidazolium, pyridinium, ammonium, and phosphonium, was investigated (Munoz et al., 2015a, 2015b).", "Fenton oxidation gave total mineralization and effluents with almost negligible ecotoxicity (below 0.01 TU in most cases).", "It was observed that by increasing the temperature, reaction time and consumption of excess H2O2 could be reduced.", "It was demonstrated that the aromatic ILs can be easily oxidized into nontoxic short chain organic acid in comparison to aliphatic ILs.", "However, cyano-based ILs showed toxicity in treated effluents.", "The toxicity was because of toxic cyanide species.", "Poza-Nogueiras et al. studied the heterogeneous electro Fenton process for the degradation of imidazolinium-based ionic liquids 1,3-Bis(2,4,6-trimethylphenyl) imidazolinium chloride, using iron alginate spheres (Poza-Nogueiras et al., 2018).", "The effect of Cl−, C(CN)3− and CF3SO3− as a counter ions, while C6F11O2−, C8F15O2− and C10F19O2− as background ions were selected to demonstrate the effect of these ions on degradation of 1-butyl-3-methylimidazolium cation (bmim+) (Siedlecka et al., 2009).", "It was demonstrated that the counter ion can affect ILs degradation because hydroxyl radicals can compete with bmim+ or can also make a complex with Fe3+ and Fe2+ ions.", "Further, it was revealed if perfluoric acids is present then there might be strong interaction between the bmim+ and negatively charged perfluocarboxylic anions (PFCA).", "It was studied that imidazolium based IL (EmimCl) can be removed by coupling of Fenton with a biological approach (Gomez-Herrero et al., 2019).", "It was observed that by combining two approaches, the high TOC conversion - COD removal efficiency was obtained.", "Bocos et al. used electro Fenton treatment for the treatment of 1-Butyl-3-methylimidazolium triflate ([C4mim][CF3SO3]) ionic liquids and investigated the effect of different electrolyte viz.NaNO3,NaSO4 and NaCl on removal of ([C4mim][CF3SO3] (Bocos et al., 2017).", "Among all, NaNO3 was reported to have high mineralization rate and 87% of TOC reduction was observed after 240 min of degradation time.", "The total mineralization of 1-butyl-4-methylpyridinium chloride and 1-ethyl-3-methylimidazolium chloride was demonstrated in anodic oxidation with electrogenerated H2O2(AO-H2O2), electro-Fenton and photoelectro-Fenton degradations by using boron-doped diamond BDD/air-diffusion cell (Garcia-Segura et al., 2016).", "Siedlecka et al. investigated the electrocatalytic degradation of imidazolium-based ionic liquids using a lead dioxide (PbO2) anode (Siedlecka et al., 2012).", "Approximately 97% mineralization was recorded in this study.", "It was demonstrated that the degradation involves the oxidative cleavage of the imidazolium ring.", "In another study, the electrolytic degradation of imidazolium and pyridinium ionic liquids in an aqueous solution was assessed by using boron-doped diamond (BDD) electrode (Pieczyńska et al., 2015).", "Degradation was due to the oxidative opening of imidazolium or pyridinium ring.", "It was reported that presence of electrolyte strongly affects the toxicity of treated effluent.", "However, treated effluent was less toxic than the parent solution, but in the presence of NaCl, increased toxicity was reported.", "An ultrasonic assisted degradation of ILs in presence of zero-valent iron/activated carbon was applied for the degradation of 1-butyl-3-methyl benzimidazolium bromide ([BMBIM]Br) and 1-allyl-3-methylimidazolium chloride ([AMIM]Cl) (Zhou et al., 2018).", "In this study TOC removal was >87%.", "The coupling of two different technologies viz. Fenton and biological oxidation processes assessed for the treatment of imidazolium-based ionic liquid 1-Ethyl-3-methylimidazolium chloride (EmimCl) (Gomez-Herrero et al., 2019).", "The effluent was more biodegradable and also reduced ecotoxicity was measured in comparison to the parent ionic liquid.", "By combining the biological oxidation with Fenton process, 90% removal of COD was obtained.", "The adsorption technology has been widely used for the treatment of variety of pollutant species.", "Adsorption technology has been successfully applied for the ILs removal/recovery from wastewater.", "Some of them have been discussed in this section.", "Adsorption of several imidazolium-based ILs on different commercial modified activated carbon adsorbents was investigated (Lemus et al., 2012).", "It was observed that adsorbent natures such as porosity and surface nature have a significant effect on ILs removal.", "It was demonstrated that the ACs with high polar groups at the surface is good for hydrophilic ILs because polar group supports for the hydrogen bonding while the ACs with low polarity showed better efficiency for the hydrophobic ILs.", "Loaded ILs can be easily recovered by regenerating the adsorbent by acetone followed by atmospheric distillation to separate ILs from acetone.", "Further, biochars derived from straw and wood-based were used for the removal of imidazolium-type ionic liquids [C2mim][BF4] (Shi et al., 2016).", "It was demonstrated that the ILs removal is highly pH dependent and can be enhanced by the phosphate anions where the phosphate anions act as a promoter for water soluble ILs.", "As previously reported, carbonaceous materials have been proved as an efficient adsorbent materials ILs removal and their properties strongly affect ILs adsorption.", "In another study, the carbonaceous material based adsorbent was tailored by HTC method for the removal of imidazolium-based ILs (Wang et al., 2015).", "Synthesis condition such as activation temperature and chemical activation can strongly affect the properties.", "It was demonstrated that with the temperature increase, the surface oxygenated group can be reduced which supports for the hydrophilic ILs adsorption.", "Tailored adsorbents showed following trend for the selected ILs [Cl− < TFA− < [BF4]− < OTf− < [PF6]− < NTf2−].", "Reinert et al. studied the adsorption of imidazolium and pyridinium ionic liquids on Na-montmorillonite (Reinert et al., 2012).", "RTILs viz. BMImCl, OMImCl, AMImCl, BpyBr, and OpyBr were selected in this study.", "A cation exchange mechanism was observed for RTIL removal and it was demonstrated that the alkyl chain length and organic cations in RTILs have a significant effect on the adsorption efficiency.", "Endothermic nature was reported for the RTILs removal in this study.", "Further, the adsorption efficiency of activated carbon was evaluated for the ionic liquids (Neves et al., 2014).", "In this study, the salting-out ability of Na2SO4 on the adsorption of ionic liquids was demonstrated and it was observed that use Na2SO4 could enhance the adsorption of ILs.", "Lemus et al. used the activated carbon for the removal of ILs in fixed bed adsorption (Lemus et al., 2017).", "The factors which influence the adsorption of ILs were superficial liquid velocity, particle size and the nature of ILs.", "Higher removal was observed in case of hydrophobic ILs.", "The adsorption of ILs with different cationic and anionic structure was also studied to investigate the effect of cations and anion.", "The particle size of AC can affect the recovery of ILs and low particle size was recommended in this study.", "Further, in another study, the adsorption of imidazolium-based ionic liquids ([Omim]Cl, [Hmim]Cl and [Bmim]Cl by using activated carbon was studied and it was revealed that the hydrophobicity and the length of alkyl chain strongly affect the ILs adsorption (Ushiki et al., 2017).", "It was found that the adsorption capacity of AC increased was greatly influenced by the alkyl chain length of cation and increased adsorption capacity was recorded in case of increased alkyl chain length of cation.", "ILs adsorption was temperature dependent which suggested for the physical adsorption.", "Guedidi et al. studied the adsorption of pyridinium ionic liquids activated carbon cloth.", "Physical adsorption was demonstrated for both 4-tert-butyl-1-propylpyridinium bromide and 4-tert-butyl-1-(2-carboxyethyl) pyridinium bromide ionic liquids (Guedidi et al., 2017).", "The ILs adsorption increased with the increase of pH from acidic to alkaline range.", "They demonstrated that the ILs adsorption can be governed by different mechanism as π-π interactions or by the electrostatic interaction.", "Qi et al. synthesized carbonaceous material for the adsorption of imidazolium-based hydrophilic ionic liquids.", "Carbonaceous material was prepared by chemical activation and hydrothermal treatment (Qi et al., 2014).", "The efficiency of synthesized material was compared with commercial activated carbon and it was demonstrated that synthesized carbonaceous material showed very higher removal of ILs due to the high number of oxygenated groups and increased adsorption capacity was observed when the hydrophobicity of the anions and alkyl chain length of the imidazolium cation was increased.", "It was proposed that the adsorbent material with high oxygenated groups are better for hydrophobic ionic liquids while commercial activated carbon is better for the hydrophobic ionic liquids.", "A clay material, bentonite was used for the removal of ionic liquid (1-butyl-3-methyl imidazolium chloride: BMImCl) from the aqueous solutions (Min et al., 2015).", "The bentonite was modified for the enhancement of its adsorption efficiency for selected ionic liquid.", "Bare, acid-activated and organic-modified bentonite were used as the adsorbent materials in batch adsorption experiments and the parameters viz. pH, ionic strength, humic acid, and IL concentration were studied.", "It was proposed that engineered bentonites samples might be applied for the removal of BMImCl containing wastewater.", "Membrane-based technologies are also investigated for the potential recovery of ILs.", "Gan et al., used the nucleopore microfiltration membranes for two ionic liquids C10-min[NTf2] and N8881[NTf2] (Gan et al., 2006).", "In this study, before membrane filtration, the ILs viscosity was decreased by using diluting agents' methanol/ethanol and after separation, methanol and ethanol were separated from ILs by evaporation.", "Evaporation doesn't affect the ILs due to their non-volatile properties ILs.", "Nanofiltration was also used for the IL separation from bromophenol blue/[C4mim][BF4] and [C1mim[MeSO4]/lactose system (Mai et al., 2014).", "The recovery of BmimBr (1-Butyl-3-methylimidazolium bromide) was obtained by a hybrid membrane-based technology (electrodialysis with ultrafiltration), which showed IL recovery ratio 75.2% and the current efficiency of ED process 79.1% (Liang et al., 2016).", "With the hybrid system, approximately 514.1 g/kw·h of energy consumption was reported.", "Application of ED technology was also reported for the recovery of 1-hexyl-3-methylimidazolium chloride ([Hmim]Cl) and 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4) (Wang et al., 2012).", "Effect of different parameters viz. initial concentration, applied voltage and initial volume on recovery ratio and the concentration ratio (ω), and the current efficiency (η) were examined.", "The highest specific energy consumption (Re) could reach about 1350 g/kW h. Birdwell et al., reported separation of three hydrophobic ionic liquids by Centrifugal Solvent Extraction (Birdwell et al., 2006).", "To make easy separation of ILs, Yang et al., reported a poly(ionic liquid) (PIL) functionalized magnetic materials (Fe3O4@SiO2@PIL) which could be easily separated by applying magnetic field (Yang et al., 2019b).", "Lee et al. reported the recovery of 1-butyl-3-methylimidazolium tetrachloroferrate ([bmim]FeCl4) from its mixture with water by applying a magnetic field (Lee et al., 2007).", "IL-based separation techniques have been extensively applied for the extraction and adsorption of different types of organic pollutants including phenol, chloro and nitrophenols, toluene, bisphenol-A, phthalates, pesticides, dyes and pharmaceuticals from wastewater.", "Still, this is just a small sample of the organic pollutants that have been separated for the purposes of chemical analytics, which indicates wider applicability.", "Several papers have also been published on the separation of organic compounds that are not generally regarded as environmental pollutants but contribute to the chemical and biological oxygen demand of wastewater (Fortunato et al., 2005; Hameister et al., 2014; Pei et al., 2012).", "And in the removal of metals and metalloids, IL-based solvent extraction techniques are particularly well established.", "Despite the fact that most of the reviewed experiments have been performed on clean water spiked with pollutants, there is also evidence on the usability of IL-based separations for dirty wastewater, including sewage effluent (Chaouchi and Hamdaoui, 2014).", "Extractions of pollutants from soil and sediment samples, as carried out for chemical analyses, have demonstrated that ILs are compatible for samples with high contents of organic matter and particles (Asensio-Ramos et al., 2012; Delgado et al., 2012; Johansson and Johansson, 2013; Wang et al., 2013).", "Therefore, there is no apparent technological obstacle for using ILs for the removal of specific pollutants even from municipal wastewater.", "However, the high costs of ILs may render them more suitable for the treatment of smaller wastewater volumes, such as segregated industrial effluents.", "The high specificity of ILs makes it possible to cover a part of the treatment costs through the recovery of spent chemicals and other valuable wastewater components for reuse, which is an important field of IL application in such.", "Since wastewater applications are still at a hypothetical stage, there are not even pilot-scale demonstrations of wastewater treatment that would resolve feasibility issues related to the treatment efficiency, economics and ultimate fate or disposal of the pollutants and ILs.", "In the development of wastewater treatment technologies, experience from analytical separations of organic pollutants can be utilized to some extent, but investigations under realistic treatment conditions and scaling up the process would be essential.", "Short equilibration times in the experiments imply savings in treatment times and space.", "However, in larger-scale implementation, it would be difficult to establish similar contact between the solute and solvent as in small-scale, even if advanced dispersal techniques were applied (Freire et al., 2012; Zhang et al., 2010).", "Large-scale application for the implementation of hydrophobic ionic liquids based extraction processes has its limitations due to non-stable fluorinated anions which may pose an environmental threat if disposed of in waste stream without proper treatment.", "So more focus can be concentrated on the application of hydrophilic ionic liquids and the use of efficient techniques by coupling to provide a safer and environmental friendly approach for the treatment of ILs contaminated wastewater effluents.", "Though ionic liquid-based water purification has many advantages, but due to the expensive reagents in the synthesis of ILs make a major hurdle in the commercialization of ILs based treatment technologies.", "Recently many approaches have been developed, which include the application of less expensive salt, recovery for reuse of ILs for different treatment cycle, which can somehow reduce the overall cost of the treatment process.", "In 2002, the low-cost ionic liquid based on methyl and ethyl sulfate ions was synthesized by using 1-methylimidazolium (US$ 65/250 g) with dimethyl sulfate (US$ 20/250 mL) and diethyl sulfate (US$ 30/250 mL) (Holbrey et al., 2002).", "The choline-based salts are not very expensive and it was reported that the choline chloride cost US$ 25 (250 g) could reduce the IL synthesis price (Andreani and Rocha, 2012).", "The cost of bulk production of triethylammonium hydrogen sulfate and 1-methylimidazolium hydrogen sulfate was reported as $1.24/kg and $2.96–5.88/kg respectively, which were rather comparable with the traditional solvent, for example, ethyl acetate ($1.30–1.40/kg) (Chen et al., 2014).", "Ionic liquid based water treatment technologies have great potential and by utilization of less expensive salt, varying the ILs cations and anions, the price could be somehow decreased.", "Furthermore, recovery by an efficient method and reuse many times can further reduce the overall treatment cost.", "Several factors are associated with ILs based separation technologies.", "For large scale application, there will be the need for enough efforts for the process engineering changes and requirement of new equipment and also competition with conventional technologies by means of economic feasibility and sustainability.", "Environmental issues associated with the IL based separation must be addressed during large-scale application by recovery and treatment of ILs wastewater effluents.", "Though at very low concentrations (<1000 ppm) extraction usually becomes highly uneconomical because of the removal of solvent residues and recovery of solvents from the extract stream (Haan and Bosch, 2013), but from an environmental point of view, it is a necessary step for ILs application by any industries.", "Some of the major concern with the commercialization of ILs based separation technologies are ILs purity, stability, recycling, reuse, and disposal.", "Variety of ionic liquids have been developed for different applications by changing cation and anion and alkyl chain according to the requirement.", "IL based technology for the removal of organic and inorganic pollutants has been investigated by several researchers.", "Major challenges in solvent extraction are related to sufficient dispersal of the IL during extraction, separation, and regeneration of the IL after extraction, although advanced techniques are available for these purposes, too.", "The possibility to decompose some environmental pollutants directly in the IL can be an advantage of solvent extraction.", "Membrane-based technologies offer high selectivity with reduced leakages of ILs into the aqueous phase, and supported IL membranes, in particular, could be feasible for wastewater treatment.", "Separation processes are highly dependent on the pollutant, IL and experimental conditions.", "Furthermore, IL based adsorbents have been developed for the removal of organic pollutants and in most of the cases, IL modified adsorbent showed enhanced efficiency.", "Though ILs are very efficient in water treatment, at the same time ILs also pose an environmental threat due to its high solubility in water and release the toxic anions and cations.", "Toxicity assessment and recovery/treatment is needed before IL contaminated water is being discharged in water bodies.", "The benefits of using ILs for the removal or organics need to be clearly established if there's any risk of contaminating the water with a compound that is even more difficult to degrade.", "Adsorption techniques especially bio-waste, activated carbon and magnetic material was reported to be effective for the reclamation of ILs without generation of by-products.", "AOPs base technologies have been investigated by several researchers for the degradation of ILs.", "However, sometime the degraded by-product of ILs may enhance the eco-toxicity.", "By coupling of different technology with Fenton process or other AOPs, this drawback can be overcome.", "Research should be focused on this direction to avoid secondary pollutant during IL degradation.", "So, there is a need to develop a systematic approach for the recovery of ILs for reuse and proper treatment of remaining ILs in wastewater.", "As the current experience of using IL-based processes for wastewater treatment relies strongly on analytical applications, strong efforts should be made to demonstrate efficient and environmentally sound water treatment processes for large scale application by giving more focus on developing the non- hazardous ionic liquids." ]	[ "Ionic liquids have a potential for removal of wide range of organic pollutant.", "Scale-up of IL-based separation technologies requires process intensification.", "Presence of toxic anions in ILs is a great issue for large-scale application.", "Coupling of different technologies for the treatment of IL contaminated wastewater." ]
Grasp preparation modulates early visual processing of size and detection of local/global stimulus features	Preparing to grasp objects facilitates visual processing of object location, orientation and size, compared to preparing actions such as pointing. This influence of action on perception reflects mechanisms of selection in visual perception tuned to current action goals, such that action relevant sensory information is prioritized relative to less relevant information. In three experiments, rather than varying movement type (grasp vs point), the magnitude of a prepared movement (power vs precision grasps) was manipulated while visual processing of object size, as well as local/global target detection was measured. Early event-related potentials (ERP) elicited by task-irrelevant visual probes were enhanced for larger probes during power grasp preparation and smaller probes during precision grasp preparation. Local targets were detected faster following precision, relative to power grasp cues. The results demonstrate a direct influence of grasp preparation on sensory processing of size and suggest that the hierarchical dimension of objects may be a relevant perceptual feature for grasp programming. To our knowledge, this is the first evidence that preparing different magnitudes of the same basic action has systematic effects on visual processing.	[ "NEUR", "PSYC" ]	[ "Action", "Global", "Local", "Perception", "Size" ]	[ "2805", "3205", "3206" ]	[ "It is well known that the visual properties of objects can influence subsequent motor processing, for example features such as spatial location, orientation and size of objects can automatically prime corresponding motor parameters (Craighero, Fadiga, Umilta, & Rizzolatti, 1996; Hommel & Prinz, 1997; Tucker & Ellis, 1998).", "These findings are interpreted as evidence for a tight coupling of action and perception and current theories suggest that this coupling is bi-directional (Hommel, Müsseler, Aschersleben, & Prinz, 2001).", "The theory of event coding proposes a shared representation for perception and action, resulting in bidirectional effects between the two domains such that observing events activates associated motor actions, and performing actions also activates associated perceptual events (Prinz, 1984).", "The ways in which perception can influence action have been widely investigated (for a review see Vogt & Thomaschke, 2007), however much less is known about how exactly the planning of simple actions can modulate online visual perception.", "Most of the evidence for effects of action on perception comes from motor-visual priming paradigms, which investigate perceptual processing of stimuli that share relevant features to a planned action, and have revealed that perception can indeed be biased toward action relevant features.", "Early behavioural experiments (Craighero, Fadiga, Rizzolatti, & Umilta, 1999) demonstrated that the processing of a visual stimulus is facilitated if the stimulus has the same orientation as a prepared grasping action.", "Subsequent evidence for motor-visual priming has compared grasping and pointing movements and demonstrated that the processing of object size is selectively enhanced during grasp preparation (Fagioli, Hommel, & Schubotz, 2007) as well as processing of object orientation (Bekkering & Neggers, 2002; Gutteling, Kenemans, & Neggers, 2011; Hannus, Cornelissen, Lindemann, & Bekkering, 2005).", "These findings suggest that action preparation may tune incoming sensory information to the perceptual features relevant for the upcoming action, resulting in a bias in visual processing to match the prepared action.", "Whereas there are now numerous behavioural studies showing effects of action preparation on vision, the neural correlates of motor-visual priming remain largely uninvestigated.", "One exception using electrophysiology (Wykowska & Schubö, 2012) combined a movement task (grasping vs pointing) and a visual search for size and luminance targets and found facilitated performance on action-perception ‘congruent’ conditions (i.e., grasping facilitated size targets and pointing facilitated luminance targets).", "This pattern was reflected by a modulation of early event-related potential (ERP) components, providing supporting evidence that action affects early perceptual processing.", "That study found that qualitatively different actions (i.e., grasping vs pointing) can prime different aspects of visual processing, demonstrating a large-scale effect of action preparation on visual processing.", "However, it remains unclear whether preparing different versions of the same basic action can also lead to subsequent differences in visual processing.", "If action preparation indeed tunes incoming sensory information toward features relevant to the prepared action, then specific perceptual features (e.g., large vs small objects) should be modulated, as well as the broader feature dimensions (e.g., size vs luminance targets) previously investigated (Wykowska & Schubö, 2012).", "The key aims of the current study were i) to further investigate to extent to which action preparation can influence visual processing, specifically whether varying preparation of the same grasping action in terms of grasp magnitude and force (i.e., a precision vs a power grasp) would selectively enhance the processing for local vs global aspects of a compound visual stimulus (Navon, 1977), respectively, and ii) to measure visual evoked potentials associated with a small or large probe object in a context of a prepared small (precision) or large (power) grasping action.", "A number of findings suggest that the hierarchical structure of stimuli may be influenced by grasp preparation.", "For example Vainio, Ellis, Tucker, and Symes (2006) found an object affordance size effect (size of task irrelevant objects modulates power/precision grasp responses), however the effect only occurred when holding a precision device in the right hand and the power device in the left hand.", "In a further study (Vainio, Ellis, Tucker, & Symes, 2007), right hand responses to the ‘local’ component of an object (e.g., the stalk of a fruit) were facilitated when it was part of a precision-graspable ‘global’ object (e.g., a strawberry) while left hand responses to the same object were facilitated when it was part of a power-graspable object (e.g., an apple).", "These findings suggest that object information pertaining to power and precision grasping is predominantly processed in the right and left hemispheres, respectively, and that the processing of hierarchical structure of objects is linked to power/precision grasping actions.", "More recently Gable, Poole, and Cook (2013) also used unilateral hand contractions to activate the right or left central parietal hemispheres and observed behavioural facilitation of global and local processing, respectively.", "Local/global processing is also modulated when stimuli are presented near the hands (Davoli, Brockmole, Du, & Abrams, 2012; Langerak, La Mantia, & Brown, 2013; Thomas, 2015), effects often interpreted as a facilitation of perceptual information relevant to covert manual action preparation (Gozli, West, & Pratt, 2012; Makin, Holmes, Brozzoli, & Farnè, 2012; Reed, Betz, Garza, & Roberts, 2010).", "Additionally, evidence from a variety of approaches suggests that both precision/power grasping and local/global processing may share a similar pattern of hemispheric lateralization.", "Findings have long supported the notion that the local and global levels of hierarchical stimuli are predominantly processed in the left and right hemispheres, respectively.", "Behavioural (Hübner, 1998; Van Kleeck, 1989) as well as imaging studies using Positron Emission Tomography (PET) (Fink, Marshall, Halligan, & Dolan, 1998), functional Magnetic Resonance Imaging (fMRI) (Fink et al., 1996, 1997) and electrophysiology (Evans, Shedden, Hevenor, & Hahn, 2000; Malinowski, Hübner, Keil, & Gruber, 2002) support the lateralization of global (right hemisphere) and local (left hemisphere) processing.", "Some causal evidence is provided from neuropsychological studies of patients with left/right temporal-parietal lesions exhibiting selective impairment in local/global stimulus processing (Robertson & Lamb, 1991; Robertson, Lamb, & Knight, 1988).", "Although, some inconsistencies exist within the neuropsychological literature, as a common feature of Bálint's syndrome is simultanagnosia, a selective impairment in global stimulus processing with intact local processing, which results from bilateral damage to parieto-occipital junction (Farah, 1990), However in healthy subjects, causal evidence for an asymmetry was provided by Romei, Thut, Mok, Schyns, and Driver (2012) who impaired global processing with right-parietal repetitive Transcranial Magnetic Stimulation (rTMS) and local processing with left-parietal rTMS.", "In the current study, motor-visual priming of local/global stimulus features was investigated in three experiments.", "In Experiment 1, participants were required to detect a target stimulus presented at the local or global level of a compound stimulus following a cue to prepare either a power or precision grasp.", "If the magnitude of grasp preparation biases visual processing toward stimulus features relevant for the upcoming action, then detection of local targets should be facilitated during the preparation of precision grasping relative to power grasping, and detection of global targets should be facilitated during power grasping, relative to precision grasping.", "Experiment 2 sought to replicate findings from Experiment 1 while also reducing the number of local elements within the compound stimuli, a manipulation known to reduce the commonly observed global bias in tasks using hierarchical stimuli (Kimchi, 1988; Martin, 1979; Yovel, Yovel, & Levy, 2001).", "This was done in order to investigate whether effects of action preparation on local/global processing are dependent on the commonly observed global bias in visual processing.", "Experiment 3 utilized the fine temporal resolution of electroencephalography (EEG) to investigate the effects of grasp preparation on processing of visual size, aiming to directly demonstrate a selective effect of grasp preparation on early stages of processing in visual cortices.", "This experiment also enabled a further investigation of the behavioural effects of global/local processing during grasp preparation using this adapted experimental design.", "Similarly to Experiments 1 and 2, participants were cued to prepare a power or precision grasp before being presented with a compound stimulus.", "Also as in Experiments 1 and 2, they were instructed to detect a target shape that could appear at either the local or the global level of the compound stimulus, or it could be absent from the display.", "On two thirds of trials, during the cue-target interval, a task-irrelevant visual probe was presented that could either be relatively small or large.", "In visual processing, enhanced ERP components have been observed in response to a task-irrelevant visual probe stimulus presented in an attended area relative to an unattended area of space (Hillyard & Anllo-Vento, 1998; Hillyard, Vogel, & Luck, 1998).", "Similar effects have been observed at the goal location of eye movements (Eimer, Van Velzen, Gherri, & Press, 2006; Eimer, Van Velzen, Gherri, & Press, 2007) and at effector and goal locations of reaching movements during movement preparation (Gherri, Van Velzen, & Eimer, 2009; Job, de Fockert, & van Velzen, 2016; Mason, Linnell, Davis, & Van Velzen, 2015) reflecting adaptive modulation of sensory processing tailored to the specific movement being prepared.", "Recent data suggest that the early P1/N1 components can also reflect a biasing mechanism operating on processing of other stimulus features, not just spatial locations (see Zhang & Luck, 2009).", "If the behavioural effects of grasp preparation reflect a similar adaptive sensory modulation we would expect that early ERPs elicited by the visual probes should be modulated in line with the probes' compatibility with the prepared grasp.", "This would mean that early components (P1 and N1) elicited by the large probe should be enhanced in amplitude during the preparation of a power grasp relative to a precision grasp, while the components elicited by the small probe should be enhanced during precision relative to power grasps.", "A total of 16 participants (13 female) with a mean age of 21 years (SD = 4.02) participated in return for course credits or £10.", "All participants were right handed (mean laterality quotient (Veale, 2014) = 92.21, SD = 11.06) and reported normal or corrected to normal vision.", "Participants provided written informed consent in accordance with the ethical guidelines presented in the 1964 declaration of Helsinki.", "A local/global target detection task run with E-prime software (Schneider, Eschman, & Zuccolotto, 2002) required participants to respond when presented with a target shape stimulus.", "The target shape could appear at either the local or the global dimension of a compound stimulus (target present trials) or the stimulus could be comprised only of non-target shapes (target absent trials).", "Compound stimuli were composed of 13–20 local shapes (squares, circles or crosses) (.76° visual angle) arranged into a global configuration (square, circle or cross) (3.8° visual angle).", "All shape combinations at the local and global dimensions were presented, excluding same-shape combinations.", "On each trial, before target presentation, a coloured fixation cross (.91° visual angle) instructed participants to prepare either a power or a precision grasp (see Figs. 1 and 2 for schematics of the response devices and trial procedure, respectively).", "At 1000 msec following the grasp cue a compound stimulus was presented for 250 msec.", "Participants were instructed to execute the prepared grasp as fast as possible if a pre-specified target shape (square) was present at either the local or the global dimension and to withhold the grasp in the absence of the target shape.", "A black fixation cross was presented during the inter-trial interval (ITI) for either 800 msec, 900 msec or 1000 msec, randomly.", "Following provision of written consent and the revised Edinburgh Handedness Inventory (Veale, 2014), participants completed a practice block of 20 randomised trials, followed by six blocks of 60 randomised trials with self-timed breaks between blocks.", "The entire session lasted approximately 25 min.", "The mapping between the colour of the cue (blue/green) and the grasp (power/precision) was counterbalanced across participants.", "The hand used to execute the grasps was also counterbalanced such that half of participants used their right hand for even numbered blocks and their left hand for odd numbered blocks while the reverse was true for the remaining half of participants.", "Reaction times (RTs) in response to local and global targets were compared using a 2 × 2 × 2 repeated measures Analysis of Variance (ANOVA) with factors of target level (local/global), grasp (power/precision) and hand (left/right).", "Table 1 shows the mean percentage errors made across conditions.", "As errors were made on an average of only 3.5% of trials, they were not further analysed and only trials in which a correct response was recorded were included in the reaction time analysis.", "Fig. 3 shows the mean RTs to global and local targets using power and precision grasps with the left (a) and right (b) hand.", "A main effect of grasp magnitude was observed with faster execution of precision grasps (M = 421, SD = 79.06) than power grasps (M = 439, SD = 88.94) by 18 msec (SE = 6.73), F(1, 15) = 7.32, p = .016, ηp2 = .328.", "A main effect of target level was also observed with faster responses for global (M = 418, SD = 85.59), compared to local targets (M = 442, SD = 82.42) by 24 msec (SE = 5.18), F(1, 15) = 22.74, p < .001, ηp2 = .603.", "There was no main effect of the hand used, F(1, 15) = .58, p = 456, ηp2 = .048 or interactions involving the factor of hand.", "An interaction between grasp magnitude and target level was observed (F(1, 15) = 6.40, p = .023, ηp2 = .299) indicating that the cued grasp magnitude influenced RTs to local and global targets.", "Post-hoc t-tests confirmed that responses to local targets were faster using precision grasps (M = 427, SD = 72.68) compared to power grasps (M = 456, SD = 87.32) by 29 msec [standard error of the mean (SEM) = 7.99], t(15) = 3.63, p = .002.", "The effect of grasp was not significant for global targets (t(15) = .93, p = .365).", "The results of Experiment 1 show that the preparation of either a precision or power grasp can selectively modulate the processing local visual information.", "Local level targets were detected faster following preparation of a precision grasp, relative to a power grasp.", "This finding suggests that variation in the same basic action (a grasp) can selectively influence detection of a subsequent visual local target.", "In contrast to previous evidence suggesting a hemispheric asymmetry for power and precision grasping (Vainio et al., 2006, 2007) the hand used to execute the movements had no influence on power/precision response times in Experiment 1.", "The effect of the prepared grasp was only present in terms of detection of local targets, whereas global target detection was unaffected by grasp preparation.", "A possible factor contributing to this asymmetry is that we used compound stimuli that consisted of a relatively large number of densely organized local elements.", "Previous work has shown that the magnitude of global bias is dependent on stimulus features such as size and density (Kimchi, 1988; Martin, 1979; Yovel et al., 2001).", "For example displays with densely arranged local elements spaced close together promote a strong global precedence (Caparos, Linnell, Bremner, de Fockert, & Davidoff, 2013; Enns & Kingstone, 1995; Martin, 1979), meaning that global target detection is greatly facilitated relative to local target detection.", "In the current study, responses were indeed substantially faster to global, compared to local, targets, which may have obscured any subtle effects of grasp preparation on detection of global targets.", "Experiment 2 was therefore run in order to replicate the findings of Experiment 1 and to investigate the effects of action preparation on detection of local and global targets in displays in which the global level of the compound stimulus was made less salient by using fewer and less densely organized local elements.", "A total of 16 adults (12 female) with a mean age of 24 years (SD = 4.83) participated in return for £10.", "All participants were right handed (mean laterality quotient (Veale, 2014) = 87.5, SD = 23), and reported normal or corrected to normal vision.", "Participants provided written informed consent in accordance with the ethical guidelines presented in the 1964 declaration of Helsinki.", "The task was identical to Experiment 1, except the compound stimuli consisted of fewer local shapes (9–12) spaced further apart (see Fig. 4).", "This manipulation of the saliency of the global level has been previously successfully used in order to reduce the relative saliency of the global level (Caparos et al., 2013).", "Errors were made on an average of only 2.9% of trials, so were not further analysed.", "RTs were analysed using the same 2 (target level: local vs global) × 2 (grasp: power vs precision) × 2 (hand: left vs right) ANOVA as Experiment 1.", "Fig. 5 shows the mean RTs to global and local targets using power and precision grasps with the left (a) and right (b) hand.", "A main effect of grasp magnitude was observed with faster precision responses (M = 468, SD = 83.68) compared to power (M = 489, SD = 83.25) by 21 msec (SE = 5.37), F(1, 15) = 15.82, p = .001, ηp2 = .513.", "No main effect of target level (F(1, 15) = .838, p = .374, ηp2 = .053) or hand (F(1, 15) = .008, p = .931, ηp2 = .001) was found.", "A marginally significant interaction between the target level (local/global), the grasp magnitude (power/precision) and the hand used (left/right) was observed, F(1, 15) = 4.56, p = .050, ηp2 = .233.", "To investigate this interaction further, separate two-way ANOVAs with factors of level (local/global) and grasp magnitude (power/precision) for each hand confirmed a significant interaction between level and grasp, F(1, 15) = 7.97, p = .013, ηp2 = .347 for right hand responses.", "Post-hoc t-tests revealed that right hand responses to local targets were faster using precision (M = 460, SD = 68.61) relative to power (M = 498, SD = 79.85) grasps by 38 msec (SEM = 9.21), t(15) = 4.09, p = .001.", "This difference was not significant for left hand responses to global targets,1 t(15) = −.42, p = .684.", "The two-way interaction between target level and grasp magnitude was not significant for left hand responses, F(1, 15) = .409, p = .532, ηp2 = .027.", "The results from Experiment 2 replicated the core finding of Experiment 1 that local level targets are detected faster following preparation of a precision grasp, relative to a power grasp.", "This time the effect of grasp preparation was present in the absence of a main effect of target level.", "The influence that grasp preparation has on the processing of local/global information is therefore not dependent on the commonly observed global bias in visual processing.", "Unexpectedly, the effect of grasp magnitude on local/global processing in Experiment 2 was only observed in the dominant right hand, compared to Experiment 1 where the effect was not influenced by the hand used to execute the movement.", "It is unclear why reducing the number of local elements, and in turn the global bias, would limit the effect of grasp preparation on local processing to the right hand.", "Perhaps the relative increase in the saliency of the local level, predominantly processed by left hemisphere structures (Hübner, 1998; Van Kleeck, 1989), resulted in the right hand specificity of the effect.", "In addition to this, regardless of the hand used to execute the grasps, precision responses were faster than power responses across both Experiments 1 and 2.", "This is not in line with the notion that, relative to the right hand, the left hand may be specialized for power grasping (Guiard, 1987).", "Experiment 3 was run to obtain a direct measure of the selective effects of grasp preparation on early stages of visual processing in the brain and to replicate the action preparation effect observed in Experiments 1 and 2 using a version of the local/global paradigm adapted for this purpose: We used a similar design to Experiments 1 and 2, combining an action preparation task with a Navon task (1977) requiring detection of a visual target at either the local or global level of a compound stimulus.", "Additionally, on each trial a task-irrelevant visual probe was presented following action preparation.", "The probe could be either small or large in size, and the key prediction was that early probe-evoked visual ERPs would be enhanced for small cues following preparation of a precision (vs power) grasp, and enhanced for large probes following preparation of a power (vs precision) grasp.", "A total of 16 adults (13 female) with a mean age of 25 years (SD = 3.85) participated in return for £10.", "All participants were right handed (mean laterality quotient = 87.06, SD = 17.00) and reported normal or corrected to normal vision.", "Participants provided written informed consent in accordance with the ethical guidelines presented in the 1964 declaration of Helsinki.", "Stimuli and task were identical to Experiment 2, except for the following aspects: At 1000 msec following the cue prompting participants to prepare either a precision or a power response, a task-irrelevant visual probe stimulus was presented that could be either large (4.8° visual angle), small (1.6° visual angle) or absent with equal probability.", "Probes were presented for 100 msec, and 600 msec after probe onset, the compound stimulus was presented for 250 msec.", "Following an error (incorrect grasp) or a time-out (no response within 1200 msec) a feedback tone was delivered via two speakers symmetrically aligned in front of participants.", "The contrast of the presentation was also reversed in Experiment 3 with white stimuli presented on a black background.", "Stimuli were presented using the Psychophysics Toolbox extensions (Brainard, 1997) version 3.0.12 implemented in MATLAB (R2014b, version: 8.4).", "Fig. 6.", "Following provision of written consent and the (revised) Edinburgh Handedness Inventory (Veale, 2014), participants completed a practice block of 30 randomised trials, followed by eight blocks of 100 randomised trials with self-timed breaks between blocks.", "The experimental task lasted approximately 45 min.", "The mapping between the colour of the cue (blue/green) and the grasp (power/precision) was counterbalanced across participants.", "Given that effects of interest were limited to the right hand in Experiment 2, only right hand responses were recorded in Experiment 3.", "EEG was recorded using a BioSemi ActiveTwo amplifier from 64 Ag–AgCl electrodes placed according to the extended 10–20 system at a digitisation rate of 2048 Hz and down sampled offline to 1024 Hz.", "Electrodes were referenced to the average of electrodes placed on the left and right earlobes.", "Activity from horizontal eye movements was recorded from a pair of electrodes placed on the outer canthi of the eyes.", "Vertical eye movement activity was recorded from electrodes placed above and below the left eye.", "Offline pre-processing of EEG data was conducted using EEGLAB toolbox version 13.4.4b (Delorme & Makeig, 2004).", "Analysis was conducted using a combination of FieldTrip toolbox (Oostenveld, Fries, Maris, & Schoffelen, 2011) and custom MATLAB scripts.", "For analysis of the probe-evoked potentials, continuous EEG data were divided into 700 msec epochs locked to the onset of the probe including a 100 msec pre-stimulus baseline.", "Epochs including voltages exceeding + and/or −100 μV were automatically rejected prior to analysis.", "Eye-blink artefacts were corrected for using Independent Component Analysis (ICA).", "The mean amplitudes of ERP components within pre-defined time windows were extracted for analysis.", "The mean positive amplitude between 70 and 110 msec post probe onset was extracted as the P1 mean amplitude.", "The mean of negative amplitudes between 80 and 120 msec post probe onset was extracted as the N1 mean amplitude.", "Peak measures were extracted from electrode sites PO7 and PO8, which elicited the largest ERPs as observed in scalp maps of averages over all conditions.", "The difference between the mean P1 and N1 values was computed to obtain a mean peak-to-peak amplitude measure of the N1 component.", "For the ERP analysis, the mean peak-to-peak amplitudes of the N1 component evoked by probe stimuli were analysed in a 2 × 2 × 2 repeated measures ANOVA with factors of grasp magnitude (power/precision), probe size (large/small) and electrode hemisphere (PO7/PO8).", "For the behavioural analysis, correct RTs to targets presented at the global and local dimension of the compound stimuli were compared using a 2 × 2 × 2 repeated measures ANOVA with factors of target level (local/global), grasp (power/precision) and probe (large/small).", "Fig. 7 shows the mean RTs for local and global targets presented after a large (a) or small (b) probe stimulus as well as after no probe was presented (c).", "A main effect of grasp magnitude was found with faster precision (M = 440, SD = 74.15) compared to power (M = 473, SD = 68.68) grasp responses by 33 msec (SE = 5.46), F(1, 15) = 35.55, p < .001, ηp2 = .703.", "A main effect of target level was also found with faster responses for global (M = 448, SD = 77.77), compared to local targets (M = 465, SD = 78.26) by 17 msec (SE = 5.63), F(1, 15) = 8.85, p = .009, ηp2 = .371.", "A significant interaction between target level and size of the probe was observed, F(1, 15) = 14.56, p = .002, ηp2 = .493.", "The interaction between grasp magnitude and target level did not reach statistical significance (F(1, 15) = 2.09, p = .169, ηp2 = .122) and neither did the interaction between grasp magnitude and probe size F(1, 15) = .805, p = .384, ηp2 = .051.", "However a significant interaction between target level, grasp magnitude and probe size was found, F(1, 15) = 13.80, p = .002, ηp2 = .479.", "This indicates that the mean RTs to global/local target stimuli were influenced both by the relative size of the task-irrelevant probe preceding the target as well as the prepared grasp.", "To investigate this interaction further, ANOVAs with factors of grasp magnitude (power/precision) and target level (global/local) were used for each probe condition (large/small) separately.", "Following the presentation of a large probe stimulus (Fig. 7a), the interaction between grasp magnitude and target level was significant (F(1, 15) = 7.46, p = .015, ηp2 = .332) and post-hoc t-tests confirmed that responses to local targets were faster using a precision grasp (M = 443, SD = 61.51), compared to a power grasp (M = 489, SD = 63.92) by 46 msec (SEM = 6.15), t(15) = 7.50, p < .001.", "This difference was not significant for global targets, t(15) = 1.01, p = .331.", "The interaction between grasp magnitude and target level was also not significant following small probes (Fig. 7b), F(1, 15) = .251, p = .624, ηp2 = .016.", "Similarly, an ANOVA with factors of grasp magnitude (power/precision) and target level (global/local) was used for probe absent trials (Fig. 7c) and revealed no significant interaction between grasp magnitude and target level, F(1, 15) = 2.50, p = .135, ηp2 = .143.", "Fig. 8 shows the grand averaged event-related potentials (ERPs) elicited by the task-irrelevant probe stimuli.", "For the N1 mean peak-to-peak amplitude, no main effects of grasp magnitude (power/precision), probe (large/small) or electrode hemisphere (PO7/PO8) were observed (F(1, 15) = .92, p = .352, ηp2 = .058, F(1, 15) = 3.28, p = .090, ηp2 = .108 and F(1, 15) = 1.66, p = .217, ηp2 = .100, respectively).", "A significant interaction between the size of the probe and the grasp being prepared was observed, F(1, 15) = 8.95, p = .009, ηp2 = .374, as well as a significant interaction between the probe size, grasp magnitude and electrode hemisphere (F(1, 15) = 5.10, p = .039, ηp2 = .254).", "Separate two-way ANOVAs with factors of probe size and grasp magnitude were then run for left and right hemispheres, revealing a significant interaction at left hemisphere sites, F(1, 15) = 13.54, p = .002, ηp2 = .474.", "Post-hoc t-tests confirmed that the mean peak-to-peak size of the N1 component at left electrode sites evoked by large probes was enhanced during the preparation of power grasps (M = 4.22, SD = 4.17), relative to precision grasps (M = 3.15, SD = 4.08), by 1.07 μV (SD = 1.00), t(15) = 4.25, p = .001.", "The reverse effect was observed for the N1 at left electrode sites evoked by small probes, with marginally larger mean amplitudes during the preparation of precision grasps (M = 3.13, SD = 3.17), relative to power grasps (M = 2.37, SD = 3.76) by .76 μV (SD = 1.33), t(15) = −2.30, p = .036 (p = .072 following correction for multiple comparisons).", "At right hemisphere electrode sites the interaction between probe size and grasp magnitude was non-significant, F(1, 15) = 2.50, p = .135, ηp2 = .143.", "The results provided here are the first to show an influence of different versions of the same basic action, power and precision grasps, on visual perception of hierarchical information.", "Experiment 1 demonstrated that the preparation of either small or large grasping actions modulates RTs to local targets presented in compound stimuli.", "Faster RTs were observed for targets presented at the local level of compound stimuli with a precision grasp, relative to a power grasp.", "Experiment 2 manipulated the relative saliency of the global level by using fewer local elements in the compound stimuli and observed the same influence of grasp cueing on RTs for local targets, for right hand responses.", "This shows that the effect of action preparation is not dependent on the commonly observed global bias.", "Experiment 3 showed that grasp preparation biased early visual ERPs elicited by task-irrelevant probes of varying sizes.", "The visual N1 component was enhanced for large probes during power, relative to precision, grasp preparation and marginally enhanced for small probes during precision, relative to power, grasp preparation.", "This demonstrates a direct effect of grasp preparation on early stages of visual processing.", "Effects of manual reaching and eye movement preparation on sensory processing have been linked to overlapping brain networks involved in action and attention (Astafiev et al., 2003; Corbetta, 1998; Corbetta et al., 1998; Corbetta & Shulman, 2002).", "Whether similar links exist for grasping movements remains to be determined and future studies should elucidate the brain mechanism activated by a grasp instruction.", "Furthermore, in Experiment 3, a behavioural effect of probe size on local/global target detection was modulated by grasp, such that large (vs small) probes only facilitated global (vs local) processing during power (vs precision) grasp preparation.", "Previous evidence has shown that action preparation can influence visual perception of stimulus features such as orientation (Bekkering & Neggers, 2002; Hannus et al., 2005) and size (Fagioli et al., 2007; Wykowska & Schubö, 2012).", "These examples show that visual information is biased toward stimulus features relevant for upcoming action, representing the tight coupling of action and perception.", "Here, the bias is extended to include not only early visual perception of size, but of hierarchical stimulus features.", "In contrast to the previous evidence that compared the influence of qualitatively different actions (e.g., grasping vs pointing) on visual perception of overall feature dimensions relevant to the actions (e.g., orientation/size and colour/luminance), the findings presented here demonstrate a more fine-grained influence whereby varying the magnitude of the same basic grasping action influences subsequent visual processing.", "This is likely an important aspect of the adaptive control of movement, such that perceptual features most relevant to the upcoming action are facilitated in visual processing, compared to less relevant features.", "Additionally, previous demonstrations of action-modulated vision manipulated the prepared action in blocks, rather than randomly cueing actions on each trial, with the exception of Wykowska and Schubö (2012), who instead presented the size or luminance targets in separate blocks while varying the cued action trial-by-trial.", "Our design demonstrated modulated visual information during action preparation where both the cued action (power/precision grasp) and the target stimulus feature (local/global) are manipulated randomly on each trial.", "This demonstrates a more dynamic adjustment to visual processing as a consequence of action preparation, without the possible confound associated with participant's prior knowledge of the action and/or target stimulus feature before trial onset.", "Although grasp preparation altered RTs to the compound stimuli in Experiments 1 and 2, this was not replicated in the probe-absent trials of Experiment 3 as expected.", "An effect of approximately 35 msec in Experiments 1 and 2 was reduced to just 10 msec in Experiment 3.", "Only following a large probe stimulus, was an effect of grasping observed on RTs to global/local target stimuli in Experiment 3.", "A number of differences between the experiments, implemented to enable the use of EEG, may have contributed this loss of effect.", "For example, additional trial numbers were required for Experiment 3 and the contrast was also reversed so stimuli were white on a black background.", "Furthermore, the longer cue-target interval in Experiment 3 (+600 msec), which was used to ensure that trials had the same length as those in which a probe was presented, may have played a role.", "Modulations of sensory processing in the context of the specific action being prepared have been demonstrated to be temporally dynamic (Mason et al., 2015) and further research is required to determine how grasp preparation affects sensory processing over time.", "Finally, the mere presence of the visual probes in Experiment 3 may have affected the action-perception effect in general, even on the no-probe trials.", "Exactly how action-perception interactions are affected by the context in which they are measured is an intriguing question that warrants further work.", "Given that RTs to local/global target stimuli in the tasks are gathered from execution of the cued movement itself, it is possible that perception of the local/global target facilitated the action, rather than the inverse.", "It seems unlikely that the reaction time effects observed here reflect visual-motor, rather than motor-visual, priming for a number of reasons.", "First, participants are always cued to prepare the grasp up to 1600 msec before the onset of the compound stimulus, so visual-motor priming would imply a strategy of inhibiting a movement cue that is highly informative, until after onset of the compound stimulus.", "More importantly, the pattern of probe-evoked potential effects from Experiment 3 shows a selective influence of the movement cue on visual processing before onset of the compound stimuli.", "Nevertheless, it is conceivable that visual-motor priming is contributing to some extent to the reaction time effects of grasping on local/global target detection presented here.", "Separating the cued motor response from the perceptual decision in a dual task design may help to elucidate this further.", "It is now widely accepted that perception and action are tightly coupled, such that bidirectional influences exist between the two domains.", "While the influence of perceptual information on motor processing has been extensively studied, much less is known about how action preparation influences perceptual processing.", "Actions such as grasping appear to influence the online perceptual processing of features relevant to the upcoming action, which likely reflects a mechanism of selection in visual processing that prioritizes action relevant information in the environment.", "Previous demonstrations of modulated perception during grasp preparation compared different movement types such as grasping versus pointing.", "Here, the magnitude of the grasp (power vs precision) resulted in specific modulations of early electrophysiological markers of visual perception, as well as faster detection of targets presented at the grasp-relevant dimension of a compound stimulus.", "Overall, the results suggest overlapping brain mechanisms involved in the motor processes of grasping and perceptual processing of size as well as local/global object perception." ]	[]
The commensuration of pain: How nurses transform subjective experience into objective numbers	Commensuration—the transformation of different qualities into a common metric—has recently received increased scholarly attention. While mostly studied at the meso- or macroscale, this article extends the focus to microscale commensuration. Based on fieldwork in a Norwegian emergency medical service, the article analyses how nurses rated patients' pain intensity on a scale from zero to ten. While nurses were instructed to score pain by combining patients' self-report with their own ‘objective’ assessment, the article finds that their actual assessments relied almost solely on the latter. After exploring nurses' objections against the use of self-report, the article reconstructs the principles, methods and beliefs underlying nurses' ‘objective’ approach to pain scoring. In so doing, the article demonstrates how a fundamental aspect of human experience was filtered through nurses' professional gaze—to the advantage of some patients and the disadvantage of others. The article also advances theorising on microscale commensuration, thus providing guidance for future studies of how the world is transformed through the everyday production of numbers.	[ "ARTS", "SOCI" ]	[ "Commensuration", "Discretion", "Ethnography", "Norway", "Pain assessment", "Quantification", "Standardisation" ]	[ "1207", "3306" ]	[ "In recent decades, social scientists have begun to pay more explicit attention to commensuration, the process of transforming qualitative differences into a common metric (Espeland and Stevens, 1998, 2008).", "Commensuration is a ubiquitous and fundamental feature of modern societies, as evidenced by the proliferation of rankings, cost-benefit analyses and standardised tests across a range of institutional settings.", "While its ubiquity and apparent objectivity can make commensuration easy to overlook, Espeland and Stevens (2008, p. 406) have urged scholars not to take these processes for granted; instead, we should question the work and conventions that underpin the production of seemingly neutral numbers.", "This is particularly crucial because commensuration is a transformative process, emphasising certain aspects of the objects that it measures while downplaying others (Espeland and Stevens, 1998, pp. 314–8).", "Whereas previous research has primarily studied commensuration at the meso- or macroscale, this article extends the focus to microscale commensuration.", "It does so by exploring a puzzling case of pain scoring, drawn from my fieldwork in a Norwegian emergency medical service.", "The service in question was a walk-in clinic similar to emergency departments in other countries.", "Its frontline personnel were triage nurses, who assessed the urgency of patients' complaints.", "As part of their assessment, nurses were required to score patients' pain intensity using the Numeric Rating Scale (NRS).", "The NRS is an 11-point scale ranging from zero (‘no pain’) to 10 (‘the worst pain imaginable’); when used in triage assessments, the NRS score has consequences for priority setting, affecting whether the patient must wait for hours or is seen immediately by a physician.", "To determine a patient's NRS score, the clinic's triage guidelines specified that nurses should score pain by combining their own assessment with the patient's self-reported pain score, respectively referred to by nurses as ‘objective and ‘subjective’ NRS.", "However, nurses did not adhere to these guidelines; instead of combining the two ratings, they relied almost solely on the ‘objective’ NRS, asking patients to self-report their pain score only when the ‘objective’ NRS was considered ambiguous or inconclusive.", "Such deviations from guidelines are of course not uncommon (cf. Timmermans and Epstein, 2010), but this particular practice seemed puzzling, as self-report is widely seen as ‘the gold standard’ for pain assessment (cf. Pierik et al., 2017).", "Indeed, one influential definition states that pain is “whatever the experiencing person says it is, existing whenever the experiencing person says it does” (cited in Woodrow, 2002, p. 62).", "Additionally, nurses themselves commonly insisted that pain is a subjective phenomenon.", "This raises two important questions: Why did nurses disregard patients ‘subjective’ pain score, and what did their ‘objective’ approach entail?", "To address these questions, the present study employs an ethnographic approach that emphasises Verstehen—i.e. considering “actors' situated intentions, beliefs, and opportunities” (Turco and Zuckerman, 2017, p. 1273) to locate their actions “in an intelligible and more inclusive context of meaning” (Weber, 1978, p. 8).", "This approach serves to foreground the situated logic of nurses' commensuration of pain, which has largely been neglected in previous studies of pain assessments.", "Specifically, the article shows that nurses considered ‘subjective’ NRS to be antithetical to pain assessment, and how they instead relied on a range of ‘objective’ methods and principles they believed would provide more correct, consistent and efficient commensuration.", "While facilitating decision making in triage, this ‘objective’ approach also transformed the phenomenon of pain in significant ways, with systematic consequences concerning who and what was prioritised.", "Thus, in analysing nurses' commensuration of pain, the present article seeks both to contribute to the study of microscale commensuration and to demonstrate how a fundamental aspect of human experience is filtered through nurses' professional gaze.", "In the following, I discuss existing studies of pain and commensuration; describe the study's data and methods, and then present and discuss the study's findings.", "Studies of pain have a long history in the social sciences, dating back at least to Zborowski (1952) Cultural Components in Responses to Pain.", "Comparing different ethnic groups, Zborowski demonstrated significant differences in their attitudes towards and reactions to pain.", "His findings challenged the idea that pain is a purely physical phenomenon and the sole domain of the natural and medical sciences, showing instead that pain has a cultural component and is a suitable subject for social scientific inquiry.", "Following Zborowski, social scientists have shown a clear preference for exploring patients' perspectives on pain, often also contrasting these to how professionals comprehend patients' experiences (cf. Frank, 1997; Greenhalgh, 2001; Kleinman, 1988).", "Concerning the latter, the studies give important demonstrations of the “enormous chasm between embodied experience and professional evaluation” (Bendelow, 2006, pp. 59–60); however, as they predominantly focus on the patient-side of this relationship, they largely leave professionals’ perspectives and practices unexplored.", "Some notable exceptions include macro-oriented studies of how pain is conceptualised within the broader context of biomedical culture (Bendelow and Williams, 1995; Morris, 1993; Scarry, 1985; Vrancken, 1989), and more micro-oriented studies of how clinicians perceive and treat chronic pain patients (Baszanger, 1992; Buchbinder, 2015; Crowley-Matoka and True, 2012).", "However, while these studies provide important insight into the cultural and organisational aspects of pain assessment, they pay less attention to the question of how professionals assess pain in concrete encounters with patients.", "Insofar as the practice of pain assessment has attracted academic attention, this is predominantly in quantitative studies that measure the quality of clinicians' assessments (cf. Duignan and Dunn, 2008; Modanloo, 2010; Pierik et al., 2017; Prkachin et al., 2007; Puntillo et al., 2003; Solomon, 2001; Teanby, 2003).", "Comparing clinicians' with patients' own pain scores, these studies show that clinicians systematically give lower pain scores than patients do, and that the discrepancy is greater for experienced clinicians.", "Moreover, as many studies use patients' self-report score as yardstick, these findings are often taken as evidence of clinicians underestimating patients’ pain and becoming more prone to underestimation as their experience increases (cf. Pierik et al., 2017; Prkachin et al., 2007; Solomon, 2001).", "While giving important evidence of workers assessing pain differently from patients, these studies provide less documentation on how workers go about assessing pain or why their assessments differ from those of patients.", "A notable exception is Vuille and colleagues' (2017) interview study of nurses in a Swiss ED, which shows that ED nurses regularly perceive a difference between their own pain assessment and patients' self-report, that the nurses privilege their own assessment, and that they use different techniques to ensure greater congruence between the two assessments.", "The authors attribute these findings to nurses' pursuit of objectivity, which they in turn interpret as an expression of nurses' self-understanding as professionals.", "While closely aligned with the present article, Vuille and colleagues' study is limited by a relatively small sample (12 nurses) and the use of interviews to draw conclusions about nurses’ practice—limitations that are recognised by the authors themselves, who characterise their study as “an important first step in understanding pain management” (Vuille et al., 2017, p. 675).", "The present article seeks to take further steps, delving deeper into both how and why nurses assess pain as they do.", "In addition to unpacking nurses' pain assessments, this article also contributes to research on commensuration.", "While implicit in much social scientific work (cf. Simmel, 1978), the study of commensuration has been most explicitly formulated by Espeland and Stevens (1998, 2008), who define commensuration as “the transformation of different qualities into a common metric” (1998, p. 314).", "Commensuration is a form of classification, in which people or things are sorted into categories of ‘more or less’; it is often also treated as synonymous with quantification, but we should note a subtle difference between the two.", "Commensuration is a subtype of quantification, in which qualities are reduced to quantities and distinctions between objects become a question of magnitude (Espeland and Stevens, 1998, p. 316); this must be distinguished from quantification that is purely symbolic, as in the differentiation of football players based on their jersey number (Espeland and Stevens, 2008: 407).", "Importantly, commensuration is a transformative process, emphasising certain aspects of the objects being measured while downplaying others.", "As such, commensuration can be understood as “a system for discarding information and organizing what remains into new forms” (Espeland and Stevens, 1998, p. 317).", "To date, research on commensuration has predominantly focused on meso- and macro-level issues.", "One strand of studies traces the increasing emphasis on quantification in modern societies (cf. Desrosières, 2002; Hacking, 1990; Porter, 1996); another elucidates the broader cultural and institutional arrangements that make commensuration possible (cf. Fourcade, 2011; Huault and Rainelli-Weiss, 2011; Khaire, 2014; Samiolo, 2012).", "Representing the latter, a range of studies have explored how difficulties in commensuration have been overcome to create markets for ‘products’ such as art (Velthuis, 2003), carbon emissions (MacKenzie, 2009) and organ transplants (Healy, 2006).", "Another strand explores the unintended consequences of commensuration—a key example being Espeland and Sauder (2007) study of how law school rankings alter the workings of the law schools whose quality they are supposed to rank neutrally and non-intrusively.", "Complementing these meso- and macro-level studies, a small but significant literature has explored commensuration on the ground floor of professional practice.", "For instance, Essén and Oborn (2017) study the ‘performativity’ of numbers in a Swedish rheumatology unit; focusing on the use of metrics in clinical practice, they highlight “the role of numbers in constituting the healthcare reality they are intended to depict” (2017, p. 134).", "Similar to the present article, their study draws attention to metrics that are generated habitually by a range of actors in clinical practice—numbers that are typically produced in a more distributed and discretionary fashion than those published in authoritative reports by corporations and agencies (a dominant case in the larger-level studies).", "However, in contrast to the present article, Essén and Oborn limit their study to how “numbers do things” (2017, p. 134) for patients and physicians—thus focusing on the work numbers do rather than the work of making numbers.", "In shifting the focus to the situated production of numbers, the present article aligns more closely with a few other studies that explore number production in micro-level practice.", "Key contributions are Lamont's (2009) study of how members of peer review panels work to render applications commensurate, and Lynch's (2018) study of how legal actors accomplish—and circumvent—the numbers-based logic of federal sentencing guidelines.", "While less extensive than meso- and macro-level accounts, these microscale studies allow the researchers to zoom in on the distributed number production that occurs routinely in professional settings—often without notice, yet with great significance for peoples' life chances.", "As such, they demonstrate the importance of studying the principles, methods and beliefs that underlie microscale commensuration.", "When seen together, these studies also highlight a salient contrast between differently elaborated forms of commensuration: While Lynch's judges are required to use elaborate guidelines for sentencing, Lamont's peer reviewers have far fewer prescriptions for how to rank applications.", "The nurses in the present study are most closely aligned with the peer reviewers, in that their guidelines leave significant room for discretion (as I return to in the Findings section).", "Moreover, in contrast to the more deliberative settings of court rooms and peer review panels, the present article examines commensuration under conditions of significant time pressure and uncertainty.", "Actors have to ‘think fast’ (Kahneman, 2011) when scoring, and in so doing, they rely on a range of routines and heuristics to elaborate their guidelines (Lipsky, 1980, pp. 63–5).", "As well as exhibiting the same transformative effects as ‘slower’ commensuration, this ‘faster’ commensurative work depends largely on experiential, common-sense and unsanctioned knowledge.", "Scrutiny of fast and decentralised commensuration is therefore highly relevant—especially in professional settings, where workers typically have significant power over clients.", "Thus, in addition to addressing how and why nurses assess pain as they do, the study raises a range of questions of broader relevance for the microscale study of commensuration: How do commensurators commensurate?", "What criteria, methods, principles and broader epistemological concerns underpin their work, and how do these transform the commensurated objects?", "How is their commensuration shaped by the circumstances under which it takes place?", "And what are the broader implications of their work?", "These questions are addressed in the Findings section, but first I discuss the study's data and methods.", "The paper forms part of the author's larger ethnographic project exploring how Norwegian emergency medical workers interpret and prioritise patients (Johannessen, 2018a).", "Ethnography involves the first-hand study of people as they go about their everyday lives (Emerson et al., 2011), which enables the researcher to observe first-hand what people do rather than relying purely on people's own verbal accounts of what they do (Jerolmack and Khan, 2014).", "Accounts are not unimportant—indeed, much of nurses' work cannot be observed directly, including the knowledge and reasoning underlying their assessments—but ethnography allows the researcher to situate the actors' accounts in the context of what they do from day to day, “when all the constraints of their ordinary social situation are operative” (Becker, 1996, p. 62).", "Moreover, through first-hand participation in a social milieu for an extended period, the ethnographer can sample people's sayings and doings in a range of settings and situations, comparing these various data points to make strong inferences about people's reasons for behaving as they do (Becker, 1996; Jerolmack and Khan, 2014).", "This makes ethnography an apt method for unpacking both the hows and whys of nurses' commensuration of pain.", "The article's ethnographic data are drawn from a Norwegian emergency primary care clinic (EPCC).", "In general, EPCCs are heterogeneous institutions, ranging from single physicians on call in rural areas to large-scale urban organisations employing hundreds of workers (Hansen and Hunskår, 2016).", "The largest EPCCs are open to all patients at all times, and in contrast to Norwegian emergency departments (EDs), they allow patients to walk in at their own discretion.", "Because they also serve patients across the entire spectrum of criticality, these large-scale EPCCs resemble EDs in most other Western countries (Vassy, 2014).", "The clinic under study was one such large-scale EPCC.", "It was located in the city centre, performed more than 50,000 consultations per year, employed more than 100 nurses and physicians and was open for 24 h on every day of the week.", "The clinic was intended to serve patients with medical rather than surgical conditions.", "Fieldwork at this EPCC was conducted between April and December 2015.", "The 47 fieldwork sessions had an average duration of approximately 6 h. Of these, 20 sessions were spent observing nurses in triage and asking them about their assessments in close proximity to actual patients, which was crucial because of the ephemeral and largely unspoken nature of nurses' ‘objective’ pain scoring.", "In total, I observed 342 face-to-face assessments, performed by 2 male and 14 female nurses, most of whom were aged between 25 and 35 years.", "All of these nurses held a bachelor's degree in nursing (as is required for the protected title of ‘nurse’ in Norway); on average, they had worked in this EPCC for 3.5 years (range 1–7 years).", "I also attended two mandatory courses in triage nursing and conducted semi-structured interviews with seven nurses, two physicians and two managers, in which pain assessment was an important theme.", "For the purpose of analytical contrast, a further nine fieldwork sessions were conducted at two other emergency institutions.", "The interviews were transcribed verbatim.", "During fieldwork sessions, I scribbled keywords and near-verbatim quotes on a notepad or laptop for subsequent use in more elaborate field notes, yielding approximately 1270 single-spaced pages.", "As all notes were written in Norwegian, I have translated the extracts included here, making minor grammatical and aesthetic adjustments.", "The study was approved by Norwegian Social Scientific Data Services.", "Pseudonyms are used to secure informants' internal and external confidentiality (Tolich, 2004), and no other identifying information is disclosed.", "I signed non-disclosure agreements with the participating EPCCs and secured workers’ informed consent by distributing an information letter and delivering several short presentations on the project.", "When interacting with patients, each EPCC worker I shadowed would ask the patient whether it was acceptable that I witnessed their interaction.", "Analysis began immediately on entering the research setting.", "I learned about nurses' ‘objective’ approach to pain scoring quite early in the fieldwork and subsequently dedicated significant time and attention to understanding what that approach entailed and why they favoured it.", "For the purposes of this article, relevant field notes have been inductively differentiated and iteratively reviewed to explore the logics of nurses' pain scoring.", "However, as the analysis has also been significantly influenced by my reading of the empirical and theoretical literature, it is more appropriately referred to as an abductive process—that is, as a dialogue between data and theory, in which data influences the choice of theory, and theory facilitates the interpretation of data (Swedberg, 2017; Tavory and Timmermans, 2014).", "In considering nurses' commensuration of pain, I begin with a general overview of triage and pain assessments.", "After describing the nurses' objections against ‘subjective’ NRS, I go on to review their ‘objective’ approach, which they considered a more correct, consistent and efficient method of assessing pain.", "Working at the clinic's frontline, the EPCC triage nurses sat in booths and called patients individually.", "Triage was often carried out under hectic conditions, as the waiting room was noisy, the queue to triage could be long, and there was always the risk that those waiting to be triaged might be critically ill.", "As nurses assessed anywhere between 40 and 70 patients during busy shifts, triage was considered tiring work.", "(Having witnessed approximately 60 unique assessments on my first day in the field, I can attest to this.)", "A triage assessment typically lasted 4–8 min, during which time the nurse would record a brief medical history, collect vital parameters and perform simple examinations before assigning an urgency level.", "While all of this information had to be documented for each assessment, their geographical separation from the rest of the clinic meant that there was little direct supervision of nurses' practices.", "To regulate nurses' assessments, the clinic used the Manchester Triage System (MTS), which is currently the most widely used triage system in Europe.", "The MTS is part of a broader trend towards the standardisation of clinical practice; since the late 1980s, the drive towards evidence-based medicine has led to a mass introduction of clinical practice guidelines in healthcare (Timmermans and Berg, 2003).", "These guidelines represent a shift in how professional work is regulated; while earlier attempts sought primarily to streamline working conditions, clinical practice guidelines instead seek “to intervene at the moment of a health care provider's special expertise: medical decision making” (Timmermans and Berg, 2003, p. 13).", "The MTS is less optional in character than most clinical guidelines, aiming for near-exhaustive codification of the decision-making process.", "Designed as a paper-based manual, the MTS consists of 53 flow charts ordered by ‘chief complaints’, such as abdominal pain, allergy, ear problems and head injury.", "Nurses must assess patients using the most relevant of these charts, each of which lists ‘discriminators’ (i.e. symptoms and signs) related to the complaint in question.", "The aim is to order patients in one of five formal levels of priority; known as ‘triage codes’, these levels are referred to by colour: red (most urgent), orange, yellow, green and blue (least urgent).", "(Note: As an instrument for ranking patients' complaints, the MTS is itself a form of commensuration; see Johannessen (2018b; 2018a; 2017) for a discussion of how nurses generally use—and depart from—the MTS.)", "Pain is a central component of the MTS-prescribed triage assessment.", "For one, the system requires nurses to check for symptom-oriented aspects of patients' pain—such as its character, duration and location—as this can help reveal underlying disease (such as heart attacks or abdominal aneurisms).", "Moreover—and of particular relevance for present purposes—the MTS also instructs nurses to assess patients' pain intensity, which is listed as a separate ‘discriminator’ in 44 of the MTS′ 53 flowcharts (it is not included in charts like ‘asthma’ and ‘intoxication’).", "The fact that the MTS treats pain intensity as a separate criterion for priority setting means that it can be potentially decisive for the patient's triage code (and, consequently, for how long patients have to wait before seeing a physician).", "This is symptomatic of how EMS organisations, in response to allegations of poor pain management, have sought to make pain assessments a required and standardised aspect of triage (Teanby, 2003).", "This, in turn, reflects a broader shift in how pain is conceptualised in medicine, where it is now seen as harmful in itself rather than just as a symptom of something else (Morris, 1993, p. 74).", "To determine pain intensity, the MTS requires nurses to use the 11-point Numeric Rating Scale.", "The 11 NRS scores are distributed across the bottom four triage codes, where a score of 0 corresponds to a ‘blue’ priority, 1–4 is ‘green’, 5–7 is ‘yellow’, and 8–10 is an ‘orange’ priority.", "To arrive at a single NRS score, the MTS instructs nurses to combine two sub-measures: the patient's self-report and the nurse's own clinical pain assessment, respectively referred to by nurses as ‘objective and ‘subjective’ NRS.", "Beyond this, the system offers few instructions for combining the two measures or for clinically assessing patients' pain.", "Thus, while the MTS is a relatively elaborate tool for commensuration, the system leaves significant room for discretion in assessing pain; hence, it was up to nurses to ‘fill in the blanks’ of the system.", "Nurses were given some direction in the course they were required to attend prior to practising triage, where the instructors (who were themselves experienced triage nurses) repeatedly told participants to privilege their own ‘objective’ score over the patient's self-report.", "In observing triage assessments, I found that nurses imposed an even more hierarchical relationship between the two measures; of the 342 assessments I observed, I noted only five instances in which nurses asked patients to self-report their score.", "This was no anomaly; nurses commonly claimed that they had stopped using the ‘subjective’ NRS unless they considered the ‘objective’ score ambiguous or inconclusive.", "The next section begins to explore why.", "In the clinic under study, the ‘subjective’ NRS was widely perceived as a flawed measure.", "Nurses' objections were multiple.", "First, some patients were perceived as intentionally underreporting or exaggerating their score.", "While underreporting was attributed to a range of different motives—for example, wanting to appear brave or to avoid troubling EPCC workers—intentional exaggeration was seen primarily as an attempt to cheat the queue.", "Second, many patients were said to unknowingly distort their score—for instance, by overreporting their pain due to anxiety or fear.", "Nurses also claimed that many patients lacked imagination when comparing their pain to the NRS end-point (defined as ‘the worst pain imaginable’).", "Nurse Hanna described this with a mixture of humour and annoyance when she confided: “I had a patient who sat calmly in his chair and talking, saying ‘NRS 10’—and then I thought, ‘So, if I kick you in the nuts right now, you wouldn't feel more pain?!", "’“.", "We should note that these unimaginative patients are conceptually distinct from the ‘cheaters’ mentioned above; while cheating implies bad intentions, a lack of imagination merely suggests naïveté regarding the full spectrum of pain experiences.", "A final objection was that asking for ‘subjective’ NRS could lead to time-consuming discussions.", "Consider the following account by Nurse Helga.", "Such discussion was considered anathema to triage, as it wastes time and potentially puts sicker patients at risk (and, potentially, the nurse too; an internal annual report documented 173 reported incidences of threats and violence towards nurses in the clinic).", "In these nurses' view, then, the ‘subjective’ NRS ran counter to the aims of scoring pain consistently, correctly and efficiently.", "These are important aims across healthcare settings and may account for why other workers (e.g. in post-operative units) have been found to disregard subjective scores (cf. Solomon, 2001).", "However, nurses seemed to consider the ‘subjective’ NRS to be particularly antithetical to triage assessments, as pain scores are used here to rank patients in line to see a physician.", "Ranking requires pain scores to be spread across the whole scale—something that patients allegedly failed to do; and as Nurse Thomas put it, “There's no use having a triage system if everyone gets orange”.", "Nurses' objections stand in remarkable contrast to the notion of self-report as the ‘gold standard’ of pain assessment, and to the evaluative studies that treat self-report as the benchmark for evaluating the precision of nurses' assessments.", "Instead, nurses considered ‘subjective’ NRS unfit for the situation it is intended to standardise.", "In its place, they relied on an ‘objective’ approach to pain assessment, whose key principles are detailed in the following sections.", "The advantages of an ‘objective’ approach may seem self-evident, given that the term suggests an unbiased, factual assessment.", "However, as firmly established in science studies (cf. Daston, 1992; Porter, 1996; Shapin, 1995), what is designated ‘objective’ can vary across time, place and actors.", "It seems useful, then, to unpack what this ‘objective’ approach entailed in the present case.", "A first point of entry is nurses' reliance on so-called ‘objective’ signs.", "These were expressions of pain that are directly or indirectly observable, such as a particular form of body language (e.g. restlessness, facial contractions, gestures towards the painful area); reduced functional capacity (e.g. inability to walk upright, sit down, breathe properly, or eat and drink without feeling pain) and certain physiological changes (e.g. raised pulse, pallor or sweating).", "As the term ‘objective’ suggests, these signs were granted significant validity.", "However, ‘objectivity’ was treated in continuous rather than binary terms, which meant that some signs were seen as purer expressions of pain than others.", "The most ‘tainted’ signs were patients' self-reported pain scores, closely followed by their verbal descriptions (the latter were granted some weight, as evidenced by how nurses regularly asked patients whether they were in ‘discomfort’ or ‘pain’).", "Behavioural signs of the more familiar and easily manipulated kind were assigned somewhat greater validity, whereas the more esoteric and less governable behavioural signs (e.g. a particularly restless body language), as well as physiological signs, were considered purest.", "In addition to this hierarchy, nurses also relied on several techniques for assessing the credibility of patients' complaints.", "First, nurses appraised whether ‘objective’ signs were significant in their absence.", "As pain was assumed to express itself in at least some observable signs, patients displaying no ‘objective’ signs were taken to be in little or no pain.", "The most referenced example was patients sitting calmly in their chair, claiming to have a pain score of ten.", "Similarly, nurses typically discounted patients who claimed to be in severe pain, only to answer ‘no’ when asked if they had taken painkillers.", "Illustrating the abovementioned hierarchy of validity, Nurse Madeleine also explained, “If [you claim] your stomach really hurts and your pulse is 70, then I think, ‘Okay, you're not in that much pain’; but with a pulse of 120, then I know something's not right”.", "As these examples show, the absence of ‘objective’ signs was taken to suggest exaggeration, and nurses therefore had to adjust pain scores accordingly.", "Second, nurses also appraised signs in light of the pain interval of the patient's condition.", "By ‘pain interval’ I mean the range of pain stimuli and, consequently, of pain scores associated with a particular condition.", "Some conditions (e.g. abdominal pain) were seen to have wide intervals, potentially ranging from top to bottom of the NRS.", "Other conditions had narrower intervals; for instance, ear pains and sore throats stereotypically entailed little pain while gallstones and kidney stones were believed to elicit intense pain.", "Using these notions of inherent or ‘normal’ pain (see Sudnow, 1965), nurses could check whether patients' verbal and behavioural expressions of pain were misaligned with their condition.", "Accordingly, patients expressing pain outside their interval (e.g. by sobbing dramatically over ear pains or a sore throat) were suspected of distorting their ‘real’ level of pain.", "Third, nurses also looked for behavioural slip-ups in patients' impression management (Goffman, 1959).", "Typical examples included patients who came limping into triage only to walk flawlessly when leaving the assessment, or patients who came laughing into the clinic only to appear deadly serious in the triage booth.", "Nurses also made more elaborate inferences about how patients ‘really’ felt about their pain.", "For instance, Nurse Julie explained, “If you come in, put your purse on the floor and your coffee on the table, claiming, ‘I'm in real pain’, then I'm thinking that you're probably in pain but not so much that you couldn't manage to buy coffee first”.", "In other words, such ‘slip-ups’ were taken to suggest a discrepancy between claimed and ‘real’ pain, which nurses had to take into account.", "Finally, nurses also tried to compensate for the fact that certain patients may have a ‘different’ way of expressing their pain.", "Nurse Madeleine argued this point with reference to certain ‘cultures’.", "Madeleine's account illustrates the widespread belief that patients from certain ‘cultures’ behave in a particularly expressive way, and how this expressive behaviour might be interpreted as ‘exaggeration’.", "She did not specify the ‘cultural’ group in question, but others attributed these particular characteristics to Somali women.", "Note, however, that nurses did not assert that there are cultural differences in how patients experience or endure pain—only that certain groups express pain more strongly than others.", "‘Cultural’ differences in pain expression is a common theme in the scholarly literature (cf. the seminal studies by Zborowski, 1952; Zola, 1966), and attending to such differences is actually encouraged in the MTS handbook, which states that nurses' pain assessments must take account of how cultural groups “differ in how they respond to, or express, their pain” (Mackway-Jones et al., 2014, p. 32).", "Attending to cultural differences is supposed to increase the validity of nurses' pain scoring; put simply, if members of one group express pain more strongly than those of other groups, their information should be weighted to arrive at a more correct assessment.", "Accordingly, the most remarkable aspect of Madeleine's account is not necessarily that she holds generalised beliefs about particular patient groups; for present purposes, the more noteworthy point is that these beliefs are seen to serve an important role in calibrating nurses' commensuration.", "In addition to their reliance on objective signs and credibility checks, nurses sought to ensure consistent commensuration by using fixed reference points for the pain scale.", "While some of these have already been hinted at (e.g. sore throat, gallstones), this section foregrounds nurses' use of fixed end-points for ‘the worst pain imaginable’.", "Although not universally embraced in the EPCC, fixed end-points were a commonly claimed feature of nurses' assessments.", "In general, these were used as benchmarks for assessing the intensity of a patient's pain expression—as illustrated in the following backstage account by Nurse Natalie, concerning patients who make exaggerated claims about back pain.", "Natalie's account illustrates, first, how she equates an NRS score of 10 with the climactic moments of giving birth, and, second, how this reference point (together with the absence of certain ‘objective’ signs) is taken as grounds for discounting patients' exaggerated assertions.", "Nurses' reliance on benchmarks such as birth pains implies that they assess pain from an inter-patient perspective, in which patients' complaints are judged according to nurses' standards, rather than adopting an intra-patient perspective that relates pain intensity to the patient's own reference points.", "Opting for the latter would have been particularly challenging considering that nurses assessed unknown patients, whose pain thresholds could not be established reliably within a brief triage assessment.", "In its place, the use of nurse-determined benchmarks seemed a fitting proxy, ensuring, in nurses' view, that pain was scored according to a stable and ‘objective’ standard.", "This is not to say, however, that all nurses shared exactly the same standards.", "For instance, while Nurse Natalie equated an NRS score of 10 with birth pains, Nurse Hanna—who referred to herself as a ‘strict’ pain assessor—equated birth pains with an NRS score of 8 and reserved the 10 for ‘torture’.", "This suggests less than perfect reliability between nurses' ratings.", "Consequently, with all else being equal, the same patients would receive different pain scores depending on whether a nurse like Natalie or Hanna assessed them.", "(It is also worth noting that Hanna and other ‘strict’ nurses typically expressed their ‘strictness’ with some pride.", "This can be partly understood in light of the widespread belief that the EPCC was ‘abused’ by patients with ‘trivial’ complaints (explored more thoroughly in Johannessen, 2018c), for which reason ‘strictness’ served as an important form of ‘adult education’.", "‘Strictness’ also seemed to have a seasoned, distinguishing quality to it, which set the ‘strict’ apart from those who indiscriminately empathise with the most trivial of pains.)", "However, while there were differences in strictness, daily EPCC socialisation ensured that pain was assessed according to a similar if not identical metric.", "Working in a large clinic meant interacting regularly with colleagues, and this served to align nurses' beliefs about pain and the standards for its assessment.", "Of particular importance was nurses regular interactions with the clinic's coordinating nurse and physician, who monitored patient flow and followed up on patients post-triage.", "Among other things, coordinators sanction nurses when their pain ratings (and assessments more generally) seem too out of tune with agreed-upon standards.", "Although relatively infrequent (I observed only one case), these sanctions could have a significant preventive effect, in the sense that nurses sought to mimic the standards of their colleagues in order to avoid being reproached.", "In combination with other everyday exchanges—such as discussions about difficult, interesting or frustrating cases—these collegial interactions contributed to aligning nurses' pain assessments, creating an intersubjective agreement that was seemingly perceived as common sense and, indeed, objective (see Shapin, 2012, p. 176).", "This brings us again to the problems associated with ‘subjective’ NRS.", "While nurses could work to align each other's assessments, they had only limited influence on patients' standards.", "Educating one's clientele during brief, one-off encounters was considered a waste of time and effort and could even cause patients to feel alienated or distrusted.", "Thus, while the sociology of standardisation (Timmermans and Epstein, 2010) offers rich illustrations of the challenges in standardising professionals' behaviour, ‘subjective’ NRS highlights the additional and arguably more challenging issue of standardising patients' conduct.", "Finally, it is worth noting that some patients were considered better than others at rating their pain.", "For instance, it was suggested that the elderly are more appropriately imaginative than others; as Nurse Natalie jokingly explained, “Older people have more experience, so they're probably better at assessing, like, ‘It wasn't exactly like when I was shot during the war, but it kinda feels like it’“.", "Similarly, patients who work in healthcare were simply said to ‘get’ the NRS; as Nurse Miriam proclaimed, “It's great with healthcare workers who know the NRS—I just ask and then write what they say”.", "Thus, as long as patients were seen to follow nurses' standards, their self-report could be used for commensuration.", "To sum up thus far, we have seen how nurses disregarded patients' self-report and instead approached pain ‘objectively’ by relying on ‘objective’ signs, credibility checks and the use of nurse-determined benchmarks for ‘the worst pain imaginable’.", "In so doing, nurses rendered pain a professional concern, in which proper assessment relied on their professional gaze and standards.", "Pain was thus seen less as a subjective experience and more as an underlying ‘thing’ to be determined by reference to the absence or presence of ‘objective’ signs.", "Interestingly, this meant that nurses treated pain states in much the same way as other disease entities in biomedicine—that is, as “distinct, objective entities that are common to afflicted patients” (Chiong, 2001, p. 90).", "It should be noted that this transformation is not exclusive to the nurses under study, as a preference for ‘objective’ assessments has been reported in several of the studies reviewed above.", "Most notably, Vuille et al. (2017) found that nurses preferred ‘objective’ data to patients' self-report; had different methods for assessing patient credibility; relied on what has been described here as an inter-patient perspective; and used fixed end-points in their assessments.", "To explain this preference for an ‘objective’ approach, we can first look to the situational requirements of triage.", "Reducing pain to an observable phenomenon enables nurses to rank patients on a single dimension rather than having to weigh this against other, more subjective dimensions.", "Moreover, this reduction more easily meets the need to assess patients quickly; instead of engaging in potentially time-consuming dialogue with the patient, nurses can observe ‘objective’ signs while attending to other matters.", "Treating pain ‘objectively’ as an observable phenomenon therefore simplifies nurses' commensuration, allowing them to assign pain scores quickly and, in their view, with greater precision and consistency than if they had simply relied on patients' self-reports.", "However, the use of an ‘objective’ approach cannot be explained completely by the organisational constraints of triage, as studies have found a preference for ‘objective’ pain scoring among healthcare workers in other contexts as well, including in pain clinics and post-operative units (Baszanger, 1998; Harper et al., 2007; Solomon, 2001).", "While conducive, the structural features of triage are therefore not necessary or exhaustive conditions for assessing pain ‘objectively’.", "Beyond triage, one likely explanation for the reliance on an ‘objective’ approach can be found in Goffman's remark that people regularly “use what are considered to be the ungovernable aspects of [a person's] expressive behaviour as a check upon the validity of what is conveyed by the governable aspects” (1959, p. 18).", "In other words, as people have a general propensity for using signs that are ‘ungovernable’ or beyond conscious control to assess each other's credibility, we should expect healthcare workers to prefer ‘objective’ and assumedly ungovernable signs over patients' all too governable self-report.", "Adding to this, I believe that this propensity for validity checking is exacerbated by at least two additional aspects of healthcare work.", "Firstly, these workers control access to scarce goods (e.g. a high priority in triage, painkillers in post-operative units), meaning that patients, in order to receive said goods, have incentives to exacerbate their complaints.", "Workers are therefore likely to cultivate at least some skepticism towards patients’ self-presentation (cf. Hughes, 1988; for a more general account, see Yuille, 2013).", "Secondly, healthcare workers are employed in organisations in which biomedical culture is deeply entrenched.", "Thus, as they fill roles and are held accountable to beliefs and values that are closely associated with biomedicine, we should not be surprised to see healthcare workers approach ‘subjective’ evidence with skepticism.", "Ever since the advent of ‘scientific medicine’ in the eighteenth and nineteenth centuries, the ‘subjective’ has suggested a personal, emotional and biased account, far removed from the ‘objective’ and true nature of things (Malterud et al., 2015).", "Clinicians therefore have a long history of seeking ways “to bypass patient-narratives in their search for an ‘objective’ diagnosis” (Bourke, 2014, p. 256; see also Jewson, 1976).", "In the case of pain, the shift to ‘scientific medicine’ meant that the phenomenon was characterised less in terms of an experiencing subject and more in terms of stimuli and response—typically explained in terms of an internal ‘pain system’ that carries messages from pain receptors in the skin to a pain centre in the brain before being expressed mechanistically in observable signs (Bendelow and Williams, 1995, p. 141).", "Accompanying this, clinicians increasingly attended to the ‘gestural language’ of pain (Bourke, 2014); that is, to how pain expresses itself in a stable set of observable signs (as those mentioned above).", "While the 1960s saw a renewed theoretical emphasis on patients' subjectivity (Melzack and Wall, 1965), the mechanistic stimuli-response model can still be found in clinical practice; as Bendelow and Williams argue, clinicians often equate ‘real pain’ with “something that is acute with an easily observable physiological pathology” (1995, p. 145).", "This relates to how biomedicine privileges vision as a form of knowing, and to how ‘objectivity’ often tends to depend on the visibility of evidence (Leder, 1990, pp. 19–20).", "Or as Buchbinder remarks, “to know that symptoms are ‘real’, [clinicians] need to be able to see them” (2015, p. 11).", "Returning to the nurses under study, it was primarily such a mechanistic conception of ‘real’ physical pain that underpinned their ‘objective’ approach to pain scoring.", "This is not to say that nurses always and intentionally understood pain in mechanistic terms (i.e. as directly readable from patients' bodies and behaviour); rather, this was an assumption that they often privileged in their assessments.", "As indicated, the reasons for doing so seem closely aligned with both organisational constraints and a heuristic biomedical view of pain and the body.", "Combined with the factors mentioned in the previous sections, I therefore see their use of an ‘objective’ approach as intimately linked with having to (1) rank and (2) distribute scarce goods to (3) previously unknown patients (4) quickly by use of (5) a unidimensional measure, while (6) being part of, or accountable to, a culture that places a premium on ‘objective’ evidence.", "In short, these are situations where an ‘objective’ approach seems likely to be considered the most valid, reliable and efficient proxy for scoring patients' pain.", "However, more research is needed to determine the transferability and relative importance of these factors.", "I would particularly encourage comparative designs, exploring assessments of different types of pain (e.g. chronic or acute), assessed with varying degrees of complexity and with different goals in mind (e.g. priority setting, pain relief, diagnosis), under different treatment regimes (cf. Baszanger, 1992; Vrancken, 1989), and carried out by different actors in different settings.", "Approached this way, we could significantly improve our understanding of how pain is commensurated in various healthcare settings.", "Before concluding, it is worth reflecting briefly on how nurses' ‘objective’ approach can have systematic—and potentially problematic—consequences for their prioritisation of patients; especially in light of how nurses rendered pain a professional concern, and thus shifted the power balance of the triage encounter significantly in favour of themselves.", "To begin, we have seen that members of certain minority cultures were believed to express pain in a ‘different’ and exaggerated way.", "While such assumptions arguably play an important calibrating role in nurses' assessments, the use of perceived group characteristics to judge individual patients also means that nurses run the risk of suspecting exaggeration even when evidence of exaggeration is absent.", "This is a general problem with stereotypical reasoning, often referenced by critics of related practices such as statistical discrimination (Pager and Karafin, 2009) and racial profiling (Harris, 1999).", "Furthermore, in attending solely to patients' ‘objective’ signs, nurses privilege ‘physiological’ pain over other forms of suffering, such as ‘emotional’ pain (Bendelow and Williams, 1995).", "While this may seem an obvious priority in emergency medicine, it is worth emphasising that other logics of priority setting exists, as evidenced in the following statement by Nurse Elma:", "Nurse Elma had a background in psychiatry and was a clear outlier in my sample; nevertheless, she reveals a different logic for pain scoring.", "To judge whether this is a better approach is beyond the scope of this paper; the point is rather to highlight the contingency of the ‘objective’ approach, thus opening up a space for discussing alternative logics.", "Lastly, one can also ask whether rating pain relative to nurses' benchmarks (e.g. ‘birth pains’, ‘torture’) runs counter to the point of singling out pain intensity as a separate criterion for priority setting.", "That point is to prioritise those who are experiencing pain—and, qua experience, pain is arguably determined by patients' rather than nurses' reference points (cf. Bourke, 2014).", "These issues suggest a need for developers and implementers of guidelines to find ways of incorporating patients' voice in the pain assessment, while simultaneously allowing nurses to rank patients consistently.", "This is a challenging task indeed; as the article has shown, nurses' commensuration is shaped by organisational constraints that impose significant limitations on their pain assessments.", "Every day, triage nurses face loads of complete strangers, whose subjective experience must be assessed as quickly as possible while nurses simultaneously attend to other aspects of the assessment.", "For that reason, their scoring necessarily depends on routines and heuristics, entailing various blind spots (cf. Lipsky, 1980).", "While it is important to illuminate these blind spots and to thoroughly debate them, it is equally important to realise that nurses' working conditions place significant limits on what pain can be in this context (see also Graham and Herndl, 2013).", "Such organisational constraints are of crucial importance, both for understanding and improving nurses’ assessments and for analysing commensuration more generally.", "This article departed from a puzzling deviation from guidelines: While nurses were instructed to assess pain intensity by combining patients' self-report with their own ‘objective’ assessment, their actual assessments relied almost solely on the latter.", "This contravenes the widespread belief that self-report is the gold standard of pain assessments, thus raising the questions of how and why nurses assessed pain as they did.", "The article has shown that nurses had several objections against the use of self-report in triage.", "Most importantly, they considered patients prone to knowingly or unknowingly distort their pain score, which meant that a reliance on self-report would reduce the consistency and precision of their priority setting.", "Nurses thus considered ‘subjective’ NRS unfit for the situation it was intended to standardise—a common problem that is well documented in the sociology of standardisation (Timmermans and Epstein, 2010).", "The article has also detailed nurses' ‘objective’ approach to pain scoring, which included a reliance on ‘objective’ signs, credibility checks and the use of nurse-determined benchmarks for ‘the worst pain imaginable’.", "Nurses believed that this approach enabled more correct, consistent and efficient pain scoring, in marked contrast to ‘subjective’ NRS.", "Moreover, the article has argued that nurses' ‘objective’ approach is closely aligned with their organisational constraints and a heuristic biomedical view of pain and the body—and that prioritising patients based on this approach means emphasising some forms and expressions of pain over others, with systematic consequences for who is prioritised and treated in the EPCC.", "By delving deeply into the situated logic of nurses' approach, then, the article goes further than previous studies in unpacking how and why nurses assess pain as they do.", "In so doing, the article also contributes to research on commensuration, which has so far been dominated by more meso- and macro-oriented studies.", "Specifically, the article adds to a small strand of studies demonstrating the importance of microscale commensuration, while also extending these studies by exploring ‘fast’ commensuration—a form that is particularly characteristic of professional practice at street level (Lipsky, 1980).", "As illustrated in the analysis above, street-level professionals often work in time-pressured environments that force them to use routines and heuristics to facilitate the speedy assessment of clients.", "While aiding decision-making and making the job more manageable, this reliance on heuristics can also have systematic consequences for clients, privileging some at the expense of others (Lipsky, 1980, p. 84).", "This makes ‘fast’ commensuration a particularly apt area for exploring the transformative aspects of commensuration.", "Looking forward, I would encourage social scientists to focus more explicitly on microscale commensuration.", "Although explored implicitly in many studies (cf. Latour and Woolgar, 1986), the elusive quality of both commensuration and micro-practice can conceal the broader significance and family resemblance of these practices.", "To combat this, we must reflect more explicitly and critically—and with greater amazement—on the work of creating seemingly neutral numbers.", "There is no lack of cases; healthcare has seen an explosion of metrics (Whooley, 2016) and quantification is common in all professions, from teachers grading papers to employers ranking job applications.", "By detailing how these commensurators commensurate, we can deepen our knowledge of how the world is transformed through the production of numbers." ]	[ "Uses ethnography to unpack the situated logic of triage nurses' pain assessments.", "Deepens our knowledge of how and why nurses assess patients differently from patients.", "Frames pain assessment as a form of commensuration.", "Extends studies of commensuration to include microscale number production.", "Advances theorising on microscale commensuration." ]
Generation of Acoustic Emission from the running-in and subsequent micropitting of a mixed-elastohydrodynamic contact	This paper presents the use of Acoustic Emission to study the running-in and subsequent micropitting of a pair of hardened steel surfaces under mixed lubrication conditions. These surfaces were loaded together under rolling/sliding conditions typical of heavily loaded gearing. Relocation profilometry was used to measure the rapid running-in process and the development of micropits. Acoustic emission (AE) was found to be highly sensitive to both the initial changes in surface topography during the running-in process, and to subsequent changes caused by micropit formation. However, AE appears to be sensitive to changes in asperity interaction rather than the underlying mechanisms of plastic deformation, crack growth and fracture. It is concluded that AE can provide considerable insight into conditions in mixed-elastohydrodynamic contacts.	[ "ENGI", "MATE", "PHYS" ]	[ "Acoustic Emission", "Micropitting", "Mixed lubrication", "Running-in" ]	[ "2210", "2211", "2508", "3110" ]	[ "There is currently considerable interest in using Acoustic Emission to monitor tribological conditions in mechanical equipment, such as gears and bearings within aerospace and renewable energy power transmissions.", "AE is the release of ultrasonic stress waves within a material, caused by energy release from sources such as plastic deformation, crack growth, frictional contacts and fluid cavitation.", "AE is widely held to be a sensitive technique which offers significant advantages in terms of early fault detection and diagnosis when compared to other monitoring methods [1,2].", "Although not as well established when compared to vibration or temperature-based techniques, it has been used effectively at least in laboratory conditions to detect faults in rolling element bearings [3–5]; to study the piston ring/liner interface in diesel engines [6]; and extensively for studies of tribological conditions in wear or contact fatigue testing [7–10].", "Various workers have investigated the use of AE to monitor gear systems.", "Toutountzakis et al. [11] presented tests using spur gears which demonstrated the effects of operating conditions on AE, and also the ability of AE to detect changes in tooth surface condition.", "They subsequently undertook a series of experiments using seeded, or artificial, pitting defects on spur gears [12].", "They concluded that seeded defects introduced different source mechanisms when compared to naturally-occurring defects, and recommended the use of life tests where defects are allowed to occur naturally to develop the use of AE for gear monitoring.", "Within a gearbox, transmission paths between source (at the tooth contact) and sensor (perhaps mounted on a bearing housing for example) may be complex with multiple interfaces and complex attenuation behaviour.", "Sing et al. [13] studied these complex transmission paths using a tooth breakage test rig, whilst Crivelli et al. [14] investigated the use of advanced signal processing methods to detect AE from tooth fatigue cracking in the root fillet region.", "In life tests of precision forged gears, Scheer et al. [15] used wavelet-based techniques to detect signals related to micropitting and to tooth root fatigue, whilst Wirtz et al. [16] adopted similar signal processing techniques to identify micropitting in an FZG back-to-back gear test rig, with sensors mounted on the gear test housings.", "Barrueto Novoa and Molina Vicuna [17] reviewed the various hypotheses relating to the sources of AE in gears.", "Typical AE signals measured for spur gears consist of continuous emission, superimposed with a series of transient bursts spaced at the gear meshing frequency.", "They considered that the transient bursts could be caused by the instantaneous pure rolling conditions at the pitch point, or by the EHL exit pressure spike, or (as seems most likely) the shock loading caused by tooth engagement, whilst the continuous emission was most likely related to asperity contact.", "They conducted tests using a planetary gearbox [17,18] where the effects of load, speed and lubricant temperature were investigated, concluding that temperature and speed-related changes in the lubricant film thickness between the gears affected the levels of asperity interaction during the meshing cycle and, hence, the levels of AE produced.", "To investigate the link between specific film thickness (or lambda ratio) and AE, Rajah Hamzah and Mba [19] conducted a series of novel experiments where short bursts of liquid nitrogen were used to rapidly cool the gear surface, with the aim of increasing lubricant viscosity by a significant amount.", "They found a reduction in RMS levels of AE, which directly followed the changes in gear temperature, suggesting a strong link between specific film thickness (and therefore asperity interaction) and AE.", "This was confirmed by further experiments using spur and helical gears under a range of operating conditions [20], where they concluded that, at least in part, asperity interaction was responsible for the AE from running gears.", "Other work [21,22] also identified asperity interactions under the mixed lubrication conditions which occur in heavily loaded gearing to be a source of AE signals.", "This paper presents experimental work which is part of a wider study by the authors to investigate AE caused by asperity interactions in mixed lubrication conditions, using a power-recirculating twin disk test rig.", "This approach has many advantages as it allows the scientific study of this AE source without the complications of the variable kinematic and loading conditions within the gear meshing cycle, or the other potential sources of AE such as tooth engagement.", "In this paper, AE is found to be sensitive to subtle changes in asperity geometry, quantified using in-situ relocation profilometry, due to running-in and subsequent micropitting of the steel disk surfaces.", "The experiment was carried out on a twin disk rig used to simulate, with simplified kinematics, the contact between heavily loaded gears.", "It has been previously used to investigate how material hardness, and the lubrication regime, i.e. the specific film thickness, affect the surface wear and the contact friction [23,24].", "It has also been used to evaluate novel techniques for investigating the lubrication regime, such as electrical resistance of the lubricant film [24], and currently, acoustic emission measurements.", "Fig. 1 shows a schematic of the rig.", "The test contact is made between two geometrically identical disks mounted on shafts connected by a helical gear pair, the ratio of which determines the slide/roll ratio (SRR) of the disk contact.", "A constant SRR, as generated by this rig, differs from that of meshing gear teeth, where it varies over the tooth contact cycle; however, it significantly reduces the complexity of the measurement and analysis techniques required to study the contact.", "In order to investigate the possibility of very rapid wear and surface modification regimes, occurring in the order of ones or tens of fast disk cycles, it is necessary to minimise transients associated with starting and stopping a test.", "This is achieved by the use of a hydraulic ram which provides a rapid loading and unloading response.", "The slow shaft is supported in a pivoting housing that allows the centres-distance of the disks to vary in response to the load.", "A flexible coupling arrangement ensures the fast and slow shaft remain parallel.", "When no load is applied the disks do not make contact which allows the rig to be run up to speed before the test is started.", "The contact load, which is directly measured by a load cell, is calibrated prior to testing by adjustment of the hydraulic pressure supplied to the ram.", "During calibration a wedge is placed in-between the fast and slow shafts to keep the disks separated.", "A ball-valve is used to quickly switch the ram pressure on and off at the start and end of a test.", "The test disks are made of a case hardened gear steel meeting Rolls-Royce specification 6010.", "The disks have a diameter of 76.2 mm, a face width of 9.5 mm and a crown of radius 304.8 mm.", "This geometry results in a spheroidal contact face that generates an elliptical test contact having a 4 to 1 aspect ratio.", "The minor axis of the ellipse is aligned in the slide/roll direction.", "The crown is machined using a special grinding technique that results in a surface with the ridges and valleys aligned approximately normal to the slide/roll direction.", "This closely replicates the roughness orientation found in ground gear teeth.", "Further details of the steel specification and grinding process are given in Ref. [25].", "For the work presented in this paper a single set of previously un-used disks was tested.", "The Vickers hardness of each disk was tested and found to be 760 Hv for each.", "Before the disks were assembled in the rig the roughness of a small area from the contact face of each was measured using a Taylor Hobson Talysurf Form2 stylus profilometer.", "Fig. 2 shows the composite roughness (the sum of the roughness of each disk) made using these measurements.", "The grinding process produces ridges and valleys that are ‘extruded’ across the disk width along a slightly swept path.", "When assembled in the rig the direction of roughness sweep for one disk is opposed to that of the other thus generating the cross-hatched composite roughness evident in Fig. 2.", "The contact is lubricated by an extreme pressure gear oil conforming to Defence Standard OEP-80 [26].", "The oil is pumped from a heated bath and sprayed directly onto the inlet and outlet of the test contact to ensure it is always flooded.", "Oil from the same bath is also used to lubricate the drive gears and support bearings.", "The rig is driven by a three phase electric motor controlled by a variable frequency drive, and geared to provide fast shaft speeds between 200 and 2000 rpm.", "An encoder on the fast shaft measures its speed, counts the number of cycles and synchronises the data acquisition system.", "The temperature of each disk is measured using an embedded thermocouple located in a small hole 3 mm below its contact surface on the centre line.", "A strain gauge on a reduced diameter section of the slow shaft measures the torsion between its driven gear and the test disk, this can be used to estimate the friction at the contact, and is calibrated to isolate the frictional traction at the contact from parasitic friction losses in the bearings and shaft windage.", "The amount of direct asperity interaction at the contact is monitored by measurement of its electrical resistance.", "To facilitate this the section of slow shaft supporting its test disk is electrically isolated from the main body of the rig.", "This allows an electrical potential, termed here the Contact Voltage (CV), to be set up and measured across the disks.", "The contact resistance varies with the amount of direct, metal to metal, asperity contact that bridges the oil film.", "In full film lubrication the contact resistance is effectively infinite and the CV will be at a maximum that is equal to the source potential.", "In mixed lubrication the instantaneous CV will fluctuate rapidly with the amount of instantaneous asperity contact.", "The mean CV, over time, will give an indication of where in the mixed lubrication spectrum the contact is operating.", "In boundary lubrication the contact resistance is effectively zero and thus the CV will measure zero.", "A detailed study of CV measurements in mixed lubrication has been previously carried-out using the twin disk rig and is presented in Ref. [24], together with a more detailed description of the contact voltage measurement technique.", "For the tests presented in this paper a 44 mV potential was used.", "For clarity the CV results are presented normalised between zero, the minimum, and one, the maximum representing full film conditions.", "A Physical Acoustics Pico transducer is used to measure the AE from the contact.", "It has a nominal operating range of 200–750 kHz and resonant frequency of 250 kHz.", "This sensor was chosen primarily for its small size and ease of installation in the test rig.", "The sensor is clamped to the side of the fast disk near its periphery.", "A thin film of silicone sealant is used as an acoustic couplant between the sensor and disk face to aid signal transmission across the interface.", "This material is widely used within the AE research community as an effective acoustic couplant [5].", "Moreover, the silicone layer is extremely thin, as primary mounting of the sensor is achieved using a clamp.", "The transducer signal is routed through an air cooled silver/graphite slip ring to a 40 dB pre-amplifier with a 20 kHz high-pass filter.", "A second Pico transducer is affixed to one of the fast shaft bearing housings.", "For this, the signal attenuation is sufficient to ensure that AE from the test contact is negligible.", "Thus data from this sensor can be used to help to identify sources of noise.", "A dedicated Physical Acoustics computer performs analogue to digital conversion on the amplified signals from both sensors and saves them when triggered by a signal from the rig computer.", "The experiment consisted of a number of ‘test stages’ in-between which the rig was stopped so that the disk surfaces could be measured and changes to their roughness monitored.", "Constant and identical control parameters were used for all test stages, and are shown in Table 1.", "They were chosen, based on previous experience, to give a specific film thickness corresponding to mixed lubrication conditions that would cause micro-pitting fatigue within the time available for testing.", "The only variation between the test stages was their duration.", "It was intended to run-in the disks in a single, initial, stage, the duration of which was set at 3 min based on previous running-in experiments using similar disks and contact parameters [23].", "The length of subsequent stages was decided as the test progressed based upon the rate of surface modification.", "The schedule that resulted is shown in Table 2.", "To minimise the effect of temperature transients at the start of each running-stage, the unloaded disks were first run (out of contact) at the test speed whilst the oil was heated and pumped through the test enclosure.", "This allowed it to approach thermal equilibrium, usually after a few hours, before the test stage was started by the application of the load, bringing the disks into contact.", "The data acquisition was synchronised with the fast disk by a single shaft encoder pulse per rotation.", "Upon each pulse the load, temperatures and CV were sampled at 1.25 MHz for 75% of a single fast disk rotation, these data were then averaged to provide a single mean value for each fast disk rotation.", "(Only 75% of each rotation could be sampled due to the triggering method used).", "The acquisition of the AE signal was different dependant on the length of the test-stage.", "For the first two short stages it was continuous, with sampling started shortly before the load was applied and stopped after it was removed.", "For the remaining stages it was synchronised with the fast disk and only periodically triggered as follows: For stages 3–13 every 100th fast disk rotation, and for stages 14–19 every 500th.", "The sample period was chosen to be 5 fast disk rotations as the composite surface roughness repeats every five fast disk rotations, (the gear ratio was 5/3, that is, three slow disk rotations per five of the fast disk).", "All AE acquisition was at a sample rate of 2 MHz.", "The surface measurements of the disks, made in-between each test stage and at the start and end of the experiment, were carried out in-situ using a portable Taylor Hobson 2D profilometer, using a stylus with a 90° conical diamond tip with a 2 μm radius.", "Use of an in-situ technique allowed preservation of the disks' relative angular orientation throughout the entire experiment.", "This was important as each specific orientation generates a specific composite roughness which will run-in and wear as its topography dictates.", "If that topography suddenly changed due to a new orientation it would initiate a new running-in and wear phase as new asperity interactions occur.", "The in-situ disk profiles were measured in the circumferential direction, i.e. across the lay of the ridges and valleys, normal to the cylindrical axis of the test disk/shaft.", "It is useful to study how representative sections of surface roughness change as test progresses for which positional reference points were required.", "Score marks on the side of the disks were used as a coarse reference for circumferential position.", "These did not need to be precise as cross-correlation techniques were used to accurately re-align the profiles after measurement.", "The profilometer cannot be left in place whilst the rig is running so the side of the disk was used as a reference point for all axial measurements.", "The profilometer is mounted on an axial linear stage whose position is measured by a dial gauge.", "The position of the side of the disk was established as follows: The profilometer stylus was positioned by eye on the crest of the disk.", "It was then moved axially outwards using the linear stage until its height decreased by 60 μm, this indicated the edge of the disk.", "The error in axial positioning was likely to be at least ±5 μm dominated by the resolution of the dial gauge and error in locating the disk edge.", "For this experiment 12 mm long profiles were measured at a single circumferential position and nine axial positions per disk.", "The axial measurement positions relative to the disk centre line are shown in Fig. 2.", "The measurements were spaced, in 0.5 mm increments, over a 4 mm range centred on the disk centre line.", "Given the nominal axial width of the contact, approximately 3 mm, this resulted in 5 profiles within the contact path, two at its nominal edge and two outside the contact area.", "This section describes the changes to the contact surfaces that occurred over the course of the experiment.", "As discussed in the test procedure, the surface measurement schedule was not fixed at the start but was determined as the test progressed.", "The RMS roughness (Rq) was used to confirm nominal end of the running-in process and the start of steady state fatigue testing.", "The change in Rq, for each disk, over the test is shown in Fig. 3.", "As expected, running-in caused a rapid and large reduction in the Rq of both disks.", "For the first 6000 fast disk cycles, 97% of the reduction occurred within the first 3000 cycles and only 3% in the subsequent 3000 cycles.", "This indicates that by the 6000th cycle, (the end of the second test stage), the surfaces were at a comparatively steady state and thus were fully run-in, in agreement with previous work on surface topography changes during running-in Ref. [23].", "Comparison of relocated profiles confirmed the stability of the surfaces at 6000 cycles.", "Subsequently, over the course of the fatigue test, there was a further, much slower, decrease in Rq, however approximately 70% of the total decrease for the entire test occurred within the first 3000 cycles.", "Fig. 3, which has a displaced zero, may give the impression that the slow disk was generally rougher than the fast however this difference is not significant and is likely to be due to circumferential variation, and within the manufacturing tolerance applied to surface finish.", "On average, over the entire circumference, the disks are of similar roughness.", "The relative changes to the Rq of each disk were similar despite their different starting values, with no signs of convergence.", "This is likely because the Rq represents profile features at all heights whereas surface modification predominantly affects asperity tips.", "Thus unless there is significant wear there will always be a constant component of the Rq that is dependent on the local valley features.", "Although the Rq data provides an indication of the relative rate of surface modification it does not offer any insight into the type of modification, i.e. the wear mechanism.", "To characterise this more sophisticated measurements and analysis are required.", "Fig. 4(a) presents a high resolution areal roughness measurement of a 6 × 6 mm area of the fast disk, measured at the end of the test.", "This area covers the entire width of the contact path and some of the un-run regions on either side.", "The location of the contact path can be determined by consideration of the variation of Rq with axial position as plotted in Fig. 4(b).", "The region with low Rq values, (between 0.3 and 0.4 μm), identifies the contact path.", "It has a width of just under 3 mm which is close to that expected from a Hertzian contact calculation.", "At both edges of the contact there is a region, approximately 0.5 mm wide, where the Rq smoothly transitions between that of the contact path and that of the un-run regions.", "The areal roughness measurement shows explicitly that micro-pitting had occurred by the end of the test.", "The micro-pits are evident as dark specks within the contact path.", "They are not evenly distributed across the surface but tend to be clustered along prominent asperity ridges.", "Fig. 4(c), an enlarged view of a region within the contact path, shows an asperity ridge having a set of particularly numerous and large micro-pits.", "For comparison Fig. 4(d) shows a section of the same ridge located outside the contact path.", "The section within the contact path would have looked like this at the start of the test.", "An equivalent measurement of the slow disk showed that micropitting had also occurred on this disk by the end of the test.", "As shown by Fig. 4, micro-pits can be identified easily using areal (three-dimensional) roughness measurements.", "However as the twin disk rig is not currently equipped to make in-situ three-dimensional measurements it is not possible to use these to identify when these pits are formed and how they grow.", "Knowledge of this is required to assess if micro-pitting has AE characteristics distinct from that of other wear mechanisms, and this was assessed statistically using the two-dimensional in-situ profiles.", "Roughness profiles made at different times were precisely aligned using cross-correlation in the circumferential direction, and by minimising the mean height difference between deep valley features in the vertical direction.", "The mean line cannot be used as height reference as it changes due to wear of surface asperities whereas the deep valley features are assumed to be unaffected by wear.", "Fig. 5 (a) presents an example of the aligned profiles for a short length of the fast disk centre-line.", "In this figure, profiles measured at different stages in the test from un-run (black), to immediately after running-in (blue) and during the fatigue testing (yellow to red) are overlaid.", "This allows the effects of different surface modification mechanisms to be easily visually identified and distinguished from one another.", "It can be seen that the running-in process (black to blue) resulted in the flattening and rounding of numerous asperities, large and small.", "Much of this is suspected to be due to plastic deformation [23], however for the most prominent asperities the local material volume appears to reduce which implies some material removal as well.", "An example of the apparent reduction in volume can be seen at C. By the end of the experiment, many of the locations where running-in had caused the most initial change had subsequently pitted.", "Examples of this are at positions A to D.", "It can be seen that these micro-pits have dimensions on the scale of the previously existing roughness features.", "Thus it is not possible to identify micro-pits from 2D analysis without considering the surface change over time, i.e. the aligned profiles.", "Once a pit is identified it might be supposed that it would be a simple matter to track its growth by considering the change from one measurement time to the next.", "Unfortunately, this is not the case.", "The error in the axial position of the profile measurements is on the same scale as the width of a typical micro-pit so it cannot be assumed that a pit is consistently measured in the same location.", "This will cause the pit size to fluctuate based on how close the edge or centre it was measured.", "An example of this can be seen at D where the pit measured after 13 × 105 cycles appears shallower than that measured after 5 × 105 cycles.", "Whilst the disks were cleaned before each measurement, debris trapped within valley features may also affect the measurements.", "Visual examination of the aligned profiles cannot accurately gauge the rate of pit formation and growth and so a mathematical method has been developed instead.", "This requires the identification of all sections of the profile that had pitted by the end of the test.", "The standard deviation of the set of aligned profiles at each circumferential position is used to identify pitted locations, an example of this is shown in Fig. 5(b).", "For this the standard deviation, at each circumferential sample point, has been calculated for all profiles measured after running-in (from cycle 6000 onwards).", "The standard deviation is comparatively high at pitting locations for two reasons: firstly the growth of the pit over time, and secondly, the axial position error has a greater affect at these locations.", "A minimum standard deviation threshold is used to identify the final pitting locations.", "In Fig. 5(b), a threshold of 0.2 μm, shown in bold, has been used to identify pits A to D.", "The rate of pit formation and growth was assessed by consideration of how the mean height of the identified final pitting locations changed over the test.", "This is shown in Fig. 6 for each disk separately, with the mean height of the entire profile and the un-pitted locations shown for comparison.", "The five axial profile positions within the contact path were analysed giving a total sample length of approximately 54 mm.", "The end pitting locations were calculated using a standard deviation threshold of 0.2 μm.", "This gives the percentage of the profile that was pitted as 2.6% for the fast disk and 3.1% for the slow.", "However, these are almost certainly underestimations as it can be seen in Fig. 5(b) that a threshold of 0.2 μm excludes the edges of the large pits and some small pits entirely.", "Despite this, a value of 0.2 was found to represent a good compromise between noise induced by a small sample size, prone at a high threshold, and erroneous pit identification, prone at a low threshold.", "From Fig. 6 it can be seen that, at the start of the test, locations that subsequently pitted were, unsurprisingly, at relatively high elevations.", "Running-in caused a moderate reduction in their mean elevation.", "However the most substantial decrease, and the change from a positive to negative mean elevation, occurred in the early stages of the fatigue test, between 6000 and 2 × 105 fast disk cycles (test stages 3 and 4).", "This indicates that the majority of the pitting took place in this period.", "By about a quarter of the way through the fatigue test the mean elevation of the pitting locations appears to have stabilised at a minimum for both disks.", "This implies that, for these test conditions micro-pitting was a self-limited wear process.", "The likely explanation for this is as follows: Initially the contact load is carried by a few prominent and heavily loaded asperities.", "As these are removed by micro-pitting the load is redistributed more evenly to other asperities and the partial lubricant film, until the cyclic loads on individual asperities are not sufficient to cause fatigue failure.", "In this section the AE and CV measurements made during the running-in process are assessed with reference to the measured surface changes discussed in section 4.", "Fig. 7 shows the measurements made during test stage one, running-in, and test stage two, validation of the run-in state.", "As discussed in the test procedure, the AE for these was acquired continuously as a broadband signal which does not lend itself to easy interpretation.", "Frequency filtering and time averaging were required to produce useful results.", "The technique used here was to calculate the discrete Fourier transform (DFT) along a moving window, the width and increment of which was used to control the amount of time averaging.", "This gave a three dimensional data set of time vs frequency vs amplitude (An example of a data set of this type is presented in Fig. 11).", "This allowed for easy filtering by directly extracting a subset of the frequency axis.", "The absolute mean within this subset was then calculated to give the ‘mean AE’ vs time.", "For the AE results presented in Fig. 7 the window width was equal to the time period required for the composite roughness to repeat, 0.3 s, and the frequency range was 150 kHz–300 kHz.", "This frequency range, which has been used for all the ‘mean AE’ results in this paper, was chosen as it offers the best signal to noise ratio.", "This range was identified by comparing the spectral distribution of data from the fast disk transducer with that from the bearing housing transducer.", "The spectral distributions are presented and discussed later in the paper.", "Fig. 7 shows that when the disks were first loaded at the start of the running-in, (A), there was an extremely short transient peak in the AE to above 1.5 mV.", "After this it remains at a comparatively steady state of approximately 0.9 mV until the end of the first stage (B).", "It is known that there was a large reduction in the surface roughness during this period so a large reduction in AE is also expected.", "The only reduction occurring is the decay from the transient peak which is extremely rapid, effectively instantaneous on the time scale of the entire test stage.", "This suggests that running-in occurred almost instantly upon the start of the test, which is in agreement with previous work which suggests that initial running-in is predominantly a process of plastic-deformation [23,27].", "Subsequent modification of the surface geometry is predominantly due to micropitting rather than further running-in.", "This may be confirmed by examination of non-pitted regions of the profiles in Fig. 5, which show that there is very close agreement between profiles taken after the running-in stage and those taken at the end of the test.", "Further evidence of this is provided by consideration of the AE response at the start of the second test-stage (C).", "The decrease in surface roughness for this test stage was negligible compared to that in the first.", "If the transient AE peak is due to running-in it should therefore be absent, or greatly diminished, and the latter is the case.", "In the second stage there is a short smooth increase in the AE after the transient before it approaches steady state.", "This is due to the temperature change which affects the AE indirectly.", "Fig. 7 shows that upon initial loading, the temperature of both disks immediately starts to increase.", "This is due to frictional heating at the contact.", "The heating is relatively minor and is consistent throughout the test so a new thermal equilibrium is quickly approached.", "The effect of an increase in temperature is to lower the viscosity of the oil film in the contact and consequently decrease its thickness [24].", "This leads to greater direct asperity contact which results in an increase in AE [22].", "Fig. 7 shows the temperature response for both test stages to be similar however the expected increase in the AE is only evident in the second.", "This is attributed to the effects of running-in (reducing asperity interaction) masking the counter-effects of increased temperature (thinning films leading to increased asperity interaction) in the first test stage.", "The AE results from Fig. 7 indicate that the start of the test needs to be considered in greater detail.", "Fig. 8 presents the AE and load response at the start of the first two test stages, during the loading period.", "For this the mean AE was calculated using a narrow window having a width of 1.25 ms, equivalent to 7.5° of rotation of the fast disk.", "This results in minimal temporal averaging with the effect that rotational frequencies are not filtered out as is the case for the wider window used in Fig. 7.", "(It should be noted that the scale of the mean AE amplitude is dependent of the window width which is why it differs between Figs. 7 and 8.", "As only relative changes in the AE are considered in this paper this is of little consequence).", "There are significant oscillations in the mean AE at the frequency of the fast disk rotation.", "These may be caused by one or more of the following cyclic affects: the AE sensor position relative to the contact; the slip ring connection; dynamic rotational loads, perhaps caused by load transmission error; and most intriguingly, any variation in the circumferential roughness of the disk affecting the amount of AE from the contact.", "Further testing would be required to quantify the contribution of each of these factors to the low frequency components of the AE signal.", "Fig. 8 shows that the transient peak in the AE occurs within the period affected by loading transients so it is necessary to assess the implications of these.", "The sampled load was averaged over each fast disk rotation so the temporal resolution is not high but despite this it can be seen that at the start of both test stages there was a brief transient peak in the load which quickly settled.", "The transient peak at the start of the second stage appears to be of lower amplitude, this may have been the case or an artefact of the low sampling rate.", "It is known that AE is dependent on load so the peak in this must have contributed, at least partly, to the peak in the AE independent of the running-in process.", "Crucially however, the loading ramps during the first 0.5 s of each stage are similar whereas the AE responses are not.", "For the first stage there is a significant jump in amplitude followed by an increase to well above the subsequent steady state value, whereas for the second stage the jump is almost absent and the increase barely exceeds the subsequent steady state value.", "These differences are attributed to the presence of running-in in the first stage and its absence in the second, and support the conclusion that the running-in seen in these tests occurs very rapidly, in the first few cycles of operation.", "Indeed, this occurs in no more than two cycles of the complete set of asperity interactions and in only once cycle at the target load when the load ramp is considered.", "It is interesting to compare the AE and CV measurements, which should both be sensitive to asperity contact levels.", "Fig. 7 shows that at the start of the first test-stage (A), the CV immediately drops to zero, its minimum, where it remains for most of the stage except towards the end when it begins to increase slightly.", "The extended period when the CV is at zero reveals one of the major limitations of this measurement technique: its low range.", "In practice the electrical resistance of a contact will become negligible with even a relatively small amount of direct asperity contact.", "At this point the CV will saturate at zero and will not measure any further increases in direct contact.", "This is why there is no transient reduction in CV equivalent to the peak in the AE results.", "For the second test stage the CV drops instantly to a low value before gradually decreasing to zero and then increasing in the latter half of the stage.", "The initial short decrease to zero mirrors that of the increase in AE and is attributed to temperature effects as discussed earlier.", "The gradual increase in the CV, for both test stages, appears to indicate that there is some continual reduction in direct asperity contact over the course of each.", "However there is no corresponding decrease in the AE amplitude which is to be expected from such behaviour.", "This may simply be due to the difference in sensitivities of the two measurement techniques.", "Although the CV has a narrow measurement range, it has great sensitivity within this range [24,28].", "It is possible that the CV is responding to a change too slight to be detected in the AE signal.", "However, regardless of the AE results, the CV increases cannot represent the bulk of the running-in as this has been shown, by the roughness measurements, to have occurred predominantly in the first stage, whereas the increase in the CV is greater in the second stage.", "This suggests the presence of some unknown contact or measurement phenomenon affecting the CV independently of the amount of asperity contact.", "The comparison between AE and CV results shows the former to be more suitable for investigating running-in due to its superior range and consistency with the measured surface change.", "In this section the AE measurements made in the micro-pitting fatigue test are discussed.", "Fig. 9 shows the mean AE measurements for the whole of this test, i.e. from the 6000th fast disk cycle onwards, together with the slow disk temperature and the CV.", "The abscissa tick positions indicate points at which the test was paused for a surface measurement and the start and end of each test stage.", "For clarity, data acquired during the unloaded period before and after each test stage is not presented.", "Additionally the intermittent sampling regime used during the fatigue test filters out the loading transients and thus none of the data presented in Fig. 9 are affected by these.", "There are however temperature transients caused by the resumption of frictional heating when the disks are reloaded at the start of each new test stage.", "The asymptotic temperature response is similar to that presented in Fig. 7 for running-in but due to the significantly longer duration of the fatigue test the initial steep rise appears as an instantaneous temperature jump at the start of each stage.", "The starting temperature for the last five and longer test-stages, from 10 × 105 cumulative fast disk cycles onwards, appears to be higher.", "This was not the case and it is an artifice caused by the change to a lower sampling frequency.", "As discussed for the running-in results a temperature increase causes a thinning of the oil film, causing the initial jump in the AE amplitude at the start of each test stage.", "During this transient the change in surface roughness, if measured, would have been negligible, so the change in AE is due to thermal effects reducing the film thickness.", "Although not the main subject of this paper it is worth noting that the transients reveal the high sensitivity of AE measurement to changes in the film thickness.", "Using a commonly used formula [29], the decrease in the predicted central film thickness, for an increase in contact temperature from 80 to 90 °C, is approximately 20% or in absolute terms 0.07 μm.", "There were also CV transients associated with the temperature jump, for clarity these have been cropped out of Fig. 9 by removing the first 3 min of CV data at the start of each test stage.", "Disregarding these transients, the contact temperature during the entire fatigue test remained within a range of 2.5 °C.", "Post-processing parameters for the AE data were chosen so that the scales of Figs. 7 and 9 are comparable.", "It can therefore be seen that the AE at the start of fatigue testing was consistent with its value at the end of the running-in testing at just below 0.9 mV.", "After this there is a smooth asymptotic decrease in the AE to a value of under 0.3 mV.", "For the fatigue test, as opposed to the running-in, the relative duration of the starting transients is negligible and thus all contact parameters, except surface roughness, can be considered constant over the course of the test.", "The change in AE over the course of the test can therefore only be due to a change in surface roughness.", "This proves that the AE from the contact is dependent on the amount of asperity contact.", "This has been previously inferred from tests conducted by other researchers but the importance of the results presented here is that the surface roughness was the only variable.", "Commonly, speed and load variation are used to investigate different contact conditions, but as these are known to affect the AE independently of the amount of asperity contact [5] such experiments can be ambiguous and difficult to interpret.", "It is also interesting to note that the changes in AE are greater over the first stages.", "This corresponds to the increase in measured micropit depth which is significantly greater in the first section of the test than in the latter stages (Fig. 6).", "The CV results in Fig. 9 broadly support those of the AE.", "There is a general increase in the CV to a value just under the maximum of one.", "The decrease in AE and increase in CV both indicate decreasing asperity contact which in this case can only be due to surface wear/micropitting, the presence of which has been confirmed by the roughness measurements.", "The high final CV value indicates that, by the end of the test, the surface roughness had reduced to the extent that the lubrication conditions were near full-film.", "Given this it is highly likely that, had the test been continued, it would have run for many millions more cycles with only negligible further surface wear.", "This supports the earlier assertion that micro-pitting fatigue was, in this case, a self-limiting process.", "It could be argued that since it resulted in an improvement in the contact conditions, i.e. less direct asperity contact, it could be viewed as a secondary phase of the running-in process rather than as damage.", "The ordinates of the AE and RMS roughness were then scaled and positioned so that the values of the roughness fit and the AE amplitude are at identical heights at the beginning and end of the test.", "For clarity the portions of the AE results affected by starting transients have been omitted.", "It can be seen that the results of the RMS roughness and AE measurements are in broad agreement with both having a similar asymptotic form.", "There is some significant uncertainty in the comparison but, perhaps unexpectedly, this is due to imprecision in the RMS roughness measurements rather than in the AE.", "It is likely that the difference in precision can be attributed to the difference in the effective sample size of each.", "The AE measurements effectively sampled the roughness of the entire contact path whereas the 2D profilometry measurements only sampled a small fraction of this.", "The problems of imprecision in the RMS roughness serve to highlight the relative ease with which suitably band-pass filtered AE measurements can be used to detect minute changes in the conditions of the surfaces of a lubricated contact.", "There is considerable interest in the possibility that wear mechanisms have specific AE signatures, usually in terms of their frequency, that can be used to distinguish them from the AE ‘noise’ inherent in rotating machinery [15,16].", "The utility of such a signature is clear: it would allow incipient damage to be detected that would other-wise be drowned out by general operating noise.", "It is worth then considering if the micro-pitting induced in this testing had some AE signature that distinguishes it from the bulk signal.", "Take first, the premise that the formation of a micro-pit causes a transient increase in the broadband AE amplitude, the mostly likely cause of which would be material fracture during crack growth.", "If this were the case then in periods of significant micro-pit formation the AE amplitude might be expected to deviate to levels above that expected from direct asperity interaction alone.", "The results of the fatigue test presented thus far show no evidence of this.", "It has been shown that the majority of micro-pitting occurred in the period between 6000 and 2 × 105 fast disk cycles so this is the expected location of any deviation.", "However, as Fig. 10 shows, there is a good correlation between the AE and the RMS roughness during this period.", "This implies that any broadband AE from micro-pit formation was insignificant compared to that from general asperity interaction, and that in this experiment the AE is sensitive to changes in asperity interaction caused by micropit formation, rather than directly to the crack-growth mechanisms themselves.", "The second premise is that the frequency content of the AE from micro-pit formation is different to that from asperity interaction.", "Fig. 11 shows the spectral distribution of the AE over the duration of the fatigue test.", "The frequency band of 0.15–0.3 MHz used to calculate the mean AE amplitude results in this paper is marked.", "It can be seen that for the fast disk transducer, Fig. 11(a), there is significant and variable activity within this range.", "In comparison, for the bearing housing transducer, Fig. 11(b), there is negligible activity within this range, as indicated by the different colour scale used in this figure.", "This demonstrates that the AE measured within this range is from the test contact and not from potential sources of noise such as the support bearings.", "The fast disk transducer shows that the amplitude of frequency components above 0.3 MHz is barely above that of the background level and even when examined at a finer resolution there are no patterns in the distribution that can plausibly be related to micro-pit formation.", "However, as with all AE measurements, the frequency components of signals are not only related to the source mechanism, but also to the signal transmission path and sensor sensitivity.", "Below 0.15 MHz there are bands of significant amplitude in the signal from the fast disk transducer but there is relatively little variation in the amplitude of these over the course of the test and so it is highly unlikely that they are related to micropit formation.", "Additionally the bearing housing transducer also shows significant activity in this region which suggests that it is susceptible to noise and thus of little use for investigating asperity interactions.", "The results from the fatigue test have shown that if micro-pitting formation does have a distinct AE signature it is extremely subtle, and may only be revealed by further analysis using more complex techniques which examine changes in the AE signal over much shorter timescales, for example resolved in terms of angular position during individual disk rotations.", "It may be that advanced analysis techniques [16] could uncover it, but it is more likely that if such a signature does exist, it is indistinguishable from the background AE caused by asperity interaction.", "However, it cannot be denied that this work has demonstrated the sensitivity of AE to changes in mixed lubrication conditions due to surface topography modifications caused by micropitting and micro-wear mechanisms.", "This work has shown that Acoustic Emission is a sensitive tool for studying subtle changes in mixed lubrication conditions during both the running-in and micropitting processes.", "Detailed measurements of the evolution of surface topography have shown that running-in is a rapid process whereby prominent asperities are flattened during the first few cycles of operation.", "This is reinforced by consideration of the AE signals recorded during the initial operation of the twin disk test rig.", "During subsequent operation of the run-in surfaces, the formation of micropits was quantified using in-situ relocation profilometry.", "AE signals recorded during the micropitting test were strongly related to changes in surface topography, with a close correlation between AE signals and RMS surface roughness.", "However, examination of the frequency content of the AE signals did not show characteristic features related to the crack-growth and fracture mechanisms at work during the formation of micropits.", "Rather, this research demonstrates that AE is highly sensitive to the resulting changes in asperity interaction under mixed lubrication conditions, due to micropitting.", "In general, AE was found to be more sensitive to mixed lubrication conditions than the contact voltage technique, as the AE was still able to discern subtle changes in conditions after the contact voltage measurement had saturated.", "AE is therefore proposed as a useful tool for the experimental study of mixed lubrication conditions, which has the potential to offer detailed insight into contact behaviour under realistic operating conditions.", "Information on how to access the data that supports the results presented in this article can be found in the Cardiff University data catalogue at https://doi.org/10.17035/d.2016.0008118727." ]	[ "The acoustic emission (AE) from the running-in and micro-pitting of a mixed lubrication contact was studied.", "Surface profile and electrical resistance measurements were used to validate the AE measurements.", "The amplitude of AE was extremely sensitive to changes in the lubrication conditions induced by surface modification.", "AE measurements indicate that running-in occurs extremely quickly, within a few rotational cycles.", "Measured AE depends on the effects of micro-pits on the lubrication conditions rather than their formation mechanism." ]
VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis	The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.	[ "NEUR" ]	[]	[ "2800" ]	[ "The efficient formation of synaptic vesicles (SVs) from the plasma membrane after neurotransmitter release is critical to maintain the fidelity of neurotransmission across a wide range of stimulation intensities.", "Distinct SV endocytosis modes are present within central nerve terminals that are triggered by discrete patterns of neuronal activity.", "These are ultrafast endocytosis (Watanabe et al., 2013, 2014), clathrin-mediated endocytosis (CME), which is prevalent during mild stimulation (Granseth et al., 2006), and activity-dependent bulk endocytosis (ADBE), which is only triggered during intense neuronal activity (Clayton et al., 2008; Clayton and Cousin, 2009a).", "The route for SV formation differs between these endocytosis modes, with CME generating single SVs, whereas both ultrafast and ADBE generate endosomes directly from the plasma membrane from which SVs can then bud (Kokotos and Cousin, 2015).", "A key aspect in the formation of functional SVs is the incorporation of the correct protein cargo with the appropriate stoichiometry.", "CME utilizes both the adaptor protein complex AP-2 and a series of monomeric adaptor proteins to ensure a high fidelity of SV cargo clustering and incorporation (Diril et al., 2006; Kelly and Owen, 2011; Koo et al., 2011; Rao et al., 2012).", "In contrast, very little is known regarding the mechanism of SV cargo retrieval from the plasma membrane in either ultrafast endocytosis or ADBE.", "Recent studies have hinted that cargo sorting occurs primarily at the endosome for both modes (Cheung and Cousin, 2012; Kononenko et al., 2014; Watanabe et al., 2014), suggesting that endosomes are formed in a relatively non-specific manner and may resemble the plasma membrane in composition (Kononenko et al., 2014).", "During ADBE the generation of “bulk” endosomes is rapid and synchronous with neuronal activity (Clayton et al., 2008).", "This event is widely thought to be clathrin independent, since inactivation or knockdown of clathrin does not impact on either the formation of bulk endosomes (Heerssen et al., 2008; Kasprowicz et al., 2008) or the recovery of SV cargo during high-frequency stimulation (Kononenko et al., 2014).", "We investigated whether directed sorting of SV cargo occurs at the plasma membrane during ADBE, since this will ultimately impact on the molecular composition of SVs generated via this endocytosis mode.", "We examined the retrieval of a series of exogenously expressed SV cargo molecules, and we found that only one was preferentially trafficked via ADBE—VAMP4.", "Endogenous VAMP4 was selectively enriched on bulk endosomes and was also essential for ADBE to proceed, indicating that it is an essential ADBE cargo molecule.", "ADBE is triggered by intense neuronal stimulation and is the dominant mode of SV retrieval during such stimuli (Clayton et al., 2008).", "However, little is known regarding SV cargo selection mechanisms by this endocytosis mode.", "To investigate this, we examined the trafficking of a series of genetically encoded reporters of SV cargo that have a pH-sensitive GFP moiety (pHluorin) fused to a luminal domain.", "Such reporters are widely used to monitor both SV fusion and CME, since pHluorin fluorescence is quenched in acidic environments such as the SV interior (Kavalali and Jorgensen, 2014).", "SV exocytosis is therefore reported as an increase in fluorescence, whereas SV endocytosis is monitored as a decrease, since CME is rate limiting when compared to the rate of SV acidification (Sankaranarayanan and Ryan, 2000; Atluri and Ryan, 2006) (but see Egashira et al., 2015).", "This interpretation becomes more complicated when monitoring ADBE, however, since the initial step in this pathway is the formation of a bulk endosome, which is present in nerve terminals for at least 30 min following its generation (Cheung et al., 2010).", "From first principles the larger interior of the bulk endosome when compared to a SV (approximately 50-fold for a 150 nm endosome) should mean it takes longer to acidify.", "This should retard the rate of the post-stimulation fluorescence decay, rendering interpretation of the pHluorin signal a much more complex process.", "We first examined the trafficking of VAMP4-pHluorin during intense neuronal activity, since this reporter displays a unique activity-dependent fluorescent profile in comparison to other pHluorin-tagged SV cargo.", "VAMP4-pHluorin exhibits a fluorescence decrease when challenged with action potentials followed by a slow post-stimulation increase (Raingo et al., 2012), suggesting its activity-dependent retrieval from the plasma membrane occurs only during intense stimulation.", "The post-stimulation, activity-dependent increase is thought to be asynchronous release (Raingo et al., 2012), which is proposed to be mediated by SVs generated by ADBE (Evstratova et al., 2014); thus, VAMP4-pHluorin may be a potential ADBE cargo.", "When cultured hippocampal neurons expressing VAMP4-pHluorin were stimulated with a train of high-frequency action potentials (40 Hz, 10 s) to evoke ADBE, they displayed an average response consisting of an immediate downstroke and slow recovery to baseline (Figures 1A and 1B) (Raingo et al., 2012).", "However, when individual nerve terminal responses were assessed, the average response could be dissected into two discrete post-stimulation fluorescent profiles (Figure 1B).", "Approximately 40% of nerve terminals displayed a slow decrease in fluorescence after stimulation, whereas 60% displayed an increase (Figure 1C).", "We next assessed the evoked VAMP4-pHluorin profile in cultures of cerebellar neurons since a larger proportion of their nerve terminals display ADBE (Clayton and Cousin, 2009b), most likely due to chronic stimulation in culture (Burgoyne and Cambray-Deakin, 1988).", "In these neurons 65% of nerve terminals displayed a slow fluorescent decrease and only 35% displayed an increase, correlating with the higher prevalence of ADBE in this culture system (Figures 1D and 1E).", "We hypothesized that this slow fluorescent downstroke may represent the acidification of bulk endosomes after their rapid activity-dependent generation by ADBE.", "We tested this by performing a series of corroborating experiments.", "First we determined the number of nerve terminals displaying slow VAMP4-pHluorin downstrokes in response to 10 Hz stimulation, a protocol that primarily triggers CME and not ADBE (Clayton et al., 2008).", "Very few nerve terminals displayed a VAMP4-pHluorin downstroke in either hippocampal or cerebellar neurons under these stimulation conditions (Figures 1C and 1E).", "Thus, when ADBE is not triggered by intense stimulation, the number of slow VAMP4-pHluorin downstrokes is negligible.", "To determine whether slow VAMP4-pHluorin downstrokes reflect bulk endosome acidification after ADBE, we examined how the fluorescent response is altered when this endocytosis mode was inhibited.", "We employed a stimulation 1 (S1) and stimulation 2 (S2) protocol in cerebellar cultures, where two identical high-frequency trains (40 Hz, 10 s) were applied 10 min apart (Figures 2A and 2B).", "A very similar proportion of nerve terminals displaying evoked slow downstrokes was observed at both S1 and S2, indicating the reproducibility of the response (Figure 2C).", "We assessed the effect of arresting ADBE by inhibiting glycogen synthase kinase 3 (GSK3).", "Antagonism of this protein kinase has no effect on ADBE during an initial high-frequency stimulus; however, ADBE is inhibited during subsequent identical action potential challenges (Evans and Cousin, 2007; Clayton et al., 2010).", "This was confirmed by monitoring uptake of large fluorescent dextran molecules (tetramethylrhodamine-dextran [TMR-dextran]) that selectively report ADBE (Figure S1A).", "When S1 and S2 experiments were performed in the presence of the GSK3 antagonist CT99021 (2 μM), there was no difference in the proportion of nerve terminals displaying slow VAMP4-pHluorin downstrokes during the first stimulus train (Figure 2C).", "However, when S2 responses were monitored (at which time ADBE is inhibited), there was a significant reduction in the number of nerve terminals displaying a downward fluorescent response (Figure 2C).", "Thus, inhibition of ADBE greatly diminishes the proportion of nerve terminals displaying activity-dependent slow VAMP4-pHluorin downstrokes, suggesting this downstroke reflects bulk endosome acidification.", "To confirm that slow VAMP4-pH fluorescent downstrokes were ADBE dependent, we silenced expression of the essential ADBE molecule syndapin I using a characterized short hairpin RNA (shRNA) vector (Clayton et al., 2009).", "Inhibition of ADBE was confirmed by monitoring TMR-dextran uptake during high-frequency stimulation (Figure S1C).", "Syndapin I knockdown dramatically altered the average VAMP4-pHluorin response, with almost all nerve terminals displaying an immediate evoked increase in signal (Figure 2D).", "Indeed, syndapin I knockdown reduced the number of nerve terminals displaying slow VAMP4-pHluorin downstrokes to almost zero (Figure 2E).", "This provides compelling evidence that ADBE is responsible for these evoked downstrokes during intense neuronal activity.", "If VAMP4-pHluorin is specifically retrieved via ADBE, its fluorescent signal should be inaccessible to impermeant weak acid, since it will be trapped inside a slowly acidifying bulk endosome directly after intense stimulation.", "To test this, an impermeant acid solution was applied immediately after high-frequency (40 Hz) stimulation in either cerebellar or hippocampal neurons to quench the fluorescent signal from the surface reporter.", "Approximately 40% of the VAMP4-pHluorin response was resistant to quenching when compared to a pre-stimulus baseline in either neuronal subtype (Figures 3A and 3B).", "Importantly, complete quenching of the VAMP4-pHluorin response was observed at 10 Hz stimulation, where ADBE is not triggered (Figure 3B).", "This indicates that VAMP4-pHluorin is located inside a slowly acidifying compartment such as a bulk endosome after intense stimulation and that VAMP4 is a bone fide cargo for ADBE.", "To determine whether other SV cargoes are also recovered by ADBE, we performed a surface quenching experiment in cerebellar neurons using synaptophysin-pHluorin (syp-pHluorin) as a typical SV cargo.", "In contrast to VAMP4-pHluorin, application of acid pulses immediately after high-frequency stimulation decreased the syp-pHluorin response to near baseline levels (Figures 3C and 3D).", "Thus, virtually all syp-pHluorin fluorescence is present inside rapidly acidifying compartments (such as SVs) immediately after high-frequency stimulation.", "This suggests that syp-pHluorin is not recovered by ADBE during intense neuronal activity.", "To determine whether a series of other SV cargo molecules could be recovered by ADBE, we performed S1 and S2 experiments in cerebellar neurons with pHluorin-tagged versions of either syp-pHluorin, synaptobrevin II (sybII-pHluorin), synaptotagmin-1 (syt1-pHluorin), or the vesicular glutamate transporter (vGLUT1-pHluorin) (Figure 4A).", "In all cases the evoked fluorescent response was unaltered between the S1 and S2 challenge, in terms of the extent of the fluorescent response or the kinetics of fluorescence recovery (Figure S2).", "When these experiments were repeated in the presence of CT99021, there was no change in either the extent of the pHluorin signal (Figure S3B) or the kinetics of fluorescence recovery (Figures 4B–4F) between S1 and S2 for any reporter.", "This suggests that inhibition of ADBE does not impact on the recovery of most SV cargoes during intense stimulation, a premise supported by the fact that a second maneuver that arrests ADBE (inhibition of cyclin-dependent kinase 5 with the antagonist roscovitine; Figure S1B) also had no effect on activity-dependent syp-pHluorin retrieval (Figures S3C and S3D).", "This was confirmed by the absence of effect on the evoked syp-pHluorin response in cerebellar neurons where expression of the ADBE molecule syndapin I had been silenced (Figures 2F and 2G; Figure S1D).", "Thus, inhibition of ADBE does not affect the trafficking of exogenously expressed SV cargo.", "To ensure that the lack of retrieval of typical SV cargo by ADBE was not specific to cerebellar neurons, we repeated this experiment in primary cultures of hippocampal neurons expressing syp-pHluorin.", "These neurons responded to both stimulus trains in a very similar manner to cerebellar neurons (Figure S4B).", "When incubated with CT99021 to inhibit ADBE during the second stimulus train, there was no difference in either the extent of syp-pHluorin response or the kinetics of its fluorescence recovery (Figures S4C and S4D) compared to untreated neurons.", "Thus, inhibiting ADBE does not impact on the trafficking of a series of exogenously expressed SV cargo molecules during intense neuronal activity in multiple culture systems.", "We have shown that VAMP4-pHluorin is selectively recovered by ADBE during intense stimulation.", "However, the endocytosis mode responsible for recovery of other SV cargo remains unclear.", "CME is maximally active during intense stimulation (Clayton et al., 2008) and therefore may be responsible for the recovery of these cargo molecules.", "To test this, we examined the trafficking of syp-pHluorin after inhibition of CME in cerebellar neurons.", "CME was arrested by exposure to the clathrin inhibitor pitstop-2 (15 μM) (von Kleist et al., 2011).", "Pitstop-2 had no effect on ADBE, confirmed by its absence of effect on TMR-dextran uptake evoked by frequency stimulation (Figure 5A).", "In contrast, pitstop-2 abolished the fluorescence recovery of syp-pHluorin after an identical stimulus train (Figure 5B).", "Thus, syp-pHluorin appears to be predominantly trafficked via CME even during intense neuronal activity.", "Prolonged exposure to pitstop-2 deacidifies SVs (Hua et al., 2013), potentially explaining the arrest of the syp-pHluorin fluorescence recovery.", "However, this effect only became apparent beyond the timescale of our experiments; therefore, SV deacidification is not responsible for this block of syp-pHluorin retrieval (Figure S5).", "We next determined whether arresting CME interfered with the evoked VAMP4-pHluorin response during high-frequency stimulation.", "We predicted that the fluorescent downstroke would not be affected by this maneuver, since it would be independent of CME.", "This was the case, with the VAMP4-pHluorin response displaying an initial upstroke followed by a slow post-stimulation fluorescence decrease to below baseline levels when CME was arrested using pitstop-2 (Figure 5C).", "This response was observed in all nerve terminals investigated.", "Thus, the slow VAMP4-pHluorin downstroke is unaffected by the arrest of CME, confirming its dependence on ADBE.", "To confirm these observations, we knocked down expression of clathrin heavy chain (CHC) using shRNA oligonucleotides (Royle et al., 2005).", "This shRNA approach reduced endogenous CHC levels in cerebellar neurons, but surprisingly it also inhibited TMR-dextran uptake (Figures S6A and S6C).", "This inhibition was most likely due to chronic arrest of clathrin-dependent SV budding from bulk endosomes, since cerebellar neurons are cultured in permanently depolarizing conditions (Burgoyne and Cambray-Deakin, 1988).", "In agreement, CHC knockdown had no effect on TMR-dextran uptake in cultured hippocampal neurons (Figure 5D).", "We therefore examined the effect of CHC knockdown on syp-pHluorin retrieval in hippocampal neurons, since its effect in cerebellar neurons cannot be interpreted due to off-target effects on ADBE (Figure S6C).", "Neurons expressing scrambled shRNA exhibited a characteristic syp-pHluorin response, whereas those expressing CHC shRNA exhibited a greatly retarded fluorescence recovery after stimulation (Figure 5E).", "Thus, inhibition of CME by either CHC knockdown or a clathrin inhibitor significantly impacts on syp-pHluorin retrieval during high-frequency stimulation, indicating that CME is the dominant SV retrieval mode for this SV cargo even during intense neuronal activity.", "We also determined the effect of CHC knockdown on the VAMP4-pHluorin response in hippocampal neurons.", "Neurons expressing CHC shRNA displayed a similar VAMP4-pHluorin profile to pitstop-2-treated neurons, with an initial increase followed by a slow post-stimulation downstroke (Figure 5F).", "Thus, the slow VAMP4-pHluorin downstroke, which is only triggered during high intensity, was unaffected by two independent maneuvers that arrest CME, confirming its dependence on ADBE.", "We have shown that VAMP4-pHluorin is a genetically encoded reporter of ADBE.", "We next determined whether endogenous VAMP4 was also selectively accumulated by ADBE.", "To achieve this we performed biochemical fractionation experiments to enrich bulk endosomes from neuronal cultures.", "Cerebellar neurons are excellent for such studies, since they can be cultured to greater than 95% homogeneity (Burgoyne and Cambray-Deakin, 1988).", "We confirmed the enrichment of a bulk endosome fraction by tracking newly formed endosomes and SVs using the fluorescent dye FM1-43.", "After loading FM1-43 in the absence or presence of a strong stimulus, the neurons were lysed by mechanical disruption.", "The post-nuclear supernatant was then fractionated using discontinuous Nycodenz gradients after a short spin to remove cellular debris (Figure 6A) (Barysch et al., 2010).", "Analysis of FM1-43 fluorescence in the stimulated fractions showed two distinct peaks, corresponding to endosomes (fraction 2) and SVs (fraction 5) (Figure 6B) (Barysch et al., 2010).", "To confirm that these fractions were enriched for bulk endosomes and SVs, respectively, we performed an identical experiment using the fluid phase marker horse radish peroxidase (HRP).", "Morphological analysis confirmed the enrichment of HRP-labeled endosomes in fraction 2 and HRP-labeled SVs in fraction 5 when comparing stimulated to unstimulated cultures (Figures 6C–6G).", "Next we probed for the presence of endogenous SV cargo by western blotting.", "This was achieved by comparing the presence of these cargoes in endosome and SV fractions from the same preparation of stimulated cultures.", "The endogenous SV cargoes syt1, syp, sybII, and vGLUT were all present in both the bulk endosome and SV fraction (Figures 6H and 6I).", "In contrast, VAMP4 was almost exclusively present in the endosome fraction (Figures 6H and 6I).", "Thus, bulk endosomes do contain endogenous SV cargo; however, only VAMP4 is enriched within this compartment.", "To confirm the localization of VAMP4 to bulk endosomes in vivo, we performed ultrastructural analyses using silver-enhanced immunogold staining in either resting or stimulated (40 Hz, 10 s) cerebellar cultures.", "We observed a high degree of VAMP4 localization to bulk endosomes in stimulated nerve terminals, confirming its recovery via ADBE (Figure 7A).", "One key remaining question is whether VAMP4 simply represents an ADBE cargo or whether it is essential for ADBE to proceed.", "We determined this by using validated shRNA oligonucleotides against VAMP4 (Bal et al., 2013) (Figures S7A–S7C).", "Knockdown of VAMP4 had no effect on the syp-pHluorin response evoked by 40Hz stimulation, indicating no essential role in CME (Figure S7D).", "However, under identical stimulation conditions VAMP4 knockdown abolished TMR-dextran uptake, an inhibition fully rescued by expression of wild-type VAMP4-pHluorin (Figures 7B and 7C).", "Therefore, in addition to being the first identified ADBE cargo molecule, VAMP4 is essential for this key endocytosis mode.", "The essential requirement for VAMP4 in ADBE suggests it must share key interactions with other endocytosis molecules to direct this process.", "One potential association is with adaptor proteins, since a di-leucine motif on the cytoplasmic N terminus of VAMP4 coordinates such interactions (Peden et al., 2001).", "To test this we performed a rescue experiment in VAMP4 knockdown neurons using a VAMP4 mutant (L25A) that disrupts this interaction (Peden et al., 2001; Raingo et al., 2012).", "We observed no rescue of TMR-dextran uptake with this mutant (Figures 7B and 7C), suggesting interactions between VAMP4 and adaptor proteins are essential for progression of ADBE.", "We next examined whether disrupted adaptor protein interactions affected the trafficking of VAMP4-pHluorin.", "This mutation abolished the fast activity-dependent VAMP4-pHluorin downstroke in both cerebellar and hippocampal neurons in agreement with previous work (Raingo et al., 2012) (Figures 8A and 8B).", "Furthermore, the L25A mutant displayed a similar average trafficking profile to wild-type VAMP4-pHluorin in neurons where ADBE had been inhibited (compare Figures 8A and 8B with Figure 2C).", "This effect was not due to a dominant-negative effect on ADBE by L25A overexpression, since overexpression of this mutant or wild-type VAMP4-pHluorin has no effect on evoked TMR-dextran uptake (Figure S7E).", "Thus, adaptor interactions of VAMP4 are essential for both its trafficking via ADBE and for ADBE itself.", "The essential requirement for VAMP4 in ADBE provided us an opportunity to examine how ablation of this endocytosis mode alters presynaptic function during sustained and intense neuronal activity.", "To achieve this, we examined the trafficking of syp-pHluorin in response to four consecutive trains of high-frequency action potentials (40 Hz, 10 s) in hippocampal neurons transfected with either VAMP4 shRNA or a scrambled control.", "The syp-pHluorin response in control neurons was highly reproducible, displaying a reduction in the peak fluorescent response with each consecutive action potential train, presumably due to short-term depletion of SVs (Figures 8C and 8D).", "VAMP4 knockdown neurons displayed a greater reduction in peak height during the latter stimulus trains when compared to the scrambled shRNA controls (Figures 8C and 8D).", "Importantly, the time constant of syp-pHluorin retrieval was not significantly different between VAMP4 knockdown and control neurons (Figure 8E), indicating that the reduction in peak height was not due to modulation of CME.", "Therefore, the reduction in presynaptic performance in VAMP4 knockdown neurons is most likely due to a reduction in SVs generated via ADBE, highlighting the importance of this endocytosis mode in maintaining neurotransmission during periods of sustained neuronal activity.", "During brief bursts of intense neuronal activity, SV cargo and membrane transiently accumulate at the plasma membrane of central nerve terminals.", "ADBE is the dominant SV endocytosis mode under these conditions; however, it was unclear whether ADBE actively sorted cargo at the plasma membrane, since both clathrin and adaptor proteins are essential for SV generation from bulk endosomes (Heerssen et al., 2008; Kasprowicz et al., 2008; Cheung and Cousin, 2012; Kononenko et al., 2014).", "We have identified one SV cargo, VAMP4, which is specifically sorted into endosomes during ADBE and is also essential for ADBE to occur.", "The generation of bulk endosomes via ADBE is a clathrin-independent process (Heerssen et al., 2008; Kasprowicz et al., 2008).", "Recent studies using syt1-pHluorin appeared to support this idea, since neither AP-2 nor clathrin knockdown had no effect on the evoked fluorescent response during high-frequency stimulation (Kononenko et al., 2014).", "In contrast, we observed a robust inhibition of the syp-pHluorin response on inhibition of CME during intense activity using either the clathrin antagonist pitstop-2 or CHC knockdown.", "One potential explanation for this discrepancy is the efficiency of CHC knockdown by lentiviral shRNA (as used in Kononenko et al. [2014]), since this method displays a wide range of knockdown between individual cultured neurons (López-Murcia et al., 2014).", "Another confounding factor would be the temperature at which experiments were performed, since CME may not be dominant at 37°C (Watanabe et al., 2014).", "However, at physiological temperatures we still observed a robust arrest of the syp-pHluorin response with CHC knockdown (Figure S6E).", "Finally, under stimulation conditions identical to those in the previous study (Kononenko et al., 2014), we observed a robust inhibition of Syt1-pHluorin retrieval in cultured hippocampal neurons on CHC knockdown (Figure S8).", "We are currently unable to explain why we observe such disparate results using almost identical tools; however, we have shown identical effects on SV cargo retrieval in multiple systems using two independent maneuvers to arrest CME.", "An important point to note from our biochemical enrichment of bulk endosomes is that endogenous SV cargoes were accumulated by ADBE.", "This may be a result of ADBE accumulating excess SV cargo in a non-specific manner, due to CME reaching saturation capacity.", "An alternative explanation is that a population of SVs generated via CME may fuse with bulk endosomes to provide key molecules required for functional ADBE-derived SVs.", "Regardless, the presence of essential SV cargo on bulk endosomes will ensure that ADBE-derived SVs have the requisite complement of trafficking and fusion molecules that are essential for a functional SV.", "We have identified the first genetically encoded reporter of ADBE, VAMP4-pHluorin.", "ADBE is visualized as a slow downstroke in VAMP4-pHluorin fluorescence after challenged with a train of high-frequency action potentials.", "This downstroke reflects acidification of bulk endosomes rather than VAMP4-pHluorin retrieval, since (1) ADBE only occurs during stimulation (Clayton et al., 2008) and (2) the downstroke continues for minutes after termination of stimulation.", "We also observed an immediate drop in VAMP4-pHluorin fluorescence in hippocampal neurons and to a lesser extent in cerebellar neurons, consistent with a parallel retrieval of this reporter via CME (Raingo et al., 2012).", "In agreement this fast activity-dependent fluorescent decrease was ablated by inhibition of this endocytosis mode.", "It was also ablated by mutating the interaction motif for adaptor proteins on VAMP4 (Raingo et al., 2012), suggesting that interactions mediated via this motif are also required for VAMP4 retrieval via CME.", "It should also be stated that CME is not required for retrieval of VAMP4 via ADBE, since the slow post-stimulation downstrokes were retained when CME was inhibited.", "Thus, an adaptor-dependent, clathrin-independent retrieval process retrieves VAMP4 as a first, essential step of ADBE.", "The endosomal location of VAMP4 was confirmed by both in vivo ultrastructural analysis and the inability of impermeant acid to quench VAMP4-pHluorin fluorescence after high-intensity stimulation.", "This latter result meant that we were able to estimate the kinetics of bulk endosome acidification for the first time.", "We found VAMP4-pHluorin fluorescence decreased with a time constant of approximately 32 ± 2 s in the presence of pitstop-2 (to eliminate contaminating CME).", "This is an order of magnitude slower than SV acidification (Atluri and Ryan, 2006; Egashira et al., 2015), consistent with the larger internal volume of the bulk endosome compared to a SV (approximately 50-fold, assuming a typical bulk endosome of 150 nm diameter).", "Thus, when interpreting pHluorin responses during high-frequency stimulation, CME is rate limiting for most SV cargo, whereas bulk endosome acidification is rate limiting for VAMP4-pHluorin.", "The selective capture of VAMP4 by ADBE is not simply due to its high expression at the plasma membrane in comparison to other SV cargo (Raingo et al., 2012).", "This is because the proportion of plasma membrane VAMP4-pHluorin (23.1% ± 4.0% cerebellar neurons; 36.1% ± 7.0% hippocampal neurons) is comparable to the surface expression of both sybII-pHluorin and Syt1-pHluorin (Gordon et al., 2011; Pan et al., 2015; Zhang et al., 2015), neither of which are selectively accumulated via this endocytosis mode.", "We show that a di-leucine sorting motif on VAMP4 is essential for its recovery via ADBE and for ADBE itself.", "This motif coordinates interactions with adaptor proteins (Peden et al., 2001), suggesting that VAMP4 recruits specific adaptors to mediate both ADBE and its own recovery from the plasma membrane.", "The cytoplasmic domain of VAMP4 contains other interaction sites for adaptor proteins such as PACS-1 (Hinners et al., 2003), and its SNARE motif can interact with the CALM/AP180 family of monomeric adaptor molecules (Sahlender et al., 2013).", "It will be important to establish the role of these interactions and how they coordinate the recovery of other ADBE-specific cargoes.", "The selective accumulation of VAMP4 during ADBE suggests that SVs formed via this mode of endocytosis may have a specific molecular signature that defines their physiological function in central nerve terminals.", "It is known that ADBE-derived SVs repopulate the reserve pool, which is only released during intense stimulation after the synchronous release of the readily releasable pool (Richards et al., 2003; Cheung et al., 2010).", "In agreement we observed a rundown in presynaptic function in VAMP4 knockdown neurons, confirming the requirement for ADBE-derived SVs to maintain neurotransmitter release during intense neuronal activity (Cheung et al., 2010).", "SV pools have been proposed to have a specific molecular composition that defines their role, in particular the expression of non-canonical forms of sybII (Hua et al., 2011; Raingo et al., 2012; Ramirez et al., 2012).", "Modulation of VAMP4 expression has bidirectional effects on asynchronous release in neuronal culture (Raingo et al., 2012), suggesting ADBE-derived SVs may replenish this specific functional pool.", "In support, AP-3b2 knockout mice display large deficits in asynchronous release (Evstratova et al., 2014), agreeing with the essential role for the adaptor protein AP-3 in generating SVs from bulk endosomes (Cheung and Cousin, 2012).", "Thus, ADBE may produce SVs with a distinct molecular signature that destines them to specifically maintain a SV pool that mediates asynchronous release.", "The pHluorin expression vectors were obtained from the following sources: Syp-pHluorin, Prof. L. Lagnado (University of Sussex); Syt1-pHluorin, Prof. V. Haucke (Leibniz Institute of Molecular Pharmacology); vGLUT1-pHluorin, Prof. R. Edwards (University of California, San Francisco); sybII-pHluorin, Prof. G. Miesenbock (Oxford University); and VAMP4-pHluorin, Prof. Ege Kavalali (UT Southwestern Medical Centre).", "The sequence encoding VAMP4-pHluorin was cloned into a Clontech EGFP-N1 mammalian expression vector by first removing EGFP and then inserting VAMP4-pHluorin using AgeI and NotI enzymes.", "The L25A mutant was made via mutagenesis using the primers forward tgaaaggagaaatgctttggaagatgatg; reverse catcatcttccaaagcatttctcctttca (mutated bases underlined).", "The empty vector for mCerulean was made as described (Gordon and Cousin, 2013) as were syndapin I and empty mCerulean-tagged pSUPER shRNA vectors (Cheung et al., 2010).", "Validated shRNA oligonucleotides and their scrambled controls for both CHC (Royle et al., 2005) and VAMP4 (Raingo et al., 2012) were ligated into mCerulean-tagged pSUPER shRNA vectors as described (Cheung et al., 2010).", "FM1-43 and advasep-7 were from Biotium.", "VAMP4, synaptophysin, and vGLUT1 antibodies were from Synaptic Systems.", "Pitstop-2 and syt1 and sybII antibodies were from AbCam.", "CT92001 was from R&D Systems, and roscovitine was from Merck.", "Neurobasal media, B-27 supplement, penicillin and streptomycin, minimal essential medium (MEM), and Lipofectamine 2000 were obtained from Invitrogen.", "The silver enhancement kit was from Nanoprobes, whereas F(ab’) 2 fragment anti-rabbit antibodies conjugated to ultrasmall gold particles were from Electron Microscopy Sciences.", "Osmium tetroxide, paraformaldehyde, and glutaraldehyde were from Agar Scientific.", "All other reagents were obtained from Sigma-Aldrich.", "Primary cultures of cerebellar neurons were prepared from the cerebella of 7-day-old Sprague Dawley rat pups of both sexes (Anggono et al., 2006).", "Dissociated primary hippocampal neuronal cultures were prepared from E17.5 C56BL/6J mouse embryos of both sexes by trituration of isolated hippocampi to obtain a single cell suspension, which was plated at a density of 5 × 105 cells/coverslip on poly-D-lysine and laminin-coated 25-mm coverslips.", "Cultures were maintained in neurobasal media supplemented with B-27, 0.5 mM L-glutamine, and 1% v/v penicillin and streptomycin.", "After 72 hr, cultures were further supplemented with 1 μM cytosine β-d-arabinofuranoside to inhibit glial proliferation.", "Cerebellar neurons were transfected between 5 and 7 days in culture, whereas hippocampal neurons were transfected between 6 and 8 days in culture with Lipofectamine 2000 (Gordon et al., 2011).", "In most experiments, two constructs were co-expressed; pHluorin vectors were cotransfected with either mCerulean empty vector or mCerulean expressing shRNA vectors.", "Both sybII-pHluorin and syp-pHluorin were expressed in the absence of other vectors unless specifically stated in the legends.", "Cerebellar neurons were imaged after 8–10 days in culture, whereas hippocampal neurons were imaged after 13–16 days.", "Cerebellar neuron cultures were removed from culture medium and left for 10 min in incubation medium (170 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 20 mM TES (N-tris[hydroxy-methyl]-methyl-2-aminoethane-sulphonic acid), 5 mM NaHCO3, 5 mM glucose, 1.2 mM Na2SO4, 1.2 mM MgCl2, and 1.3 mM CaCl2 [pH 7.4]).", "They were mounted in a Warner imaging chamber with embedded parallel platinum wires (RC-21BRFS) and placed on the stage of a Zeiss Axio Observer A1 epifluorescence microscope.", "Transfected neurons were visualized with a Zeiss Plan Apochromat ×40 oil immersion objective (NA 1.3) at 430 nm excitation (to illuminate mCer), whereas pHluorin reporters were visualized at 500 nm (both using a dichroic > 525 nm and long-pass emission filter > 535 nm).", "Cultures were subjected to continuous perfusion with incubation medium and stimulated with a train of either 400 action potentials delivered at 40 Hz (100 mA, 1-ms pulse width) or 300 action potentials delivered at 10 Hz where indicated.", "At the end of the experiment, cultures were challenged with alkaline imaging buffer (50 mM NH4Cl substituted for 50 mM NaCl) to reveal total pHluorin fluorescence.", "Where indicated cultures were also challenged with acidic imaging buffer (20 mM MES substituted for 20 mM TES [pH 5.5]).", "Fluorescent images were captured at 4-s intervals using a Zeiss AxioCam MRm Rev.3 digital camera and processed offline using Image J 1.43 software.", "Regions of interest of identical size were placed over nerve terminals, and the total fluorescence intensity was monitored over time.", "Only regions that responded to action potential stimulation were selected for analysis.", "All statistical analyses were performed using Microsoft Excel and GraphPad Prism software.", "Where required, traces were decay corrected using a mono-exponential decay function fitted to the first 15 points of acquisition.", "The pHluorin fluorescence change was calculated as FΔ/F0, and n refers to the number of individual coverslips examined.", "Imaging of hippocampal cultures was performed in essentially the same manner apart from perfusion with an altered imaging buffer (136 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 10 mM glucose, and 10 mM HEPES [pH 7.4] supplemented with 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione and 50 μM DL-2-Amino-5-phosphonopentanoic acid).", "The uptake of TMR-dextran (40 kDa) was monitored as described previously (Clayton et al., 2008).", "Briefly, cerebellar neurons were removed from culture medium, left for 10 min in incubation medium, and then stimulated with a train of 400 action potentials (40 Hz, 10 s).", "TMR-dextran (50 μM) was present during the stimulus and was washed away immediately after stimulation.", "The extent of loading was determined by the number of fluorescent puncta in a defined field of view (130 × 130 μm) using a 40× oil immersion objective at 550 nm excitation and >575 nm emission.", "Thresholding analysis was performed to discount regions too large to represent individual nerve terminals (diameter greater than 2 μm).", "The average number of dextran puncta per field for each experiment (usually eight fields of view per experiment) were averaged for the same conditions and subtracted from background fluorescence.", "The final value for dextran puncta was obtained by averaging the individual averages from at least three independent experiments (n is taken as the number of experiments).", "To ensure the density of nerve terminals was consistent between fields and experimental conditions, experiments were always performed on the same set of cultures.", "Cultures were used between 8 and 10 days in vitro.", "Experiments with hippocampal neurons were performed in an almost identical manner, with the exception of the 10-min repolarization in altered imaging buffer.", "Hippocampal neurons were used between 14 and 16 days in vitro.", "Experiments using neurons transfected with either shRNA or overexpression vectors were performed in the same manner, with the number of dextran puncta per μm of axon calculated and then normalized to control values.", "In both cases n is the number neurons analyzed from at least three independent coverslips.", "Immunolabeling was performed as described (Gordon et al., 2011).", "Briefly transfected neurons were visualized at 480 nm after incubation with anti-GFP antibodies to enhance the signal from mCerulean expressing neurons.", "Endogenous CHC or VAMP4 were visualized at 550 nm (antibody dilutions were 1:250 and 1:500 for CHC and VAMP4, respectively).", "Identically sized regions of interest were placed over transfected neurons in the same field of view, along with background regions.", "The level of either CHC or VAMP4 expression was calculated by subtracting background autofluorescence prior to calculating the ratio for transfected/non-transfected expression levels.", "Cerebellar neurons were removed from culture medium, left for 10 min in incubation medium, and then either stimulated with a train of 400 action potentials (40 Hz, 10 s) or left to rest.", "Cultures were immediately fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.1 M sodium phosphate buffer (PB [pH 7.4]) at room temperature.", "After three washes in 0.1 M PB, cerebellar neurons were permeabilized using 2% BSA/0.1% Tx-100 in 0.1 M PB for 1 hr.", "Cultures were then incubated with VAMP4 antibody (1:100) in 2% BSA/0.1% Tx-100 in 0.1 M PB for 1 hr.", "After three washes in 2% BSA in 0.1 M PB, cerebellar neurons were incubated with ultrasmall gold anti-rabbit conjugated Fab’antibodies for 1 hr in 2% BSA in 0.1 M PB.", "Cells were washed three times in 0.1 M PB and post-fixed in 2% glutaraldehyde in 0.1 M PB for 30 min.", "After three washes in 0.1 M PB, cultures were subjected to HQ Silver Enhancement as per the manufacturer’s instructions (Nanoprobes).", "After further washes in dH2O and then 0.1 M PB, cerebellar neurons were stained using 1% osmium tetroxide in 0.1 M PB for 30 min.", "After washing, cultures were subjected to post hoc staining with uranyl acetate before dehydration and embedding using Durcupan resin.", "Samples were sectioned at 70- to 900-nm thickness and collected on formvar-coated slot grids (Agar Scientific).", "Grids were stained with lead citrate before being viewed on a JEOL-1200 EX transmission electron microscope.", "Cerebellar neuron cultures were left to repolarize in incubation medium for 1 hr to minimize existing bulk endosomes and were then stimulated for 2 min with 50 mM KCl in the presence of 10 μM FM1-43.", "Cells were washed once with incubation medium and then twice with incubation medium supplemented with 200 nM Advasep-7.", "Cerebellar neurons were collected in buffer containing 250 mM sucrose and 3 mM imidazole [pH 7.4] and mechanically broken using a ball-bearing cell cracker (European Molecular Biology Laboratory), with the lysate spun for 15 min at 1,200 g.", "The post nuclear supernatant was deposited at the base of a discontinuous Nycodenz (Axis-Shield) gradient (12%, 20%, 25%, and 35% in 3 mM imidazole, 0.5 mM EDTA [pH 7.4]).", "The samples were centrifuged for 90 min at 170,000 g in an Optima MAX-XP Tabletop Ultracentrifuge (Beckman Coulter).", "The different fractions were collected as indicated in Figure 6A, with bulk endosomes found at the interface between 12% and 20% Nycodenz and SVs between 20% and 25% Nycodenz.", "The fluorescence of all fractions was monitored in a TD-700 fluorometer (Turner Designs) to reveal the presence of labeled compartments.", "For protein biochemistry studies, stimulated fractions 2 (bulk endosome) and 5 (SVs) were lysed in SDS sample buffer (67 mM SDS, 2 mM EGTA, 9.3% glycerol, 12% β-mercaptoethanol, bromophenol blue, and 67 mM Tris).", "Samples were resolved on SDS-PAGE and transferred onto nitrocellulose membranes for western blotting.", "Primary antibodies were used at the following dilutions: VAMP4, 1:2,000; syt1, 1:1500; syp, 1:8,000; sybII, 1:8,000; vGLUT, 1:1,500.", "These antibodies were amplified using IRDye anti-mouse, rabbit, and guinea pig secondary antibodies (all diluted at 1:10,000; LI-COR Biosciences).", "Membranes were imaged using an Odyssey 9120 Infrared Imaging System (LI-COR Biosciences) and analyzed using Image Studio Lite (LI-COR Biosciences).", "The intensity of the endosome and SVs fractions from the same membrane were calculated and expressed as an abundance ratio (endosome/SV) after normalizing to total protein content.", "Cerebellar neuron fractions were processed in an identical manner for electron microscopy except for the fact that HRP (10 mg/ml) was used instead of FM1-43 and Advasep-7 was omitted from the wash step.", "The bulk endosome and SV samples were fixed in 2% glutaraldehyde for 30 min at 37°C and washed in 100 mM Tris (pH 7.4).", "Samples were incubated in 0.1% diaminobenzidine and 0.2% H2O2 until color developed.", "The samples were stained with 1% osmium tetroxide and dehydrated using ethanol and polypropylene oxide and embedded using Durcupan resin.", "Samples were sectioned, mounted on grids, and viewed using a FEI Tecnai 12 transmission electron microscope.", "HRP-labeled structures were identified and their diameter was calculated by taking the average of the longest and shortest diameters of individual endosomes using ImageJ (NIH).", "A cutoff of 80 nm was used to separate bulk endosomes from SVs (30–60 nm).", "A Student’s t test was performed for comparisons between two datasets.", "For greater than two datasets, a one-way ANOVA was employed.", "For comparisons between fluorescence responses over time, or where greater than one variable was being compared, a two-way ANOVA was performed.", "Conceptualization, M.A.C.; Methodology, M.A.C., K.J.S., J.C.N.-F., T.H.G., and A.C.K.; Formal analysis, J.C.N.-F.", "and A.C.K.; Investigation, J.C.N.-F., T.H.G., A.C.K., and K.J.S.; Writing – original draft, M.A.C., K.J.S., J.C.N.-F., and A.C.K.; Supervision, M.A.C. and K.J.S.; Funding acquisition, M.A.C." ]	[ "VAMP4 is the first identified ADBE cargo", "VAMP4 is essential for ADBE", "Most synaptic vesicle cargoes are not selectively recovered by ADBE" ]
The importance of passive integrated transponder (PIT) tags for measuring life-history traits of sea turtles	Capture-mark-recapture studies rely on the identification of individuals through time, using markers or tags, which are assumed to be retained. This assumption, however, may be violated, having implications for population models. In sea turtles, individual identification is typically based on external flipper tags, which can be combined with internal passive integrated transponder (PIT) tags. Despite the extensive use of flipper tags, few studies have modelled tag loss using continuous functions. Using a 26-year dataset for sympatrically nesting green (Chelonia mydas) and loggerhead (Caretta caretta) turtles, this study aims to assess how PIT tag use increases the accuracy of estimates of life-history traits. The addition of PIT tags improved female identification: between 2000 and 2017, 53% of green turtles and 29% of loggerhead turtles were identified from PIT tags alone. We found flipper and PIT tag losses were best described by decreasing logistic curves with lower asymptotes. Excluding PIT tags from our dataset led to underestimation of flipper tag loss, reproductive periodicity, reproductive longevity and annual survival, and overestimation of female abundance and recruitment for both species. This shows the importance of PIT tags in improving the accuracy of estimates of life-history traits. Thus, estimates where tag loss has not been corrected for should be interpreted with caution and could bias IUCN Red List assessments. As such, long-term population monitoring programmes should aim to estimate tag loss and assess the impact of loss on life-history estimates, to provide robust estimates without which population models and stock assessments cannot be derived accurately.	[ "AGRI", "ENVI" ]	[ "Caretta caretta", "Chelonia mydas", "Flipper tag", "Green turtle", "Loggerhead turtle", "Tag loss" ]	[ "1105", "2309" ]	[ "Population-based conservation is dependent on accurate estimates of life-history traits (Hernández-Camacho et al., 2015; Yokoi et al., 2017).", "Most population studies use capture-mark-recapture (CMR), which involves the release of tagged individuals and their subsequent recapture events over time.", "These studies rely on individual recognition to provide information about demography, behaviour and survival, assuming tags are correctly identified and retained over time (Pradel, 1996).", "This assumption, however, is often violated, having implications for the interpretation of demographic and survivorship data (Arnason and Mills, 1981; González-Vicente et al., 2012; Rotella and Hines, 2005).", "In CMR models, individuals having lost all tags are no longer identifiable and are indistinguishable from dead individuals, which can lead to the overestimation of population abundance and underestimation of survival (Arnason and Mills, 1981; Cowen and Schwarz, 2006; Laake et al., 2014).", "CMR has been used extensively in sea turtle research to study behaviour (e.g. Lazar et al., 2004) and to estimate life-history traits (e.g. Stokes et al., 2014).", "Such studies generally rely on tagging individuals with two external flipper tags, reducing the probability of individual loss from the identifiable population.", "Double tagging also allows tag loss estimation, the rate of which may be influenced by tag type and size, species and tagger experience (Casale et al., 2017; Limpus, 1992).", "Flipper tags can be combined with subcutaneously injected passive integrated transponder (PIT) tags which are thought to be more durable, cannot be lost through abrasion or during courtship and have higher readability and retention than flipper tags (Gibbons and Andrews, 2004; Godley et al., 1999; McNeill et al., 2013; Rivalan et al., 2005).", "Thus, individuals having lost all external tags may nevertheless be re-identified.", "Detection failure can occur, however, if tags are expulsed from the body before wound healing (Feldheim et al., 2002; Godley et al., 1999), if tags migrate within the animal's tissue (Van Dam and Diez, 1999; Wyneken et al., 2010) or if tags become unreadable (McNeill et al., 2013; Van Dam and Diez, 1999).", "Whilst PIT tags have very little negative impact on animals overall (Gibbons and Andrews, 2004), their benefits may be outweighed by their higher financial cost.", "Furthermore, the availability and necessity of PIT tag readers for tag detection (Gibbons and Andrews, 2004) may prohibit their use, especially when recapture probability is low or when non-project personnel, such as fishermen, encounter tagged animals.", "Although tagging, and particularly flipper tagging, is considered standard practice in sea turtle research, few studies have estimated tag loss.", "Two approaches have been used to do so: proportion of tags lost (e.g. Bjorndal et al., 1996; Limpus, 1992) and, recently, modelling (e.g. Casale et al., 2017).", "Typically, individuals are tagged with two flipper tags and the shedding of one of the two tags is used to model tag loss using different scenarios.", "Only five studies have modelled flipper tag loss in such a manner at foraging (timeframe: 11 yr, McNeill et al., 2013, timeframe: 14 yr, Casale et al., 2017) and nesting (timeframe: 22 yr, Nishizawa et al., 2017; timeframe: 9 yr, Pfaller et al., 2019) grounds for loggerhead turtles (Caretta caretta), and only at nesting grounds for leatherback turtles (Dermochelys coriacea, timeframe: 7 yr, Rivalan et al., 2005).", "Tag loss was best described by a high initial loss before remaining constant thereafter (Casale et al., 2017; Nishizawa et al., 2017; Pfaller et al., 2019; Rivalan et al., 2005), except in McNeill et al. (2013), where it was constant over time.", "Only one study has used its findings to adjust survival estimates and to compare them to non-adjusted estimates (Nishizawa et al., 2017).", "Similarly, only one study has modelled PIT tag loss using continuous functions indicating that PIT tag loss is negligible in nesting loggerhead turtles (timeframe: 9 yr, Pfaller et al., 2019).", "The need for more accurate data on life-history parameters for demographic models and population assessments has been highlighted as a research priority for sea turtles globally (Hamann et al., 2010; Rees et al., 2016) and, in particular, for green turtles (Chelonia mydas) in the Mediterranean (Casale et al., 2018).", "For example, although annual survival has been calculated for subset groups of green and loggerhead nesting females in the Mediterranean (Omeyer et al., 2019; Snape et al., 2016), no estimates are available for nesting populations as a whole in the region.", "Saturation flipper tagging has been carried out since 1992 at Alagadi Beach, North Cyprus, and PIT tags were introduced in 1997.", "Using the resulting 26-year individual-based monitoring dataset for green and loggerhead turtles, this study aims (1) to accurately estimate long-term flipper and PIT tag loss for green and loggerhead turtles; and (2) to assess how the use of PIT tags has increased the accuracy of estimates of flipper tag loss, population abundance and recruitment, survival, and reproductive periodicity and longevity.", "Since 1992, sea turtle monitoring at Alagadi Beach has been carried out by the Marine Turtle Conservation Project (University of Exeter, UK), a collaboration between the Marine Turtle Research Group, the North Cyprus Department for Environmental Protection and the North Cyprus Society for the Protection of Turtles.", "Alagadi Beach, situated on the north coast of Cyprus (35°33′ N, 33°47′ E), consists of two coves of 0.8 and 1.2 km in length, separated by a rocky headland (Broderick and Godley, 1996).", "On average, 217 green and 65 loggerhead turtle clutches are laid annually (2014–2018).", "Satellite tracking of females of both species at this study site has revealed foraging sites in Syria, Egypt, Libya, Lebanon, Tunisia, Turkey and Cyprus (Bradshaw et al., 2017; Snape et al., 2016).", "Data were collected between early May and mid-August (encompassing the entire nesting season) from 1993 to 2017, except in 1992 when monitoring began in early July.", "Flipper tags were fitted on the trailing edge of the fore-flippers between the proximal second and third scales (after Balazs, 1999) during the covering phase, immediately after oviposition.", "From 1992 to 1999, plastic flipper tags were used (1992–1994: Dalton Jumbotags®, 1994–1999: Dalton Supertag®; Dalton Tags, UK).", "Because these tags became unreadable after a few years, from 1999 to 2015, titanium Stockbrands® (Australia) flipper tags were used, except in 2013, where Inconel 681/C tags were used (National Band & Tag Company, Kentucky, USA).", "In 2016 and 2017, Inconel and titanium flipper tags were used because it was decided to stop using titanium tags when females reacted negatively to the new design.", "The estimation of Inconel flipper tag loss was not possible due to their recent introduction and the resultant low level of tag returns to date.", "PIT tags were injected from 1997 onwards, as per Godley et al. (1999), to increase recapture rates of females having lost all flipper tags.", "Prior to 2014, Trovan microchips (11.50 × 2.12 mm, 0.10 g) were used, after which the newly available Trovan mini-transponders (8.00 × 1.40 mm, 0.06 g) were used due to their smaller, less invasive gauge needle.", "Until 2013, one PIT tag was given in each shoulder where time permitted, whereas from 2014 onwards, only one PIT tag was given, in preference, in the right shoulder, both to reduce costs and limit the number of invasive procedures.", "Checks of both shoulders were nevertheless maintained as standard.", "Effects of flipper and PIT tagging have been investigated at Alagadi Beach, showing no significant effects on post-ovipostional behaviour and reproductive success (Broderick and Godley, 1999).", "Two datasets were compared to assess the error associated with tag loss and to determine whether the use of PIT tags resulted in more accurate estimates of life-history parameters and flipper tag loss.", "In the ‘PIT tag’ dataset, flipper and PIT tag readings were used, whereas, in the ‘no PIT tag’ dataset, PIT tags were omitted and female identification was based solely on flipper tags.", "In the ‘no PIT tag’ dataset, if a previously known female returned to nest and was identified by PIT tag(s) alone, having lost all flipper tags, she was given a new identification number and thus treated as a neophyte female (first-time nester).", "Reproductive periodicity and longevity were calculated for each new female, except for females that were identified by PIT tag(s) alone on their first recapture at their second nesting season, as these females will not have successfully completed one remigration interval with both flipper tags.", "A new encounter history was created for each female and the process was repeated each time females had lost both flipper tags, meaning that a known female could have multiple identification numbers and thus multiple encounter histories in the ‘no PIT tag’ dataset.", "Finally, for the tag loss analysis of the ‘no PIT tag’ dataset, tag histories in which all tags were lost were excluded, as these could not be determined without the use of PIT tags (TH20 and TH10, see Section 2.5 for further details).", "While reproductive periodicity is the number of years elapsed between two consecutive nesting seasons, reproductive longevity is the time span since recruitment (year of first capture) to the nesting population, with neophytes being given year 0.", "The annual number of neophyte females and the annual proportion of misidentified remigrant females were calculated from 2000 onwards because of the increased accuracy of neophyte classification following one modal breeding cycle (3 yr) after the introduction of PIT tags.", "Linear models and generalised linear mixed effect models were used to determine whether PIT tags significantly improved estimates of reproductive longevity and periodicity respectively, using the package ‘nlme’ in R (R Core Team, 2018).", "Female ID was included to account for pseudoreplication and temporal effects were controlled for.", "Encounter histories were created based on nesting events.", "Survival probability was estimated using the multi-state model in MARK (White and Burnham, 1999), assuming a breeding state (B; observable state) and a non-breeding state (NB; unobservable state).", "The parameters estimated were annual survival probability (S), encounter probability (p) and transition probabilities between states (ψB→NB and ψNB→B).", "A ‘time-since-marking’ approach with two ‘age’ classes was used to allow survival the first year after initial tagging (hereafter S1) to differ from that in subsequent years (hereafter S2; Chaloupka and Limpus, 2002; Kendall et al., 2018; Pradel et al., 1997; Sasso et al., 2006).", "This allows us to account for imperfect fidelity, assuming that some neophytes are transient individuals, i.e. those being individuals that are never seen again after their initial capture.", "While S1 confounds permanent emigration and mortality, S2 is more likely to reflect true survival.", "Goodness of fit was assessed using U-CARE (Choquet et al., 2005).", "Model selection was based on the lowest qAICc value (corrected quasi-likelihood Akaike information criterion).", "Parameters were estimated using the Markov chain Monte Carlo method and were based on posterior distributions.", "95% highest posterior density credibility intervals were reported (see supplementary material for further details).", "Tag returns were ascribed to five types of tag histories (TH), using days as the unit, and were defined as follows: females released/resighted with two tags and resighted with both tags (TH22), with one tag (TH21) or with no tags (TH20); and, females released/resighted with one tag and resighted with one tag (TH11) or no tag (TH10).", "PIT tag(s) permitted the identification of females having lost both flipper tags and thus the calculation of TH20 and TH10.", "Tag histories can be combined such that a female released with two tags, resighted with two tags, and resighted again with no tags would have the following tag history: TH22 + TH20.", "Tag history probabilities were defined as per Casale et al. (2017), adapted to include TH20 and TH10.", "Maximum likelihood estimation was performed using the package ‘bbmle’.", "The same five models as in Casale et al. (2017, see supplementary material for further details) were compared.", "Model selection was based on the lowest AICc (corrected Akaike Information Criterion) value.", "The analysis was conducted separately for each tag type, species and dataset.", "Both types of plastic flipper tags and PIT tags were grouped due to small sample size.", "Because new tags were fitted if lost, the datasets included multiple tags per female.", "All previously published studies which calculated tag loss using continuous functions were reviewed and presented in Table 1 for comparison.", "Cumulative tag loss probabilities after 1 and 5 yr were calculated when absent from those original studies (see Table 1 for details).", "Of the females previously tagged with both flipper and PIT tags at Alagadi Beach, 53% of green turtles (n = 305) and 29% of loggerhead turtles (n = 132) were identified in subsequent nesting seasons by PIT tag(s) alone between 2000 and 2017, resulting in the overestimation of neophytes, particularly for green turtles (Fig. 1, Table S1).", "On average, over that period, without PIT tags, we would have assumed that 74% of green turtles (n = 671) and 78% of loggerhead turtles (n = 492) nesting were neophyte females, instead of the 44% and 69% respectively using PIT tags (Table S1).", "Simultaneously, nesting female abundance would have been overestimated by 37% for green turtles (2000-2017, PIT tags: n = 389, no PIT tags: n = 533) and 9% for loggerhead turtles (PIT tags: n = 371, no PIT tags: n = 405).", "The median reproductive periodicity was 3.0 yr for both species at this study site, with a mean of 3.5 yr for green turtles and 3.0 yr for loggerhead turtles, and intervals of up to 12 and 10 yr for green and loggerhead turtles respectively (Fig. 2, Table S1).", "Omitting PIT tags resulted in less accurate estimates of reproductive periodicity, although not significantly, for both species (green turtles: χ21 = 1.98, P = 0.159; loggerhead turtles: χ21 = 0.15, P = 0.701).", "For green turtles, the median reproductive periodicity remained unchanged when omitting PIT tags, but the mean was slightly reduced to 3.2 yr, whereas, for loggerhead turtles, it resulted in underestimation of the median (2.0 yr) and the mean (2.8 yr; Fig. 2, Table S1).", "While PIT tags captured intervals of up to 12 yr, rarely were intervals of >5 yr captured using flipper tags only at this study site (Fig. 2).", "Median and mean reproductive longevity were 6.0 and 8.0 yr for green turtles and 4.0 and 5.5 yr for loggerhead turtles, with females breeding for up to 24 and 25 yr respectively at Alagadi Beach (Fig. 2, Table S1).", "Estimates of reproductive longevity were significantly improved by PIT tags for green turtles (F1,266 = 15.76, P < 0.0001) but not for loggerhead turtles (F1,182 = 1.25, P = 0.265).", "Without PIT tags, the median and mean reproductive longevity were reduced by 2.0 and 2.7 yr respectively for green turtles (Table S1).", "For loggerhead turtles, the median reproductive longevity remained unchanged, whereas the mean was reduced by 1.0 yr without PIT tags (Table S1).", "While females remained identifiable for up to 25 yr after first nesting at this study site using both flipper and PIT tags, rarely were females of either species still identifiable after 10 yr of breeding using flipper tags only (Fig. 2).", "Model results are summarised in Tables S2–5.", "Without PIT tags, some females would have been misidentified as up to six different females, therefore underestimating annual survival at Alagadi Beach (Table S6).", "Mean annual survival was 0.48 for the first year after initial capture and 0.84 thereafter for green turtles, instead of 0.66 and 0.97 respectively estimated using PIT tags.", "For loggerhead turtles, the difference was not as large: 0.36 instead of 0.44 for the first ‘age’ class and 0.76 instead of 0.83 for the second ‘age’ class.", "Tag histories, tag retention and model results are summarised in Tables S7–10.", "Tag retention is illustrated in Fig. S1.", "Initial daily tag loss probability rapidly decreased before remaining constant 200 and 126 days after initial tagging for plastic and titanium flipper tags respectively for loggerhead turtles at Alagadi Beach (Table 1, Fig. S2).", "Projections indicated that over half of double-tagged females (58% and 56% respectively) had retained both of their plastic and titanium flipper tags after 1 remigration interval (3 yr), and over four-fifths of single-tagged females (83% and 81% respectively) had retained their only flipper tag (Fig. 3ab).", "Half of all tagged females had lost their flipper tag(s) and were no longer identifiable after <3 remigration intervals (∼8 yr) for plastic flipper tags, and after just over 2 remigration intervals (∼6.5 yr) for titanium flipper tags (Fig. 3ab).", "For both flipper tag types, daily tag loss probability was underestimated when PIT tag readings were omitted (Fig. S2), resulting in the overestimation of the proportion of females (single- or double-tagged) having retained their tag(s) over the course of the study period at this study site (Fig. 3ab).", "Although initial daily tag loss probability was higher for PIT tags than for both flipper tag types, daily tag loss probability was lower for PIT tags once it plateaued for loggerhead turtles at Alagadi Beach (Fig. S3).", "Tag retention was higher for PIT tags than for both flipper tag types.", "After 1 remigration interval, 79% of double-tagged females had retained both PIT tags, and 95% of single-tagged females had retained their only PIT tag (Fig. 3c).", "Half of double-tagged females still retained both PIT tags 17 yr after initial tagging, and two-thirds of single-tagged females still retained their PIT tag 22 yr after initial tagging (Fig. 3c).", "Initial daily tag loss probability increased before remaining stable 9 yr after initial tagging for plastic flipper tags, whereas it rapidly decreased before remaining constant 192 days after initial tagging for titanium flipper tags for green turtles at Alagadi Beach (Table 1, Fig. S4).", "Whereas daily tag loss probability was higher in green turtles than in loggerhead turtles at this study site for plastic flipper tags, it plateaued around similar values for titanium flipper tags.", "Projections indicated that half of double-tagged females (50% for both flipper tag types) had retained both of their plastic and titanium flipper tags after 1 remigration interval (3 yr), and three-quarters of single-tagged females (76% and 75% respectively) had retained their only flipper tag at Alagadi Beach (Fig. 3de).", "Half of all tagged females had lost their flipper tag(s) and were no longer identifiable after <2 remigration intervals (∼5 yr) for plastic flipper tags, and after 2 remigration intervals (6 yr) for titanium flipper tags (Fig. 3de).", "Similarly to loggerhead turtles at this study site, the omission of PIT tags resulted in the underestimation of daily tag loss probability for green turtles and, therefore, the overestimation of the proportion of females having retained their flipper tags over the course of the study period, irrespective of tag type (Fig. 3de).", "Although initial daily tag loss probability for PIT tags and titanium flipper tags were similar for green turtles at Alagadi Beach, daily tag loss probability was lower for PIT tags once it stabilised (Fig. S3).", "Tag retention 3 yr after initial tagging was much higher for PIT tags than for both flipper tag types, and was higher in green turtles than in loggerhead turtles at this study site.", "After 1 remigration interval, 91% of double-tagged females had retained both PIT tags, and 99% of single-tagged females had retained their only PIT tag (Fig. 3f).", "Only 5% of all tagged females were no longer identifiable using PIT tags 22 yr after initial tagging (Fig. 3f).", "Table 1 compiles all previously published studies which estimate tag loss using continuous functions, including this study.", "Estimates for both flipper tag types at Alagadi Beach for green turtles were higher than those calculated in Malaysia for Inconel and titanium flipper tags.", "For loggerhead turtles, estimates at this study site were within the range of those previously reported for Inconel flipper tags in Italy and the USA.", "In contrast, the asymptotic values and the cumulative tag loss probabilities after 5 yr for PIT tags for the two species at Alagadi Beach were far lower than those corrected by the presence of PIT tags for all flipper tag types.", "Without accounting for non-independence of tag loss, on average, over a fifth (22%) of tagged individuals will have lost at least one flipper tag after 1 yr and over half (58%) of tagged individuals after 5 yr across green and loggerhead turtle studies.", "In contrast, on average, 10% of tagged individuals will have lost at least one PIT tag after 1 yr and 14% after 5 yr.", "Half of loggerhead turtles tagged at Alagadi Beach will still be identifiable with at least one tag upon recapture 2–3 yr after initial tagging using titanium flipper tags, and 3–4 yr after initial tagging using PIT tags (Fig. S5a).", "On average, irrespective of tag type, only 50% of loggerhead turtles will be identifiable upon recapture after 1 remigration interval only.", "In contrast, 50% of green turtles tagged at Alagadi Beach will still be identifiable using at least one tag upon recapture 3–5 yr after initial tagging using titanium flipper tags, and 16–21 yr after initial tagging using PIT tags (Fig. S5b).", "Here, we show the importance of PIT tags for the long-term population monitoring of two different sea turtle species.", "Combining flipper and PIT tagging at this study site allowed for greater female identification and thus more accurate estimates of tag loss, life-history traits and population parameters, in particular for green turtles.", "Variation in flipper tag loss can be observed across species, life stages, sex, tag types and geographical locations (e.g. Bradshaw et al., 2000; Chambers et al., 2015; Hastings et al., 2018; Oosthuizen et al., 2010).", "For sea turtles, a pattern appears to be emerging, whereby flipper tag loss is best described by a decreasing logistic curve with a lower asymptote (Table 1).", "While flipper tag loss was found to increase with age due to individual growth in elephant seals for example (Oosthuizen et al., 2010), this was not observed in sea turtles, potentially because estimates were mainly from adults, which have negligible growth (Omeyer et al., 2017, 2018).", "The increase in tag loss found for plastic flipper tags in green turtles here is likely due to the writing on the tag fading rather than loss per se.", "In contrast, the constant tag loss for loggerhead turtles in McNeill et al. (2013) likely resulted from the lack of short-term recaptures, preventing the detection of high initial loss.", "Additionally, while tag loss may be influenced by interspecific behavioural differences and habitat effects, as observed in fur seals (Bradshaw et al., 2000) and previously described in sea turtles (Limpus, 1992), this was not apparent from Table 1, likely due to small sample size.", "Initial and asymptotic flipper tag loss across sea turtle studies remained within the same order of magnitude (Table 1), although care should be taken when drawing conclusions from such comparisons because of the inherent differences among studies (Casale et al., 2017; McNeill et al., 2013; Nishizawa et al., 2017; Pfaller et al., 2019; Rivalan et al., 2005).", "Evidence is increasingly suggesting that loss is not independent between flipper tag pairs (Stellar sea lions: Hastings et al., 2018; elephant seals: McMahon and White, 2009; Schwarz et al., 2012; sea turtles: McNeill et al., 2013; Nishizawa et al., 2017; Pfaller et al., 2019; Rivalan et al., 2005).", "Not accounting for non-independence of tag loss overestimates loss for the first tag and largely underestimates loss for the second tag.", "Unfortunately, investigating this at Alagadi Beach was not possible because of the large number of researchers with varying tagging abilities involved over the course of the study period, the inclusion of multiple tags per female and the relatively small cohort size.", "It has, nevertheless, been documented for leatherback (Rivalan et al., 2005), loggerhead (McNeill et al., 2013; Pfaller et al., 2019) and green (Nishizawa et al., 2017) turtles.", "While tissue necrosis was proposed to be the cause for leatherback turtles, it was suggested for the other two species to result from human error, also thought to be the cause of the high initial flipper tag loss in sea turtles (Table 1).", "Such a tagger effect on tag loss estimates has also been documented in fur seals (Bradshaw et al., 2000) and bluefin tuna (Chambers et al., 2015).", "Both of these emerging patterns – high initial loss and non-independence of flipper tag loss – suggest that thorough training of the tagging research staff is key to increasing long-term flipper tag retention across sea turtle studies (McNeill et al., 2013; Pfaller et al., 2019).", "PIT tags have been proposed as permanent tags (Gibbons and Andrews, 2004), despite also being subject to loss (lemon sharks: Feldheim et al., 2002; salmon: Foldvik and Kvingedal, 2018; sea turtles: McNeill et al., 2013; Pfaller et al., 2019; spiny lobsters: O'Malley, 2008).", "In this study, for both species, PIT tag loss was found to be best described by a decreasing logistic curve with a lower asymptote, similarly to flipper tags (Table 1).", "The high initial loss is likely to be the result of faulty application or ejection from the body before wound healing, such as in lemon sharks (Feldheim et al., 2002) and loggerhead turtles (Pfaller et al., 2019).", "As opposed to fish (e.g. Onders et al., 2004) and moulting species (e.g. Frusher et al., 2009; González-Vicente et al., 2012), physical loss is highly unlikely in sea turtles once the insertion wound has healed (Pfaller et al., 2019).", "Once improperly applied tags have been shed, detection failure is more likely due to human error, although mechanical failure (i.e. failure in tag transmission or reader failure) and tag migration may also influence PIT tag detection (McNeill et al., 2013; Pfaller et al., 2019; Van Dam and Diez, 1999; Wyneken et al., 2010).", "Indeed, this can be seen in our data where PIT tags previously thought to be lost are recorded at future recapture events.", "This phenomenon is bound to be exacerbated in females with multiple PIT tags in the same shoulder at this study site, in particular, if the research staff does not consistently search for the presence of more than one PIT tag.", "Similarly to flipper tags, short-term within-season PIT tag retention could be improved by providing adequate training to the tagging research staff (McNeill et al., 2013; Pfaller et al., 2019).", "Pfaller et al. (2019) also suggest the use of a temporary, fast-drying adhesive or patch at the tagging site as a method to reduce the likelihood of PIT tag expulsion before wound healing.", "While this suggestion could improve long-term PIT tag retention, it should not be seen as an alternative method to reducing high initial loss, but rather should be used in conjunction with thorough training of the tagging research staff.", "This additional step to the tagging procedure will require further training and will reduce within-season PIT tag loss only if performed correctly.", "Nevertheless, PIT tag loss is substantially less than that of flipper tags for sea turtles (Table 1; Pfaller et al., 2019; Groom et al., 2017; Schäuble et al., 2006; Parmenter, 2003, 1993).", "While PIT tags are also subject to loss, Pfaller et al. (2019) tested the assumption that PIT tags can be used as permanent markers in nesting loggerhead turtles in the USA.", "Using genetic markers, they showed that flipper tag loss estimates were not substantially biased by PIT tag loss, although this should be tested at other locations.", "Such high retention has also been observed in salmon (Foldvik and Kvingedal, 2018) and sea lions (Chilvers and MacKenzie, 2010).", "PIT tag retention is, however, highly variable between species, ranging from 100% in lobsters (Frusher et al., 2009) to as low as 3% in paddlefish (Onders et al., 2004), and is likely to be impacted by species-specific life-history traits and behaviours.", "The interspecific differences in PIT tag retention at this study site are likely to be due to a combination of different factors.", "Indeed, the accuracy of PIT tag loss estimates for loggerhead turtles is likely to have been influenced by the lower return rates of this species linked to lower nest site fidelity compared to green turtles (Snape et al., 2018, 2016) thus, reducing sample size, as well as associated length of capture histories (Tables S7–S8, Fig. S1; Omeyer et al., 2019, 2018).", "Increasing PIT tag reporting rates at a basin-wide scale would improve, and likely reduce, long-term PIT tag loss estimates for loggerhead turtles but is dependent on the availability of PIT tag readers at other monitoring sites.", "The rate at which flipper tags and other external tags are being lost in sea turtles, sea lions (Hastings et al., 2018) and spiny lobsters (González-Vicente et al., 2012) for example, is of concern for long-term individual-based population monitoring relying solely on one set of identification markers.", "The estimation of life-history traits and population parameters may, however, be improved by the use of additional identification methods, such as those making use of natural patterns (e.g. Smout et al., 2011), human-made marks (e.g. branding, Smout et al., 2011) or genetic markers (Pfaller et al., 2019).", "Here, results highlighted that the typical method used to estimate tag loss by quantifying the loss of one of the two tags in double-tagged individuals largely underestimated tag loss in the absence of PIT tags.", "As such, tag loss estimates which are not corrected for using at least one other identification method should be interpreted with caution.", "PIT tags allowed for a larger number of individual females to be identified after a longer period of time compared to flipper tags, increasing the accuracy of life-history traits and population parameters, as also demonstrated in sea lions (Chilvers and MacKenzie, 2010; Hastings et al., 2018).", "As such, the use of PIT tags over flipper tags is far preferable for the long-term individual monitoring of sea turtles (Pfaller et al., 2019), and particularly for green turtles at this study site.", "Nevertheless, both types of tags are still being used at Alagadi Beach as the presence of flipper tags may encourage researchers to look more thoroughly for PIT tags.", "Flipper tags should also improve short-term identification of individual females at recapture opportunities elsewhere when PIT tag readers are not available, such as when individuals are bycaught.", "The difference in the efficacy of PIT tags was likely influenced by interspecific differences in nest site fidelity at this study site (Snape et al., 2018).", "Indeed, considering the high flipper tag loss observed at Alagadi Beach, the proportion of loggerhead turtle females identified by PIT tags alone, as well as the accuracy of parameter estimates for this species, would likely increase should return rates be higher.", "The ‘time-since-marking’ modelling approach allowed the decoupling of annual survival estimates for sea turtles.", "Survival was lower the first year after initial tagging (S1) than in subsequent years (S2) and was likely underestimated as mortality and permanent emigration were confounded.", "However, there is currently no way to assess permanent emigration given the coverage of CMR programmes in North Cyprus and in the Mediterranean.", "Although imperfect fidelity to our study site was accounted for by the modelling approach, the misclassification of remigrant nesters having nested undetected elsewhere as neophytes could have further influenced estimates.", "While S1 estimates are low, S2 estimates are more likely to reflect true survival of remigrant females.", "Estimates calculated here highlight yet again the consistently lower estimates for loggerhead turtles globally compared to those of green turtles (Pfaller et al., 2018), which is thought to be the result of interspecific differences in foraging behaviour (Broderick et al., 2006).", "Apparent annual survival estimates for loggerhead turtles (0.83, CI: 0.78–0.87) are comparable to those of Casale et al. (2015, 2007) for large juveniles and adults in the Mediterranean, to those of adults from subset groups at this study site (Omeyer et al., 2019; Snape et al., 2016), and fall within the predictions for loggerhead turtles globally (0.82, 0.79–0.85; Pfaller et al., 2018).", "For green turtles, S2 estimates (0.97, CI: 0.95–0.99) are also comparable to those from subset groups at Alagadi Beach (Omeyer et al., 2019) and exceed the global predictions for green turtles (0.88, CI: 0.80–0.93; Pfaller et al., 2018).", "The difference in PIT tag loss estimates, combined with interspecific differences in annual survival, resulted in large differences in the proportion of individuals still alive and identifiable using PIT tags over time between the two species at this study site.", "It is, however, likely that the values presented here for loggerhead turtles are an underestimate due to low nest site fidelity (Snape et al., 2018, 2016).", "Excluding PIT tags resulted in the underestimation of both S1 and S2 survival estimates for both species.", "As Wilkinson et al. (2011) highlighted for sea lions using branding marks, the presence of PIT tags allowed for more robust estimates of annual survival to be calculated at this study site.", "Because life-history data are often not available, sea turtle population assessments have been based on temporal changes in nest counts rather than individual counts.", "Therefore, providing new or improved estimates of life-history traits and population parameters, such as those calculated here, will be key in generating population models for IUCN Red List assessments for both species in the Mediterranean, which will contribute to the conservation of these threatened species.", "Indeed, such population models will inform our understanding of population demography and dynamics and can be used to identify life-history parameters at which to target conservation actions (sea turtles: Casale and Heppell, 2016; Crouse et al., 1987; Crowder et al., 1994; Mazaris et al., 2005; spotted turtles: Enneson and Litzgus, 2008).", "However, these models are dependent on high-quality, long-term data on all life stages to be accurately parametrised, yet they are necessary to avoid erroneous or detrimental management decisions from being made (Hernández-Camacho et al., 2015; Winker and Sherley, 2019; Yokoi et al., 2017).", "Life-history data can be acquired using multiple techniques, however, the accuracy of parameter estimates is dependent on the inherent biases of each method as highlighted in this study.", "Therefore, data used for IUCN Red List assessments should systematically be updated for each species and sub-populations when tag loss estimates become available.", "While we have shown how PIT tags have improved the accuracy of female identification and life-history and population parameter estimates at this study site, there is still scope in improving these estimates further.", "Indeed, for example, satellite tracking, which has been used extensively across marine megafauna to provide information on animal behaviour, distribution and movement (e.g. Hart et al., 2019; Heerah et al., 2019; Snape et al., 2018), could be used to refine estimates of clutch frequency at this study site without relying on tag returns.", "This would be particularly relevant for loggerhead turtles as they have been shown to nest in multiple countries within the same nesting season (Snape et al., 2018, 2016).", "Tracking devices, however, are costly, have a shorter lifespan compared to PIT tags especially (e.g. max <2 yr in Hart et al., 2019; and in Stokes et al., 2015), and therefore cannot be used to obtain individual-based long-term life-history data for the species.", "While CMR programmes rarely set out to quantify tag loss, less invasive monitoring techniques, such as natural marks (sea turtles: Araujo et al., 2016; whale sharks: McCoy et al., 2018), human-made marks (hawksbill turtles: Richardson et al., 2006) or genetic markers (lemon sharks: Feldheim et al., 2002; sea turtles: Pfaller et al., 2019; giant salamanders: Unger et al., 2012), will be useful in further improving tag loss estimates and determining biological and physical factors driving this loss.", "Indeed, an improved understanding of flipper and PIT tag loss in sea turtles will help to determine better tagging practices.", "Although photo-identification and genetic tagging can be used to track individuals through time without being subject to tag loss (bottlenose dolphins: Diaz-Aguirre et al., 2018; short-finned pilot whales: Hill et al., 2018; whale sharks: McCoy et al., 2018), physical tags will currently remain necessary in the wild for sea turtles when individual-specific procedures are required as neither method allow for near real-time individual identification.", "The CMR programme at Alagadi Beach currently lacks a photo-identification library, however, genetic samples of both species have been collected for almost two decades.", "Therefore, genetic tagging at this study site could be used to assess the accuracy of our PIT tag loss estimates, as per Pfaller et al. (2019), as well as estimates of life-history parameters (e.g. Shamblin et al., 2017).", "In conclusion, we showed the importance of PIT tags for long-term individual-based population monitoring for two different sea turtle species.", "Permanent marks or long-lasting tags, such as PIT tags, are invaluable to provide more accurate estimates of tag loss and life-history parameters.", "Not accounting for tag loss has large implications for the interpretation of population demography, such as population abundance and recruitment.", "Thus, estimates where tag loss has not been corrected for should be interpreted with caution and could bias IUCN Red List assessments.", "Long-term population monitoring programmes should aim to estimate tag loss using continuous functions and multiple identification methods and assess its impact on life-history traits, in order to provide robust parameter estimates without which population models and stock assessments cannot be derived accurately.", "This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.", "All applicable international, national and/or institutional guidelines of the care and use of animals were followed.", "Fieldwork was approved by the University of Exeter Ethics Committee." ]	[ "Importance of PIT tags for gaining accurate estimates of life-history traits and population parameters for sea turtles.", "Excluding PIT tags led to underestimation of reproductive periodicity, reproductive longevity and annual survival.", "Female abundance and recruitment to the nesting population were overestimated in the absence of PIT tags.", "Flipper tag loss underestimated by a factor of two without the use of PIT tags at Alagadi Beach." ]
Photochemically modified diamond-like carbon surfaces for neural interfaces	Diamond-like carbon (DLC) was modified using a UV functionalization method to introduce surface-bound amine and aldehyde groups. The functionalization process rendered the DLC more hydrophilic and significantly increased the viability of neurons seeded to the surface. The amine functionalized DLC promoted adhesion of neurons and fostered neurite outgrowth to a degree indistinguishable from positive control substrates (glass coated with poly-L-lysine). The aldehyde-functionalized surfaces performed comparably to the amine functionalized surfaces and both additionally supported the adhesion and growth of primary rat Schwann cells. DLC has many properties that are desirable in biomaterials. With the UV functionalization method demonstrated here it may be possible to harness these properties for the development of implantable devices to interface with the nervous system.	[ "ENGI", "MATE", "PHYS" ]	[ "Aldehyde", "Amine", "Diamond-like carbon", "Neurons", "Schwann cells" ]	[ "2210", "2211", "2500", "3104" ]	[ "Diamond possesses a range of valuable mechanical and tribological properties, which offer interesting opportunities for the development of future applications.", "One such property, which is useful within biological research, is the ease with which it can be doped or functionalized whilst retaining its stability and biocompatibility [1].", "Its mechanical strength and hardness are also particularly desirable especially where device longevity is a major concern.", "However, synthetic diamond is prepared under harsh conditions, usually requiring high temperatures and pressures, prohibiting its deposition upon biological or soft materials or upon common tissue culture substrates such as polystyrene or glass.", "In contrast diamond-like carbon (DLC), a form of amorphous carbon, has similar properties to diamond yet may be synthesised directly upon substrates under relatively mild deposition conditions and even at ambient temperature [2].", "DLC films may be produced by a variety of methods including argon ion sputtering and cathodic arc spray or, as in this study, by pulsed laser deposition (PLD).", "The diamond-like character of the DLC produced is dependent upon the ratio of sp2 and sp3 carbon, with a higher sp3 content resulting in more diamond-like properties.", "Importantly, both DLC and diamond may also be readily photo-functionalized with the aid of ultraviolet (UV) radiation allowing the attachment of a wide range of organic functional groups at the surface of the material [3].", "Such an approach is ideal for modifying DLC for biomedical applications where the surface termination strongly affects protein and cell adhesion.", "We have previously shown that photo-functionalized detonation nanodiamond is an effective mimic for poly-L-lysine (PLL), a synthetic adhesion factor commonly used in neural cell culture [4].", "The neuroadhesive properties of PLL are thought to originate from the primary amine group on the side chain of the L-lysine residue, creating a highly polar molecule which assists in cell adhesion.", "Other molecules containing primary amines, such as 10-amino-dec-1-ene, are known to exert a similar neuroadhesive effect and may be covalently bound to DLC using the facile UV irradiation method.", "In this study we have created a neurocompatible substrate from DLC, which possesses the positive attributes of conventional neuroadhesive surface treatments such as PLL.", "However, as the DLC is covalently functionalized, it is envisaged that the DLC substrates will exhibit lower cytotoxicity issues and greater stability in applications in vivo.", "This is the first time that covalently functionalized DLC has been evaluated as a growth substrate for neurons and Schwann cells.", "We anticipate that the techniques demonstrated here will find applications in surface coatings and device fabrication for the development of central nervous system implants such as electrodes for deep brain stimulation and in brain-computer-interfaces.", "Unless otherwise stated, all chemicals and bioreagents were obtained from Sigma-Aldrich (Dorset, UK) and used as received.", "Experimental samples were prepared and analysed in triplicate, unless otherwise stated, and error bars and statistical tests were calculated from these replicate measurements.", "Experiments were repeated at least once in order to ensure reproducibility.", "In all cases the presented micrographs were representative of the whole sample.", "In order to coat glass cover-slips with a thin coating of DLC, a pulsed laser deposition (PLD) method was used.", "The output of an argon fluoride (ArF) excimer laser (Lambda Physik, Compex 201) with a wavelength of 193 nm was focused (lens focal length: 200 mm, angle of incidence: 45°) on a graphite disc target (Poco Graphite Inc., DFP-3–2 grade) in a stainless steel chamber maintained at a pressure of approximately 10− 6 Torr.", "The graphite was ablated using a laser fluence of 12 J/cm2.", "A thin film of DLC of approximately 20 nm thickness was achieved with 1200 laser shots at a target-substrate distance of 5 cm at 20 °C.", "The DLC-treated cover-slips were then sonicated in methanol for 15 min and then washed in distilled water overnight.", "Once complete, the cover-slips were allowed to dry in air before being stored in an airtight container in preparation for use.", "Trifluoroacetate-protected 10-amino-dec-1-ene (TFAAD) was synthesised according to a method described elsewhere [3].", "Firstly, 10-undecenoyl chloride (50.0 g, 0.25 mol) was added to tetrahexylammonium bromide (Acros Organics, Loughborough, UK, 0.6 mmol) in an ice bath.", "An aqueous solution of sodium azide (16.6 g in 50 ml) was then gradually added to the mixture within the ice bath and agitated using a magnetic stirrer.", "Once the reaction was complete, the organic fraction was separated, washed twice with deionised water, dried over anhydrous magnesium sulphate for 24 h and filtered.", "An excess of trifluoroacetic acid was then added to the acyl azide and the reaction mixture was heated to reflux under nitrogen for 6 h.", "The organic mixture was washed with a saturated solution of sodium bicarbonate, collected and subsequently dried over anhydrous magnesium sulphate for 24 h.", "The product was stored for later use at 4 °C.", "In order to ensure a uniform hydrogen-terminated surface, the glass cover-slips coated with DLC were hydrogenated by exposure to a hydrogen plasma flow for a total duration of 10 min prior to functionalization using the UV method.", "Hydrogenation was carried out in 2 cycles of 5 min with microwave power of 0.8 kW and 500 sccm H₂ total gas flow, followed by 2 min in cold H₂ flow under a pressure of 100 Torr.", "The coated cover-slips were then placed on top of a quartz slide, upon which a sufficient volume of either TFAAD (to prepare amine functional DLC) or undec-10-enal (to prepare aldehyde functional DLC) was applied to cover the samples.", "A further quartz slide was placed on top and the samples were exposed to UV radiation (λmax = 254 nm) from a Hg–Xe lamp (500 W, Hamamatsu Photonics) for a period of 15 min in the case of undec-10-enal and 4 h in the case of TFAAD.", "Excess alkene was removed by rinsing in methanol.", "Where the alkene used was TFAAD, the protected amine was deprotected in acidified methanol (0.36 M HCl in methanol) at 65 °C over 24 h.", "The samples were washed in methanol, dried with a stream of nitrogen gas, and stored in sterile containers until required.", "For the sake of brevity the amine functional DLC will henceforth be designated “DLC-NH2” and the aldehyde functional DLC will be designated “DLC-CHO”.", "The surface chemistry of the functionalized samples was analysed using X-ray photoelectron spectroscopy (XPS).", "Samples were analysed using a Kratos AXIS Ultra DLD instrument.", "Spectra were recorded using a monochromatised Al Kα X-ray source (1486.6 eV) operating at a power of 150 W, whilst charging of the sample during irradiation was reduced by an internal flood gun.", "Each sample was analysed at an emission angle normal to the sample surface.", "Data processing, analysis and charge correction were carried out using CasaXPS software (ver.2.3.12 Casa Software Ltd.).", "Component peaks within the recorded C (1 s) spectra were deconvoluted and fitted to a mixed peak shape of 70% Gaussian/30% Lorentzian composition.", "The aliphatic hydrocarbon component of the C (1 s) was set to 285.0 eV as an internal reference.", "The relative hydrophobicity/hydrophilicity of the functionalized samples was determined by measuring the water contact angle.", "To ascertain the water contact angle of the DLC surfaces, one drop of deionised water (1 μl) was deposited on each surface.", "The contact angle was measured as the angle formed by the baseline and the tangent to the drop profile at the three-phase point using a Rame-Hart contact angle goniometer at a temperature of 21 °C.", "For each condition, four measurements were carried out on each of replicate samples, giving a total of 20 measurements for each condition (n = 20).", "NG108-15 neuronal cells were supplied by the American Type Culture Collection (ATCC).", "The cells were maintained and expanded in a growth medium comprising of Dulbecco's modified Eagle's medium (high glucose) supplemented with foetal bovine serum (10% v/v), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (0.25 μg/ml).", "Cells were not used experimentally beyond passage number 20.", "Neuronal cells were seeded upon DLC samples or glass coated with PLL at a density of 1 × 105 cells per ml in the complete growth medium without foetal bovine serum in order to induce terminal differentiation.", "The cells were cultured for 1, 2, 5 or 7 days at 37 °C and 5% CO2 after which they were analysed for viability or fixed for microscopy as detailed in the following sections.", "Primary Schwann cells were isolated from male Wistar rats using the selective D-valine method reported elsewhere [5].", "Animals were sacrificed in compliance with the Animals (Scientific Procedures) Act 1986.", "Briefly, sciatic nerves were removed from male Wistar rats (2–3 months) and the connective tissue was subsequently discarded.", "The nerves were teased and cut into 2–3 mm segments and incubated with 0.05% (w/v) collagenase (Sigma, UK) at 37 °C for 1 h.", "This cell suspension was then filtered through a 40 μm Falcon filter (Becton Dickinson, USA) and centrifuged at 400 RCF for 5 min.", "The resultant cell pellet was washed with DMEM (including 10% (v/v) foetal calf serum) and resuspended in Schwann cell growth medium containing DMEM-D-valine (PAA, UK), 2 mM glutamine, 10% (v/v) FCS, 1% (v/v) N2 supplement (Gibco BRL, UK), 20 μg/ml bovine pituitary extract, 5 μM forskolin (Sigma, UK), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B.", "The Schwann cell suspension was plated within 35 mm Petri dishes which had been previously coated with 0.5 mg poly-L-lysine/7 μg laminin (Sigma, UK).", "The cultures were maintained at 37 °C with 5% CO2.", "At each time point the DMEM was removed from the NG108-15 neuronal cells, which were then washed once with phosphate buffered saline (PBS).", "To each well 500 μl of formalin (3.7% v/v in PBS) was applied for 15 min after which the cells were washed twice more with PBS.", "Cells were then permeabilised with Triton X-100 (0.1% v/v in PBS) for 5 min, before being washed two further times with PBS.", "A stock solution of phalloidin-FITC (Sigma) was prepared at a concentration of 0.5 mg/ml in dimethyl sulfoxide (DMSO) and further diluted to a working concentration of 5 μg/ml in PBS.", "DAPI (Sigma) was added as a counterstain (final concentration of 10 μg/ml) and the combined stain solution was applied for 1 h at room temperature before the cells were rinsed three more times with PBS.", "Stained cells were visualised via fluorescence microscopy in PBS using a high throughput imaging and image analysis system (Image Xpress by Axon Instruments).", "Image analysis was carried out using the automated functionality of the imaging platform.", "The analysis process was conducted with oversight from the operator to ensure that erroneous measurements did not occur.", "Immunofluorescent labelling was carried out to reveal S100β expression in Schwann cells cultured upon the functionalized DLC surfaces.", "S100β is a marker of the Schwann cell phenotype and continuous expression over time may indicate maintenance of the Schwann cell phenotype.", "After 21 days culture the medium was removed from the Schwann cell cultures, which were then washed once with PBS.", "The cells were then fixed with formalin (3.7% v/v) for 15 min, washed with PBS, and permeabilised with 0.1% Triton X-100 (Sigma-Aldrich, Dorset, UK) for 20 min at 4 °C.", "Cells were washed again with PBS and then blocked against non-specific antibody binding with 3% (w/v) bovine serum albumin (BSA) for 60 min at 4 °C.", "This was followed by incubation with polyclonal rabbit anti-S100β (1:250) (Dako) diluted in 1% (w/v) BSA overnight at 4 °C.", "The cells were washed twice with PBS and incubated with a FITC-conjugated secondary goat anti-rabbit IgG antibody (1:100 dilution in 1% (w/v) BSA) (Vector Labs, USA) for 90 min at room temperature.", "Relative cell viability plus an indication of rate of proliferation was assessed indirectly by the MTT assay.", "Cells were cultured on three samples for each condition (n = 3) at a density of 10,000 cells/ml within 24 well plates (Costar) over periods of 1, 2, 5 and 7 days.", "At each time point the DMEM was removed from selected wells and the cells were washed once with PBS.", "MTT Solution (thiazolyl blue tetrazolium bromide, Sigma) was dissolved in PBS (0.5 mg/ml) and 300 μl was added to each well.", "The cells were incubated at 37 °C and 5% CO2 for 45 min after which the MTT solution was removed and the reduced product was solubilised in 300 μl acidified isopropanol (1.25 μl HCL (37% w/w) per ml of isopropanol).", "The resulting solution was transferred in triplicate 100 μl volumes to a 96-well plate where the optical density was measured at 540 nm relative to 630 nm.", "DLC was applied to the surface of glass cover-slips via pulsed laser deposition.", "The DLC-coated glass was then subjected to UV-induced functionalization with TFAAD or undec-10-enal to yield either amine (after deprotection) or aldehyde-functionalized substrates.", "Contact angle measurements were carried out to provide an initial indication of the success of the functionalization process and to provide insight into the hydrophobicity of the samples, which may have implications for subsequent cell culture studies.", "The contact angle of a surface describes its wettability.", "Smaller contact angles, namely those below 90°, characterise hydrophilic surfaces which are highly wettable, whereas large contact angles correspond to hydrophobic surfaces which exhibit reduced wettability.", "Contact angle measurements on the pristine DLC and functionalized DLC surfaces are shown in Fig. 1.", "The contact angle of glass (52.1° ± 4.8°) was also measured as a comparison.", "Compared to glass, which is regarded as a relatively hydrophilic material, the pristine DLC was more hydrophobic with a higher contact angle (64.3° ± 3.1°).", "Upon functionalization, however, the contact angle decreased on both the DLC-NH2 (48.4° ± 1.7°) and DLC-CHO (49.7° ± 2.9°) surfaces.", "The functionalized surfaces were significantly more hydrophilic than the pristine DLC with contact angles comparable to glass.", "The functionalized DLC samples were further characterised by XPS to reveal the composition of their surface chemistry.", "High-resolution spectra of the C 1 s region are shown in Fig. 2.", "Because carbon bound to fluorine gives rise to a distinct chemical shift in the C 1 s peak, the DLC-NH2 samples were analysed prior to removal of the trifluoroacetate protecting group in order to aid in confirmation of successful functionalization.", "In each case the C 1 s peak was deconvoluted into component peaks arising from the different chemical environments of carbon in the first few nanometres depth of the material, including any modification of the surface chemistry.", "The percentage of the total C 1 s attributable to each component peak is given in the supplementary information, accessible online.", "In each of the spectra in Fig. 2 the C 1 s peak is dominated by the component at 285 eV corresponding to carbon in an aliphatic hydrocarbon environment or an equivalent environment without heteroatoms (e.g. DLC).", "In addition, several components form a shoulder at higher binding energies in each spectrum.", "In pristine DLC (Fig. 2a) these components collectively contribute to just over 8% of the total C 1 s peak area and arise from the native oxide layer on the surface of the material.", "For the functionalized samples, however, the components forming the shoulder correspond to the surface layer arising from the UV functionalization in addition to any native oxide layer on the DLC.", "On the DLC-NH2 sample the components at higher binding energy than the aliphatic component account for just under 22% of the total C 1 s peak area, with significantly larger peaks centred at 286.32 eV and 288.94 eV corresponding to carbon singly bound to nitrogen (or oxygen) and carbonyl-containing groups respectively.", "In addition, a new peak centred at 293.17 eV corresponding to carbon bound to fluorine was observed, in contrast to the pristine DLC or DLC-CHO samples, confirming successful binding of the trifluoroacetate-protected amine.", "On the DLC-CHO samples, just over a two-fold increase in the area of the peak centred at 286.31 eV and a four-fold increase in the area of the peak centred at 288.94 eV was observed relative to the pristine DLC.", "Respectively, these peaks likely correspond to carbon singly bound to a heteroatom (e.g. alcohol or ether functionalities) and a carbonyl group bound to an additional heteroatom (e.g. ester, amide or carboxylic acid).", "Although it is clear that successful addition of an organic layer had taken place in these samples, from the spectra it seems likely that some oxidation of the aldehyde functionality occurred during the UV functionalization process.", "To provide an indication of neuronal cell adhesion and viability upon the functionalized surfaces, an MTT assay was performed 7 days post cell-seeding on the pristine DLC, DLC-NH2 and DLC-CHO surfaces plus uncoated glass as a negative control and glass coated with PLL as a positive control (Fig. 3).", "Statistically significant differences were observed between certain samples.", "The summary of the statistical analysis is given in Table 1.", "MTT absorbance values were comparatively low on both the glass (negative control) and the pristine DLC, indicating a low level of viable neuronal cell attachment and accompanying low levels of cell proliferation.", "In contrast, on both the functionalized DLC surfaces relatively high MTT absorbance values were observed, comparable to the PLL (positive control) surfaces.", "For example the value for DLC-NH2 (0.87) was over 4 times higher than the corresponding reading from pristine DLC (0.17).", "Although cells grown upon PLL gave a slightly higher average reading (0.97) than those grown upon DLC-NH2 surfaces, there was no statistical difference between the two conditions.", "The reading from cells grown upon the DLC-CHO surfaces was also significantly higher than on the glass and pristine DLC, although statistically lower than on the PLL and DLC-NH2 surfaces.", "NG108-15 neuronal cells were allowed to terminally differentiate upon pristine DLC, DLC-CHO and DLC-NH2 surfaces.", "In addition, uncoated glass and glass coated PLL were used as negative and positive controls respectively.", "The cells were cultured for 7 days, after which they were stained with FITC-conjugated phalloidin to reveal filamentous actin and hence their cytoskeletal morphology.", "Fluorescence micrographs are shown in Fig. 4.", "Although cells adhered to all the surfaces tested, comparatively few were observed on glass (Fig. 4a) or pristine DLC (Fig. 4c).", "Cells on these surfaces displayed few dendrites and those that had developed were considerably shorter than on the other surfaces.", "The cells adopted a spherical morphology with little spreading and minimal contact area with the substrate.", "A much greater degree of cell adhesion was observed on the PLL surfaces (Fig. 4b) as well as upon the functionalized DLC surfaces (Fig. 4d and e).", "The cells were cultured in the absence of serum or mitogens in order to promote terminal differentiation so a significant degree of proliferation was unlikely.", "The differences in cell density are therefore likely due to differences in initial adhesion given that cells were seeded at the same concentration on all samples.", "Significant numbers of much longer dendrites were also observed on the PLL and functionalized DLC surfaces and numerous cells displayed a bipolar or even tripolar morphology, some possessing dendrites with lengths exceeding 300 μm.", "Upon the DLC-NH2 surfaces especially, cells appeared to adhere and develop similarly to those cultured upon PLL resulting in almost identical morphology and degree of confluence.", "Automated image analysis of the fluorescence micrographs was also carried out in order to quantify the dendricity of the cells at various time points to give an indication of the degree of terminal differentiation and the suitability of the different surfaces to support outgrowth of neurites.", "Graphs summarising the mean number of neurites per cell and average neurite length are shown in Fig. 5a and b respectively.", "Full statistical analysis was also carried out and the results are given in the supplementary information which is available online.", "In terms of mean number of neurites per cell, two trends were observed.", "On the glass and pristine DLC samples a moderate increase in the number of dendrites was observed over time followed by a decrease in neurite number culminating in almost complete absence of neurites by day 7.", "In contrast, on the PLL surfaces and the functionalized DLC surfaces, a steady increase in the number of neurites per cell was observed over time.", "By day 7 the largest number of neurites per cell was observed on the DLC-NH2 surfaces, a difference which was statistically significant in comparison with the PLL and DLC-CHO surfaces (p < 0.01).", "No statistically significant difference was observed between the DLC-CHO surfaces and PLL.", "Similar trends were also observed in the average neurite length (Fig. 5b).", "A steady decrease in neurite length was observed over time on both the glass and pristine DLC surfaces.", "Although neurite length appeared slightly greater on DLC compared to glass at all time points, the observed difference was not statistically significant except at day 2 (p < 0.01).", "In contrast, the average neurite length increased steadily over time on the functionalized DLC and PLL samples.", "By day 7 there was no statistically significant difference between the DLC-NH2 samples and the PLL positive controls.", "Neurite length was slightly lower on the DLC-CHO samples compared to the DLC-NH2 and PLL samples, although this difference was statistically significant only in comparison with PLL (p < 0.001).", "Primary Schwann cells were cultured upon glass, pristine DLC, DLC-NH2 and PLL surfaces for 21 days after which they were stained for the glial marker S100β.", "Fluorescence micrographs are shown in Fig. 6.", "Clear differences in Schwann cell morphology were observed across the different surfaces.", "In contrast to the NG108-15 neuronal cells, a considerable number of Schwann cells attached to the glass substrate (Fig. 6a).", "On glass the cell morphology was either bipolar or tripolar with thin processes that showed no particular orientational preference.", "A small degree of Schwann cell attachment was also observed on the pristine DLC surfaces (Fig. 6c).", "However, in contrast to cells on the glass surfaces, the cells on pristine DLC adopted a variety of uncharacteristic morphologies, some resembling fibroblasts whilst others adopted a highly spread polygonal morphology.", "Nonetheless, positive expression of the glial marker S100β was still observed on both the glass and pristine DLC surfaces.", "In contrast to those on glass and pristine DLC, Schwann cells cultured upon DLC-NH2 and PLL surfaces formed confluent monolayers and exhibited similar morphologies.", "The cells adopted a bipolar, elongated morphology, which is characteristic of Schwann cells, and nearly all processes were aligned alongside adjacent cells.", "The confluent cell monolayers appeared stable and showed no sign of delamination from the substrate.", "We have prepared DLC substrates which were subsequently modified by a UV functionalization method to covalently bond amine and aldehyde-containing molecules to the surface in order to improve neurocompatibility.", "DLC and related materials, such as CVD diamond, have several interesting properties which support their use as biomaterials.", "Firstly they may be readily doped or functionalized to alter their bulk characteristics, for example electrical properties, to suit the desired application [2,6–10].", "Diamond-based materials have also been tested exhaustively to prove their biocompatibility and low toxicity [11–16].", "With respect to manufacturing, DLC is also advantageous in that the size or shape of objects to be coated is of little consequence to the coating procedure.", "Most components and devices may be treated swiftly and at relatively low expense to create a stable and robust surface layer [2,17].", "Additionally, although of less relevance in neurosurgical applications, the wear-resistance of DLC could provide comparatively high longevity for any potential implants, potentially reducing the need for revision surgery in certain applications [2].", "Hydrogen-terminated diamond-like materials, such as the DLC produced by pulsed laser deposition used in this study, are known to inhibit neuronal growth due to the lack of adequate interaction sites to facilitate cell adhesion [18].", "By suitable surface modification, however, biocompatibility and bioactivity may be significantly improved.", "Upon modification with surface-bound amine and aldehyde groups DLC was shown to promote adhesion of NG108-15 neuronal cells and primary rat Schwann cells.", "Furthermore, the NG108-15 neuronal cells produced long neurites and in the case of the amine functional DLC were indistinguishable from the positive control coating (PLL).", "Although there are several variables that dictate the survival, growth and differentiation of neuronal cells in vitro, one of the most important is the culture substrate [19,20].", "Substrates that have been widely used in the culture of neuronal cells include glass and tissue culture plastic that have been coated with attachment factors of some kind.", "The most common attachment factors are cationic polypeptides of basic amino acids such as poly-L-lysine (PLL) and poly-L-ornithine [21,22], whilst extracellular matrix components including laminin have also been applied, with collagen and fibronectin also being used to a lesser extent [23–26].", "Such coatings have become widespread as a pre-treatment for neuronal cell culture since they promote cellular adhesion, survival and neurite outgrowth [19,20,22,27].", "It is not entirely clear how substances such as PLL, which contain amine groups, improve cell attachment and viability.", "The effect may arise from interactions with specific receptors on neuronal cell membranes enhancing adhesion to the substrate.", "Alternatively, a cationic polypeptide may act as a positively charged interlayer, electrostatically binding the negatively charged cell membrane to the substrate surface [22,28,29].", "Proteins originating from the serum component of the cell medium have also been identified as assisting cell adhesion, acting in a similar manner to extracellular matrix proteins [29].", "Substrates coated with PLL have been shown to lead to the development of a greater thickness of adsorbed proteins during cell culture compared to control surfaces, these either being secreted by the cells or adsorbed from the constituents of the culture medium [20].", "Proteins which adsorb initially to PLL undergo denaturation and do not encourage neuronal adhesion; however subsequent proteins which bind to this denatured layer retain their structure intact, thus encouraging subsequent cell adhesion.", "Although PLL is often used as the standard attachment factor to support the growth of Schwann cells in vitro, it is not envisaged to be desirable or permissible to use such synthetic polypeptides for in vivo applications such as neural prosthetics due to concerns about toxicity [30].", "Covalent functionalization of culture substrates is therefore an appealing alternative in developing such applications.", "It has previously been demonstrated that surface-bound amine groups can support neuronal adhesion and differentiation similarly without detrimental effects [31].", "Although research in this area is ongoing it is thought that amines interact with cells through membrane-bound chondroitin sulphate proteoglycans (CSPGs).", "These proteoglycans exert significant influence over neuronal differentiation via integrin-mediated signalling which may explain the neurocompatibility of amines and amine-containing substances such as PLL [32,33].", "It is possible that by covalently functionalizing surfaces with amine groups the longevity of the neurocompatibility may be enhanced in comparison to PLL.", "Indeed it has been shown that significantly higher shear stresses were required to detach NG108-15 cells from covalently-modified amine surfaces, in comparison to those functionalized with ECM proteins [19].", "This was corroborated by further research which concluded that covalently bound PLL was far more effective at retaining adhered cells than PLL that was passively adsorbed to the substrate [22].", "An additional benefit of covalently bound functionalities is their lack of leaching into solution, contrasting with non-covalently bound PLL the gradual desorption of which can result in cell dissociation [22].", "All these factors demonstrate the advantages of the UV functionalization approach demonstrated here.", "In addition to amine groups, aldehyde groups were also introduced to the surface of the DLC using a similar UV functionalization method (with undec-10-enal).", "In the cell culture studies the aldehyde functional group performed broadly comparably to the amine group with similar levels of neuronal viability, attachment and neurite development and indistinguishable Schwann cell attachment and morphology.", "Although relatively little research has been undertaken regarding such aldehyde-containing functionalities for cell growth, aldehyde terminated surfaces are regarded as being hydrophilic.", "In addition, plasma polymerised surfaces formed from aldehyde-containing monomers have been shown to support the growth and attachment of cells to a similar degree as those cultured upon tissue culture modified polystyrene [34].", "Aldehyde-functionalized substrates have been shown to be capable of immobilising proteins upon their surface to produce self-assembled monolayers (SAMs) of protein, indicating that they may be used for applications within the biosensor research field as well as for direct interface with cell and tissues [35].", "A similar study has also shown that surfaces bearing aldehyde groups may immobilise proteins from solution (in this case bFGF) which may then act as ligands for specific receptors allowing the capture and adhesion of cells [36].", "It seems highly likely that a similar mechanism could occur in which serum proteins such as fibronectin could be captured by surface-bound aldehydes, leading to increased cell adhesion.", "As it is known that adhesion receptors within the cell membrane not only influence the adherence of cells but also mediate cellular growth and differentiation, the importance of engineering substrates that promote cellular adhesion is clear [37,38].", "Although the DLC-CHO surfaces supported neuron and Schwann cell adhesion and differentiation of NG108 cells, by some measures (neurite length and viability) these surfaces did not perform as well as the amine-bearing surfaces.", "The reason for this is not clear but may be due to differences in both the degree of protein adsorption and the conformation of adsorbed proteins.", "Elucidation of this mechanism would require further study.", "The surface of DLC was modified using a UV functionalization method to introduce amine and aldehyde groups to improve neurocompatibility.", "The functionalization process rendered the DLC more hydrophilic and significantly increased the viability of neuron-like cells seeded to the surfaces.", "The amine-functionalized DLC promoted neuron adhesion and neurite outgrowth to a degree indistinguishable from positive control substrates.", "The aldehyde-functionalized surfaces performed comparably to the amine-functionalized surfaces and both additionally supported the adhesion and growth of primary rat Schwann cells.", "With the UV functionalization method demonstrated here it may be possible to harness the unique properties of DLC for the development of implantable devices to interface with the nervous system." ]	[ "Diamond-like carbon was functionalized in order to present surface-bound aldehyde or amine groups.", "The functionalized surfaces promoted cell adhesion and growth of neurites in a neuronal cell line (NG108).", "The functionalized surfaces also promoted adhesion of primary Schwann cells with maintenance of phenotype." ]

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