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The effect of cool water pack preparation on vaccine vial temperatures in refrigerators	Cool water packs are a useful alternative to ice packs for preventing unintentional freezing of vaccines during outreach in some situations. Current guidelines recommend the use of a separate refrigerator for cooling water packs from ambient temperatures to prevent possible heat degradation of adjacent vaccine vials. To investigate whether this additional equipment is necessary, we measured the temperatures that vaccine vials were exposed to when warm water packs were placed next to vials in a refrigerator. We then calculated the effect of repeated vial exposure to those temperatures on vaccine vial monitor status to estimate the impact to the vaccine. Vials were tested in a variety of configurations, varying the number and locations of vials and water packs in the refrigerator. The calculated average percentage life lost during a month of repeated warming ranged from 20.0% to 30.3% for a category 2 (least stable) vaccine vial monitor and from 3.8% to 6.0% for a category 7 (moderate stability) vaccine vial monitor, compared to 17.0% for category 2 vaccine vial monitors and 3.1% for category 7 vaccine vial monitors at a constant 5 °C. The number of vials, number of water packs, and locations of each impacted vial warming and therefore percentage life lost, but the vaccine vial monitor category had a higher impact on the average percentage life lost than any of the other parameters. The results suggest that damage to vaccines from repeated warming over the course of a month is not certain and that cooling water packs in a refrigerator where vaccines are being stored may be a useful practice if safe procedures are established.	[ "BIOC", "IMMU", "MEDI", "VETE" ]	[ "Cold chain", "Cool water pack", "Freeze-sensitive vaccine", "Supply chain", "Vaccine", "Vaccine vial monitor" ]	[ "1313", "2400", "2725", "2739", "3400" ]	[ "Loss of vaccine due to damage from freezing is an ongoing problem.", "Studies from 2006 to 2015 reported that 19% of vaccine shipments in lower-income countries and 38% in higher-income countries were exposed to temperatures below recommended values [1].", "Freeze damage carries two risks: that a freeze-damaged vial will be detected and therefore must be discarded, and that a freeze-damaged vial will not be detected and therefore might be administered, offering lower protection than expected.", "Freeze damage to a single vial can be detected using a simple shake test for aluminum-adjuvanted vaccines [2].", "Wastage from freeze damage can be expensive; in 2015, freeze-sensitive vaccines worth US$1.2 billion were procured through UNICEF [1].", "With the increasing use of freeze-sensitive vaccines that do not contain adjuvants and do not respond to the shake test, such as inactivated polio vaccine [3], there is increasing risk of freeze damage going unnoticed.", "Studies have associated vaccine exposure to freezing temperatures during transport with lower immune response [4–6].", "Inadvertent freezing can occur when vaccine vials are transported in insulated carriers with ice packs to protect them from high ambient temperatures; unless the packs are partially melted first (conditioned), they can freeze adjacent vials.", "To combat this problem, should the commensurate reduction in cool life be acceptable, WHO recommends using water packs cooled to 2–8 °C as an alternative to conditioned ice packs for transporting freeze-sensitive vaccines.", "However, WHO also states that these water packs should never be cooled in a refrigerator that contains vaccines to avoid raising temperatures and compromising vaccine potency [7].", "Complying with this requires a second refrigerator in clinics and health posts—an additional cost not easily absorbed in low-resource settings.", "While this recommendation assumes that placing warm water packs into a refrigerator is likely to damage vaccines, the thermal impact of such a practice has not been investigated.", "Evidence about the effect of recooling water packs alongside vaccines could enable better decisions about the need for using a separate refrigerator.", "The impact of heat exposure on potency is unique to each vaccine and manufacturer.", "A tool that can be used to generalize this impact is the vaccine vial monitor (VVM), a heat-sensitive label required on WHO prequalified vaccines.", "Similar methods have been used in previous studies [8,9] but have not been documented in detail.", "Each prequalified vaccine is assigned to one of four categories of VVM based on its heat stability; these are designated VVM2, VVM7, VVM14, and VVM30 (Table 1), with VVM2 vaccines being the least heat stable [10].", "The goal of our study was to understand the impact on vaccine life of cooling warm water packs in a refrigerator used to store vaccines by using VVM life as a proxy for vaccine life and the Arrhenius equation for calculation of VVM life.", "This would give us information on the value of having a dedicated refrigerator for cooling water packs.", "The overall design of testing was to place water packs at 43 °C into a refrigerator with vaccine vials at 2–8 °C and measure the temperatures of the vials over time to generate warming curves.", "These curves were subsequently analyzed using calculated time-temperature curves for each category of VVM to estimate the impact of cyclic warming on vaccine life.", "To prepare for testing, 10-mL vials were filled with water and some were instrumented with thermocouples (OMEGA Engineering, Inc., 5SRTC-TT-T-36, Stamford, CT, USA).", "Before the start of each test, the selected number of vials was arranged in a PQS-prequalified refrigerator (SunDanzer, model BFRV15, Tucson, AZ, USA) to cool.", "PQS-prequalified water packs (Blowkings, model BK 6, Mumbai, India) instrumented with thermocouples were conditioned to 43 °C, the “hot zone” temperature for prequalifying cold chain equipment [13], and placed inside the refrigerator.", "Several arrangements (Fig. 1) of varying numbers of vials and water packs were tested in duplicate or triplicate.", "Each test contained up to 18 instrumented vials; locations of these vials in each arrangement are available in Appendix 1.", "Temperature collection at a rate of two samples/minute (NI cDAQ-9172 chassis, 9211 thermocouple input module, NI SignalExpress software, National Instruments Corporation, Austin, TX, USA) began immediately following placement of the water packs in the refrigerator and continued until the vials and water packs reached at least 5 °C.", "Warming curves were plotted and then trimmed to include only the values from the first measurement to the last one where the vial temperature exceeded 5 °C.", "In this way, excursion curves were generated so that a low refrigerator set point or a long test would not artificially deflate the impact of warming.", "A threshold of 5 °C was chosen as it is the middle of the cold chain temperature range and is a standard testing point for VVMs [14].", "The known Arrhenius equation for each VVM must then be applied to the warming curves to estimate the impact of warming.", "To do this, the percentage life lost for each data point is calculated by finding the daily life lost at that temperature from the Arrhenius equation and then calculating the life lost in 30 s at that temperature.", "The total life lost due to warming is the sum of the values for life lost at each 30-s data point.", "This process is demonstrated in Fig. 2.", "It is likely that vaccines would be exposed to excursions from 5 °C more than once from placement of warm water packs, but only at the last stage before use.", "This would be at the final health post or clinic, where cool water packs would be recooled in the vaccine refrigerator.", "Thus, our aim was to understand the impact of one month of repeated, daily cycling of water packs on vaccine lifetime.", "Because VVMs (and vaccines) continue to decay even within the cold chain, this would not be only the life lost during one excursion multiplied by 30, but rather would be life lost over 30 daily cycles, each consisting of one excursion from 5 °C plus the remainder of the day at 5 °C.", "This could be reported as the percentage life lost during this daily cycling, or as the amount of time the vaccines would have to be kept at a constant 5 °C for the repeated excursions to cause the VVM on the vial to reach endpoint before the end of the month.", "A month has been simplified here to 30 days.", "Average and maximum values for life lost during 30 days of cyclic warming in eight different test cases are shown in Table 2.", "These results demonstrate that while vials in all test setups were exposed to temperatures above 5 °C, 30 days of cyclic warming would not inevitably cause early conversion to endpoint for any category of VVM.", "The life lost during a month for all four categories of VVM using the worst-case vial temperature curve, from setup 8, is shown in Fig. 3, where the importance of vaccine heat stability by proxy of VVM is evident.", "The VVM7 curve in Fig. 3 shows a degradation of 8.56% over a month.", "In order for a VVM7 vial in the same location to reach endpoint during that month, it would have to be at least 91.44% decayed at the start of the month, a time period equivalent to 29.3 months at 5 °C.", "VVM14 and VVM30 categories were affected even less.", "A VVM2 vial in the same location might have reached endpoint early if it had been kept at a constant 5 °C for 3.5 months prior to the start of temperature cycling.", "The lifetime of a VVM2 at a constant 5 °C is only 5.9 months (using the middle of the VVM endpoint tolerance range, VVM2 life at 5 °C is 177 days, not 225 days), so its lifetime would be shortened by 2.4 months due to cooling water packs in the same refrigerator.", "This is well within the shelf life of prequalified vaccines, so extra caution would be necessary when evaluating the usefulness of this practice in a refrigerator containing low-stability vaccines (VVM2).", "We investigated several factors that could influence the level of warming a vial would be exposed to, including the number of water packs cooling at a time, the arrangement and spacing of a fixed number of vials in the refrigerator, and the total number of vaccine vials in the refrigerator.", "From setup 1 to setup 2, the water pack load was increased from four to eight water packs, enough to fill one or two vaccine carriers.", "The average percentage life lost for the most sensitive category—VVM2—over a month increased 2.92%—from 19.98% to 22.90%—due to the increase in water pack load, and the maximum for any measured vial increased 7.33%.", "To investigate the impact of vial arrangement, ten vials were tested in identical arrangements on the floor and on a shelf in the refrigerator in setup 3 and setup 4.", "The average life lost increased 10.59% and the maximum increased 9.66% due to moving the vials from the floor to the shelf.", "The spacing of vials was tested by arranging ten vials close together in setup 4, then distributing them more loosely in setups 6 and 7.", "Results for the VVM2 category were similar for these tests, the largest difference among the three being a decrease of 3.62% in the average life lost from setup 4 to setup 7.", "However, there was an increase of 0.61% in the maximum life lost between the same tests despite the decrease in the average life lost, indicating that a larger spatial distribution leads to a wider range of life lost, due to varying proximity to the water packs (Fig. 4).", "In setup 7, the average life lost for the four vials closest to the water packs was 9.32% higher than the vial furthest.", "To investigate the impact of vial load on vial warming, tests were run with 1 or 10 vials on a shelf in setups 5 and 4, respectively, and with 20 and 168 vials on the refrigerator floor in setups 2 and 8, respectively.", "The average calculated life lost across 10 vials for VVM2 in setup 4 was 4.16% lower than that for the single vial in setup 5, and the maximum single vial life lost in setup 4 was 3.81% lower than that for the single vial in setup 5 (Fig. 1).", "Comparing 20 vials in setup 2 and 168 vials in setup 8, the average VVM2 percentage life lost across 30 days increased 3.15% from setup 2 to setup 8, but the maximum increased 9.72%, indicating that there was again higher variability in the larger vial load.", "This is evident when comparing the vial arrangements layer to layer: the average VVM2 percentage life lost decreased 1.51% from setup 2 to setup 8 when comparing only the bottom layer of vials on the refrigerator floor.", "The percentage life lost for the second layer decreased 0.25%.", "Only the top two layers of the setup 8 vial arrangement contributed to the overall increase in life lost from setup 2 to setup 8.", "This stratification across layers, as well as the temperature gradient due to proximity to the water packs seen in setup 7, demonstrates that location in the refrigerator has a higher impact on VVM life lost than the number of vials, though the number and location of vials in the refrigerator are dependent.", "However, as it might be expected, VVM category was shown to have a greater effect on VVM life than any of the other factors investigated here.", "Some limitations of this study were that testing was only carried out in one refrigerator and that many tests were carried out with lower vial loads than would likely be found in a vaccine refrigerator in a low-resource setting.", "Other factors that would be valuable to study include the volume of vaccine in the vials, the impact of refrigerator air flows, and the impact of warming in different types of refrigerators.", "In addition, there is a lack of data on the impact of thermal cycling on the relationship between vaccine potency and VVM behavior within this temperature range.", "Another caution is that if the vaccines had not returned to equilibrium before cooled water packs were exchanged for additional warm ones, the thermal impact could intensify and it might take fewer cycles before the VVMs on the vaccines reached endpoint.", "One further concern regarding the use of VVMs as a proxy for vaccine life is the correlation between VVM decay and vaccine decay.", "VVMs do not provide direct information on stability [12] or potency [15] of a vaccine; however, their stated purpose is to indicate when the cumulative heat exposure has reached a preset limit, and to that end, they have been well validated at temperatures lower than 37 °C [16].", "One study noted a lack of correlation between loss of vaccine potency and VVM performance at higher temperatures [17], but the warmest vial throughout our testing was well within the validated range.", "Our study suggests that serious damage to vaccines cannot be assumed from a single excursion event or from repeated exposure to warm water packs in the same refrigerator over the course of a month.", "Several factors result in loss of VVM life due to warming, but the inherent heat stability represented by the different categories of VVMs was found to be most important; i.e., vaccines in vials with a VVM2 are most likely to be damaged from cyclic warming.", "The other important factor was location in the refrigerator, with the phase-change material-lined floor and walls of the testing refrigerator better at resisting warming of vials.", "In many situations, the practice of placing warm water packs in a vaccine refrigerator would not cause any serious loss of vaccines beyond that occurring at cold chain temperatures over time.", "Further research is necessary to define safe ratios of and distances between vaccine vials and warm water packs in varying refrigerators.", "If the decrease in cool life due to using cool water-packs is acceptable, and if there is extra space available in vaccine refrigerators, cooling water packs in a refrigerator with vaccines may be a beneficial practice in cold chain management.", "By using space in already installed and available equipment, this practice could reduce loss of vaccines due to freezing without additional cost.", "The data presented here and the process outlined for evaluating the effects of the practice are useful tools to support cold chain management and regulatory entities in making these decisions." ]	[]
Semi-automatic extraction of liana stems from terrestrial LiDAR point clouds of tropical rainforests	Lianas are key structural elements of tropical forests having a large impact on the global carbon cycle by reducing tree growth and increasing tree mortality. Despite the reported increasing abundance of lianas across neotropics, very few studies have attempted to quantify the impact of lianas on tree and forest structure. Recent advances in high resolution terrestrial laser scanning (TLS) systems have enabled us to quantify the forest structure, in an unprecedented detail. However, the uptake of TLS technology to study lianas has not kept up with the same pace as it has for trees. The slower technological adoption of TLS to study lianas is due to the lack of methods to study these complex growth forms. In this study, we present a semi-automatic method to extract liana woody components from plot-level TLS data of a tropical rainforest. We tested the method in eight plots from two different tropical rainforest sites (two in Gigante Peninsula, Panama and six in Nouragues, French Guiana) along an increasing gradient of liana infestation (from plots with low liana density to plots with very high liana density). Our method uses a machine learning model based on the Random Forest (RF) algorithm. The RF algorithm is trained on the eigen features extracted from the points in 3D at multiple spatial scales. The RF based liana stem extraction method successfully extracts on average 58% of liana woody points in our dataset with a high precision of 88%. We also present simple post-processing steps that increase the percentage of extracted liana stems from 54% to 90% in Nouragues and 65% to 70% in Gigante Peninsula without compromising on the precision. We provide the entire processing pipeline as an open source python package. Our method will facilitate new research to study lianas as it enables the monitoring of liana abundance, growth and biomass in forest plots. In addition, the method facilitates the easier processing of 3D data to study tree structure from a liana-infested forest.	[ "COMP", "EART", "ENGI", "PHYS" ]	[ "Automated liana extraction", "Lianas", "Machine learning", "Python package", "Terrestial LiDAR", "Tropical forests" ]	[ "1706", "1903", "2201", "3107" ]	[ "Attempts to disentangle aboveground and belowground competition between lianas and trees have shown that aboveground competition affected the allocation of biomass in trees and resulted in poorly developed crowns (Schnitzer, 2005).", "A recent study has shown that liana load on trees can alter the allometry of trees by decreasing tree slenderness resulting in shorter and thicker stems (Dias et al., 2017).", "Recent advances in remote sensing technologies have enabled us to view the forest structural complexity in new and unprecedented ways.", "Terrestrial laser scanning (TLS) is an active remote sensing technique and can measure various forest structural parameters non-destructively with high spatial accuracy (Danson et al., 2007; Jupp et al., 2009; Calders et al., 2014, 2015).", "Because of the millimeter-level spatial resolution, TLS is potentially useful to study the plot-level impact of lianas on forest structure.", "In the past decade, there have been significant advancements in the methods developed for 3D point cloud classification using machine learning (Brodu and Lague, 2012).", "In particular, progress has been made to separate leaf and wood points from 3D data of forests using supervised and/or unsupervised machine learning algorithms.", "The methods developed were either based on the radiometric features Béland et al. (2014) or geometric features Ma et al. (2016),Wang et al. (2017), Yun et al. (2016),Belton et al. (2013), Wang et al. (2018),Vicari et al. (2019) or a combination of both Zhu et al. (2018).", "Despite the increasing importance of lianas, very few studies have attempted to quantitatively study the liana structure and the impact of lianas on individual tree and forest structure using TLS or other such remote sensing technologies (Rodríguez-Ronderos et al., 2016; Sánchez-Azofeifa et al., 2015, 2017; Krishna Moorthy et al., 2018).", "A recent study has attempted to classify liana stems using a machine learning algorithm in a point cloud using intensity values from the sensor and geometric features, which makes the proposed method sensor-specific (Bao et al., 2018).", "One of the main reasons for the slower uptake of TLS for studying lianas is that, unlike trees, lianas have irregular growth forms making them harder to study (Bongers et al., 2005).", "There are multiple reasons why the existing tree segmentation algorithms (Burt et al., 2018; Hackenberg et al., 2015) or machine learning-based leaf vs wood segmentation algorithms (Ma et al., 2016; Wang et al., 2017) do not work for segmenting or identifying lianas from a plot.", "The main reasons are listed as follows:", "lianas are in general very small with almost all of the stems falling within 10 cm diameter range.", "(Dewalt et al., 2000; Phillips et al., 2005)", "unlike trees, lianas are generally not self-supporting and hence are generally thinner, longer and have irregular growth patterns.", "Our visual observations in the field show that they can extend in almost all the directions and tend to have branches that loop to the ground multiple times before shooting up to the canopy.", "unlike trees, lianas deploy their leaves in the mid- and upper-crown region of their host trees thus avoiding the need for a tree crown-like structure (Rodríguez-Ronderos et al., 2016; Krishna Moorthy et al., 2018)", "The main objective of the study is to build a novel machine-learning based method for extracting liana woody points from 3D point clouds of tropical rainforests, based only on the geometrical features extracted from the points.", "The method builds on and extends the existing approaches for 3D point cloud classification, especially that of (Brodu and Lague, 2012).", "In this manuscript, we explain in detail the TLS data used for developing and testing the method (Section 2), entire processing pipeline for liana stem point extraction (Section 3), critical evaluation of the proposed approach on field data from two rainforest sites (Section 4).", "In addition, we provide the entire processing pipeline for liana woody point extraction as an open source python package.", "With increasing liana abundance across pan-tropical regions, the presented method will accelerate the research to study lianas with data from remote sensing platforms like TLS (Phillips et al., 2002; Campbell et al., 2018).", "By extracting liana stems from 3D point clouds, the method provides an easier and consistent way to monitor liana stem abundance and potentially stem biomass over time in forest plots.", "In addition, the method would enable the easy processing of 3D data to study trees from liana-rich sites.", "All the data were collected either using RIEGL VZ-400 (Gigante Peninsula) or RIEGL VZ-1000 (Nouragues) scanners (RIEGL Laser Measurement Systems GmbH, Horn, Austria).", "Both are multiple return time-of-flight based scanners using a narrow infrared laser beam of wavelength 1550 nm and a beam divergence of 0.35 mrad.", "Based on the manufacturer’s specifications, the data from the two scanners are interoperable (Calders et al., 2017).", "The registration of data from all scan positions for each plot was done using the RISCAN Pro software (version 2.5.3, RIEGL Laser Measurement Systems GmbH, Horn, Austria) provided by RIEGL.", "Detailed description of the number of plots per site, plot size, liana and tree density of the plots, proportion of liana points, the TLS scanner and the angular resolution used for the specific plot is given in Table 1.", "In this section, we explain in detail about the RF model built to classify the liana points.", "We also outline the workflow to use the trained RF model to extract lianas from new previously unseen forest plots as shown in Fig. 1.", "This section explains in detail all the steps involved in building the RF model for liana woody point classification.", "We define the extraction of liana woody points from 3D point clouds of tropical rainforest plots as a binary classification problem.", "We define the two classes as follows:", "Class 1 refers to liana woody components in the forest plot", "Class 2 refers to other vegetation in the forest plots, including but not limited to liana leaves, tree stems and leaves, palms, etc.", "The first step to build an automated liana extraction model using a supervised machine learning classifier is to have labelled training data from which a classifier can learn.", "The data should be as representative of the real problem as possible to create a robust classifier.", "The co-registered TLS data from the two plots (plots GIG_1 and GIG_2) of Gigante Peninsula and the six plots from Nouragues were manually labelled into two different classes: 1 being liana woody points and 2 being non-liana points (including liana leaves, trees, etc.).", "The manual labelling was the most intensive part of the machine learning pipeline.", "We labelled the liana woody points in the tree crowns as far as we could visually follow them or up until the liana stems started to branch out to leaves.", "In dense canopies, it was indeed difficult to follow them all the way up to the top owing to occlusion and the sensor limitation to resolve an object of smaller size at increasing distance from the LiDAR.", "The manual labelling was done in CloudCompare by an experienced person and took almost 120 man-hours (version 2.8.1, CloudCompare, GPL software) (Girardeau-Montaut, 2011).", "After manual labelling, we downsampled the TLS data using a voxel grid filter of size 0.04 m.", "We chose 0.04 m as the size for voxel grid filter to make a trade-off between retaining the important information in the point cloud and reducing the computational complexity (Burt et al., 2018).", "Downsampling ensures a uniform distribution of points and aids faster computation time by reducing the number of redundant points, especially the points closer to the scanner.", "Though it is not absolutely necessary to perform downsampling, it is recommended as it would improve the computational and memory efficiency of the presented algorithm by few-folds.", "However, the voxel grid size should be chosen based on the problem at hand and the beam divergence of the scanner used.", "Since our dataset is limited, we used all the points for training and validation of the model.", "Sample training data from these two different sites are shown in Fig. 2.", "In this study, we use the normalized eigen values of every point in the TLS data from a plot, across multiple spatial scales, as the predictor variables to classify liana woody points.", "We base our idea on the work of Brodu and Lague (2012), who used eigen values to determine the local dimensionality at multiple scales of a point in 3D space to separate riparian vegetation from ground.", "Previous studies have used eigen values from TLS data at single spatial scale in forest ecosystems to separate leaf from wood points, where the choice of the right spatial scale was domain-specific (Ma et al., 2016; Wang et al., 2017).", "Few other studies have proposed to choose the optimal spatial scale from multiple scales for leaf-wood classification (Zhu et al., 2018).", "Here, we show that the same concept of eigen values, but combined at multiple scales, is effective in highly complex natural scenes like tropical forests to separate liana growth forms from the rest.", "Fig. 3 illustrates the eigen vectors calculated for a set of tree, liana and leaf points.", "We selected the Random Forest (RF) machine learning classifier to build the model for the automated liana woody points extraction algorithm.", "The RF has been widely used in different fields including remote sensing mainly because of its ability to limit overfitting without compromising on the accuracy of the classifier.", "The algorithm achieves this ability by training random subsets of the data and random subsets of the features for each decision tree.", "In addition, the RF has been proved when using TLS point clouds as the most robust learner to separate photosynthetic vs non-photosynthetic elements in forest ecosystems (Wang et al., 2017).", "Since our problem is a binary classification problem (liana stem points vs leaves and other non-liana points) in a similar ecosystem, we chose the RF algorithm to build the classifier.", "For more details on RF classifier, we refer to the original paper of Breiman (2001).", "There are two important hyperparameters to be specified when employing RF for training: number of decision trees to be constructed and number of random features to be chosen while constructing a tree.", "We performed an exhaustive grid search with the following parameter values for estimating the best combination of these two hyperparameters: Number of decision trees: 10, 20, …100 and Number of random features per decision tree: 2, 3, …8.", "The model performance for the hyperparameter optimization was assessed by k-fold cross-validation approach and the model that had the best performance was chosen.", "The performance metrics used for the assessment are described in the following Section 3.1.5.", "Once the hyperparameters are optimized, we built the final model on the entire dataset (all eight plots from two sites indicated in Table 1).", "However, since our dataset is quite limited, we assessed the performance of the model via k-fold spatial cross-validation, thus eliminating the need for an independent test set.", "The performance of the liana woody points classification model is assessed by k-fold spatial cross-validation approach (Goetz et al., 2015).", "This ensures that the model’s fit on the dataset that is independent of the dataset used for training is estimated, while still using all the available information for training.", "In normal k-fold cross-validation, the data is randomly split into k disjoint sets.", "Out of the k sets, one set at a time is used for validating the model and the other k-1 sets are used for training.", "The model performance is then reported as the average performance of the k different model runs.", "In k-fold spatial cross-validation approach, instead of splitting the data into k random sub-sets, we split the data into k spatially disjoint sub-areas.", "We evaluate the model performance in the following three different categories:", "self-site spatial cross-validation, where the models are trained and validated on the same site.", "For Gigante site, we split the two 15 by 15 m plots into four equal disjoint sub-areas of size 7.5 by 7.5 m each and performed a eightfold spatial cross-validation as shown in Fig. 5.", "Since the plots in Nouraugues had only one to three liana individuals in a plot, we simply performed a sixfold cross-validation with one plot retained for testing and the rest of the five plots used for training.", "cross-site spatial cross-validation, where the models are trained on one site (eg.", "Gigante Peninsula) and validated on the other site (eg.", "Nouragues) and vice versa resulting in a twofold cross-validation approach.", "mixed-site spatial cross-validation, where the models are trained and validated on data from both sites.", "For Gigante site, we split the two 15 by 15 m plots into six equal disjoint sub-areas of size 7.5 by 5 m each and for Nouragues, we did not split the plots and had six areas in total.", "As a results, we performed a sixfold cross-validation by using one sub-area from each of the plots in Gigante and one plot from Nouragues as validation set and the rest of the areas as training set as shown in Fig. 6", "We chose these three different validation schemes to evaluate the performance of the method on different real-world usage scenarios.", "Self-site cross-validation evaluates the performance of the method in the base case scenario when new data comes from the same site as the data used for model building.", "Cross-site cross-validation indicates how the model would perform when the data comes from previously unseen site.", "Mixed-site cross-validation indicates the performance of the more sophisticated model, which has used data from multiple sites for building the model.", "As liana woody point classification problem is an imbalanced classification problem with liana woody points belonging to the minority class, the traditional assessment metrics like accuracy and Receiver Operating Characteristics (ROC) curves may provide an overly optimistic view of the classifier performance (He and Garcia, 2008; Ozenne et al., 2015; Saito and Rehmsmeier, 2015).", "For instance, a classifier which always predicts a given point as a Class 2 point (leaves,tree or other vegetation) might yield an accuracy of 95% in a plot with only 5% liana woody points.", "As a result, to estimate the performance of the classifier in an unbiased manner, we used precision, recall, F1 score, precision-recall (PR) curves and False Positive Rate (FPR) as metrics using liana woody points as the positive class.", "Precision-Recall (PR) curve shows the trade off between precision and recall for different liana woody point classification threshold values.", "The threshold values are derived from the probability with which the positive class (liana woody points in this case) is predicted by the model and could range from 0 to 100%.", "A very high threshold value indicates a model with very high precision but low recall and vice versa.", "As a result, a high area under the PR curve indicates a model with both high precision and recall.", "We use Average Precision (AP) to summarize the result of the PR curve.", "AP is calculated as the weighted average of precision at each threshold with the increase in recall from the previous threshold used as the weight.", "An ideal PR curve has a precision of 1 for every increase in recall resulting in an AP of 1.", "We explained above the steps involved in the development and assessment of RF model for extracting liana woody points from forest plots in the previous section.", "In this section, we propose the following simple post-processing steps, which are an essential part of the processing pipeline (Fig. 1), to correct the misclassified liana woody points resulting from the RF model predictions.", "The misclassified points belong to two different categories: False Positives (FP) and False Negatives (FN).", "We propose four post-processing steps to correct these misclassified points.", "Steps 1 and 2 are mainly focused on removing FPs, Class 2 points that are misclassified as liana woody points, with basic filtering and with very minimal manual intervention as explained below.", "Steps 3 and 4 focus on correcting the FNs.", "This means that these steps mainly correct the liana woody points that are misclassified by the model as belonging to Class 2 (trees, leaves or other vegetation).", "Step 1 This step aims at filtering noisy or ghost points from the Class 1 (liana woody points) model predictions.", "We apply the Statistical Outlier Removal (SOR) filter implemented in the CloudCompare software (Girardeau-Montaut, 2011) to remove the noisy points that are too far from their neighbors in the point cloud classified as belonging to liana woody points by the model (Rusu and Cousins, 2011).", "SOR filter has two parameters: number of points to be considered for distance mean distance (nPoints) estimation and standard deviation multiplier threshold to be used for filtering the points (nSigma).", "We used the following default settings of the CloudCompare: nPoints = 6 and nSigma = 1.00", "Step 2 This step aims at removing the FPs from Class 1 model predictions.", "After SOR filtering, we manually remove the obvious sections belonging to Class 2 (points that are not liana woody components) from the resulting point cloud from Step 1 using the polygonal selection tool in CloudCompare (Girardeau-Montaut, 2011).", "We identify the isolated cluster of points among the points classified as Class 2 by the RF model.", "We achieve this by applying the DBSCAN on the predicted Class 2 points.", "In section, we first report the results for hyperparameter optimization to build the RF model.", "Then, we report the results of the model performance for three different cross-validation categories explained above.", "Finally, we report the results of the post-processing steps at individual plot-level for the self-site cross-validation models.", "The model performance was assessed by a fivefold cross-validation approach for hyperparameter optimization.", "The entire data from all eight plots in Table 1 was put together and then split into five random spatially disjoint sets, out of which four sets were used for training and one set was used for validation.", "The resulting F1 is the average result of all five model runs.", "We report all the performance metrics for all three cross-validation categories in Table 2 and show the PR curves for all them in Fig. 8.", "FPR was very low with values ranging between 1 to 3% across all three categories of spatial cross-validation.", "The model performed with a high precision of 0.85 and 0.91 for Gigante and Nouragues respectively.", "However, the recall values were much lower for Nouragues compared to Gigante.", "This was mainly due to the thick straight liana stems (> 5 cm) having a geometrical structure similar to that of trees closer to the ground.", "The high AP values from the PR curves of 0.79 and 0.81 for Gigante and Nouragues respectively indicate that the model has a good discriminatory power.", "The corresponding PR curves are shown in Fig. 8a and b.", "As it can be seen from the figure, precision retains a value of 80% while the recall is increased to 70% for most of the models resulting in the higher AP value.", "Both the independent and average performance metrics of the cross-site cross-validation is shown in Table 2.", "Training on data from Gigante site and testing on Nouragues gives better results than both training on Nouragues and testing on Gigante and training and testing on Nouragues.", "This is in line with our expectations, as the plots GIG_1 and GIG_2 combined have more lianas then the plots in Nouragues resulting in more robust training data for the model to learn from.", "Similarly the AP of the model trained with the data from Nouragues site is only 0.65, indicating a much lower discriminatory power than the model trained with data from Gigante site with a high AP of 0.84.", "The corresponding PR curves can be seen in Fig. 8c.", "As the PR curve indicates, the recall of the model drops to a low value of close to 60% for a recall of 70% explaining the lower AP value.", "We indicate the average performance metrics of the sixfold cross-validation (Fig. 6) in Table 2.", "As the results indicate, the model has a high precision of 0.89, a rather low recall of 0.62 and an F1 score of 0.73.", "The PR curves indicate a good discriminatory power of the model in general with an AP of 0.77 (Fig. 8d).", "The liana woody points can be extracted from the 3D data of a tropical rainforest plot by the final model trained on the entire dataset including all eight plots from two sites.", "As the results indicate, the models validated using the different cross-validation categories have on average high precision and very low FPR across all the plots from the two sites used in the study.", "This means that when the model predicts a point as a liana woody point, the model is 88% sure that its correct (indicated by the average from Table 2).", "This explains the very low FPR of 1.4% of the model.", "The model’s high predictive performance is further confirmed by analyzing the PR curves of the different models built.", "AP, which summarizes the PR curve of the models, is high with an average value of 0.78 indicating a good discriminatory power of the model.", "However the recall is rather low with an average value of 0.58.", "This means that only 58% of all liana woody points in the plot are retrieved by the model.", "The recall values are much lower especially for plots in Nouragues because of the thick straight stems having geometrical structures similar to trees closer to the ground.", "In addition, parts of liana tangles are mis-classified as they look like a cluster of points with geometrically similar features as leaves (Fig. 9a).", "Since the FPR is much lower and is mainly caused by small stems that do not grow beyond a few meters from the ground and small segments of tree branches, we recommend a basic SOR filtering and manual removal of these small segments below the canopy as explained in Section 3.2.", "This manual removal is straightforward as it involves removing some isolated segments that belongs to trees.", "After the removal of FPs, we perform DBSCAN clustering and connectivity analysis to correct the FNs.", "We report the increased recall from the post-processing steps on the predicted liana woody points from all the plots of Gigante Peninsula and Nouragues sites from the self-site cross-validation models in Table 3.", "While the cross-site models report the results for all the plots combined for a site and mixed-site models report the results for plots from the two sites combined, self-site models let us evaluate the effectiveness of the post-processing at individual plot-level.", "The different post-processing steps are illustrated in Fig. 9.", "These post-processing steps increased the recall from 54% to 90% in Nouragues plots without compromising on the precision.", "However, in Gigante the recall only increased by 5% from the original 65% resulting in a 70% recall overall.", "This was mainly because, the misclassified liana woody points were too close to the other vegetation to be considered as separate clusters as seen in Fig. 10.", "These 30% missing liana woody points could be manually corrected in a straightforward way using CloudCompare.", "We provide an open source python software package executing the steps in Fig. 1 for the plot-level near-automated liana woody points extraction given only the xyz coordinates of the pointcloud.", "Extracting eigen values at multiple spatial scales on a small plot with approximately 500,000 points can be completed within 10 min even on a single core machine.", "However, this high computational efficiency comes at a cost of high memory requirements.", "The main specifications of the machine we used to run the algorithm are the following: dual boot (Ubuntu 16.04 LTS and Windows 10), Intel® Xeon® E5-1650 v4, 6 cores and 128 GB RAM.", "The python package has been tested on both Ubuntu and Windows operating systems.", "More details on the practical implementation and tips to work around the issue of high memory requirements of the algorithm can be found in the github page (https://github.com/sruthimoorthy/automated-liana-extraction.git).", "Our RF model performance indicates that the liana data is not site-specific as the model trained on Gigante site could be applied to data from Nouragues.", "The low F1 score of the model trained on Nouragues and tested on Gigante is mainly due to lack of sufficient training data as Nouragues had fewer lianas than Gigante.", "However, additional testing is needed on other sites to confirm this.", "The proposed post-processing steps increased the recall to 90% in Nouragues from a low value of 54% for the self-site cross-validation models mainly because of the very high precision of 91% of the models.", "It is possible that an important source of error in the RF model development might arise from the errors made while manually labelling liana points vs non-liana points for training and testing data especially in the canopy.", "In the case of TLS, minimum size of the object distinguishable by the scanner increases with increasing distance from the scanner.", "For most of the lianas, their woody component becomes almost negligible in the canopy as they are much smaller than the trees even when they are closer to the ground (Dewalt et al., 2000).", "This is a sensor limitation and not a limitation of the method as such.", "Future development of TLS instruments with smaller beam divergence could help in resolving smaller objects at greater heights in the canopy.", "The presented method is transferable to point clouds from any platform of similar high quality (i.e. minimal occlusion in the data).", "In addition, the proposed method only requires xyz coordinates of the points (refer to Fig. 1) and does not depend on any spectral properties of the points making it broadly applicable to 3D data collected from a variety of sensors.", "Another major advantage of the proposed method is combining the geometric features at multiple spatial scales, thus eliminating the need for optimal scale selection.", "Future work could focus on improving the performance of the classifier (especially the recall value), thereby reducing the amount of manual intervention required, using more sophisticated machine learning methods like deep learning.", "Recent advances in the field of machine learning have extended the deep learning for 3D point cloud classification to work directly on the unstructured point clouds (as opposed to structured voxels etc.), thereby enabling automated feature learning to build the classifier (Qi et al., 2017).", "Though deep learning requires large amount of data and substantial computing power (high-performance GPUs), it is one of the promising ways to improve the classifier performance and will be a topic of our future study.", "We presented a method for semi-automated liana extraction from plot-level TLS data with a high precision of 88%.", "The proposed method was tested on two sites: Gigante Peninsula, Panama and Nouragues, French Guiana.", "RF models had a low recall of 54% and 65% in Nouragues and Gigante, which was increased to 90% and 70% by the post-processing steps respectively.", "The method is semi-automated and the remaining 10 to 30% of the points requires manual intervention.", "Future developments of the method should focus on reducing this manual intervention by using more sophisticated methods like deep learning for liana woody point classification.", "We believe that our method will accelerate the research in high liana dense forests (e.g., Gigante Peninsula (van der Heijden et al., 2015)) and in forests where lianas continue to proliferate (Schnitzer and Carson, 2010; Schnitzer and Bongers, 2011; Schnitzer et al., 2000; Schnitzer, 2018), thereby enabling the quantification of their impact on tree and forest structure.", "The method enables researchers working on tropical forest plots to have a liana(noise)-free point cloud enabling an easier isolation of trees either manually or through an automated algorithm leading to an accurate quantification of tree structural metrics like tree volume.", "The presented method enables us to monitor and quantify liana abundance and biomass in forest plots, which is highly uncertain when calculated from allometric equations (Miao et al., 2016).", "S.M.K.M., Y.B. and H.V. conceived and designed the experiments with inputs from S.A.S. and K.C.; S.M.K.M. collected the data; S.M.K.M. and Y.B. performed the analysis; S.M.K.M. wrote the paper with critical contributions from all the authors.", "All the codes required for extracting lianas in a plot are available in the following github page: https://github.com/sruthimoorthy/automated-liana-extraction.git.", "The codes are written in Python scripting language and have been tested in both windows and ubuntu.", "The data used for building the classifier can be obtained by sending an email to the corresponding author ([email protected])." ]	[ "Semi-automated method to extract liana woody points from terrestrial LiDAR data.", "Lianas woody points are classified by a machine learning model.", "Post-processing steps improve the classification performance from 60% to 80%.", "Validation on two tropical forest sites indicates successful liana extraction." ]
Enigmatic structures within salt walls of the Santos Basin-Part 1: Geometry and kinematics from 3D seismic reflection and well data	Understanding intrasalt structure may elucidate the fundamental kinematics and, ultimately, the mechanics of diapir growth. However, there have been relatively few studies of the internal structure of salt diapirs outside the mining industry because their cores are only partly exposed in the field and poorly imaged on seismic reflection data. This study uses 3D seismic reflection and borehole data from the São Paulo Plateau, Santos Basin, offshore Brazil to document the variability in intrasalt structural style in natural salt diapirs. We document a range of intrasalt structures that record: (i) initial diapir rise; (ii) rise of lower mobile halite through an arched and thinned roof of denser, layered evaporites, and emplacement of an intrasalt sheet or canopy; (iii) formation of synclinal flaps kinematically linked to emplacement of the intrasalt allochthonous bodies; and (iv) diapir squeezing. Most salt walls contain simple internal anticlines. Only a few salt walls contain allochthonous bodies and breakout-related flaps. The latter occur in an area having a density inversion within the autochthonous salt layer, such that upper, anhydrite-rich, layered evaporites are denser than lower, more halite-rich evaporites. We thus interpret that most diapirs rose through simple fold amplification of internal salt stratigraphy but that locally, where a density inversion existed in the autochthonous salt, Rayleigh-Taylor overturn within the growing diapir resulted in the ascent of less dense evaporites into the diapir crest by breaching of the internal anticline. This resulted in the formation of steep salt-ascension zones or feeders and the emplacement of high-level intrasalt allocthonous sheets underlain by breakout-related flaps. Although regional shortening undoubtedly occurred on the São Paulo Plateau during the Late Cretaceous, we suggest this was only partly responsible for the complex intrasalt deformation. We suggest that, although based on the Santos Basin, our kinematic model may be more generally applicable to other salt-bearing sedimentary basins.	[ "EART" ]	[ "Brazil", "Internal structure", "Kinematics", "Salt diapir", "Salt tectonics", "Santos Basin" ]	[ "1907" ]	[ "A salt diapir is a mass of salt that has flowed in a ductile manner and has discordant contacts with its overburden (Fig. 1) (Hudec and Jackson, 2011).", "Salt stocks (having a plan-form axial ratio <2) and walls (plan-form axial ratio >2) are the most common types of salt diapir; they form the fundamental building blocks of many salt-bearing sedimentary basins.", "Although the controls on the external morphology of natural salt diapirs are relatively well-documented and understood (e.g. Jackson and Talbot, 1986, 1991; Vendeville and Jackson, 1992; Koyi, 1998; Davison et al., 2000; Hudec and Jackson, 2007, 2011; Stewart, 2007), considerably less is known about their internal structure and kinematics for four key reasons: (i) well-exposed, natural salt diapirs are rare because halite, a key component of many salt structures, is highly soluble and dissolves, whereas anhydrite, upon contact with water, converts to gypsum, leaving a karstic soil or crust that masks the diapir's internal structure (Jackson et al., 1990; Bruthans et al., 2009); (ii) the internal structure of exposed diapirs can be strongly deformed by gravity spreading of salt extruding at the Earth's surface (Talbot and Jackson, 1987; Talbot, 1998; Talbot and Aftabi, 2004); (iii) even where diapirs are well exposed at the Earth's surface, such as in Iran's Great Kavir, exposures are largely two-dimensional (Fig. 1A) (Jackson et al., 1990); and (iv) thick salt is typically acoustically transparent on seismic reflection data, and internal stratigraphic markers that record strain are typically poorly imaged.", "However, there are notable exceptions, which we discuss in Section 1.1 (Cobbold et al., 1995; Van Gent et al., 2011; Strozyk et al., 2012; Fiduk and Rowan, 2012; Jackson et al., 2014).", "Much of our understanding of intrasalt structure and kinematics comes from observations in mines.", "These man-made exposures suggest complex flow patterns and internal vorticity during diapir growth (e.g., Kupfer, 1976; Richter-Bernberg, 1980; Burliga et al., 2005; Bornemann et al., 2008), providing unrivalled exposures of meso- and macroscale structures.", "However, mining data yield only a quasi-3D appreciation of intrasalt structure due to the relatively limited distribution of mine galleries and boreholes (Fig. 1B).", "Scaled physical models also provide insights into salt diapir kinematics and internal structure (Fig. 1C–E).", "In such models, salt walls are typically characterised by inward flow and thickening of salt within the wall, which forms relatively simple, upright, symmetrical internal anticlines (Fig. 1D) (Dixon, 1975; Schwerdtner et al., 1978; Ramberg, 1981; Dooley et al., this volume).", "Their internal structures are thus defined by variably deformed, but nonetheless intact, stratigraphic layering.", "More-complex structures form in mushroom-shaped salt stocks in centrifuge models (Fig. 1E and F) (Jackson and Talbot, 1989).", "These diapirs contain a range of axisymmetric fold systems.", "In the simplest cases, these folds form in response to inward flow of salt from the source layer, upward flow of salt within the diapir core, and lateral spreading of salt at the diapir crest.", "More mature mushroom-like diapirs can have an overhanging bulb that is fringed by one or more skirts that may curl inward and entrain overburden at the diapir flank (Fig. 1E).", "Using sand-silicone models, Brun and Mauduit (2009) demonstrated that internal mushroom structures (sensu Jackson and Talbot, 1989) can also form in relatively immature, low-relief diapirs by shear traction exerted by translating overburden (Fig. 1C).", "Kinematic inferences from scaled physical models need to be tested by observations from natural diapirs that are exposed or imaged in three dimensions.", "Testing of physical model predictions is now feasible using modern 3D seismic data that cover large expanses and provide high-resolution (<50 m vertically) imaging of, salt-bearing sedimentary basins.", "These data yield an excellent, hitherto underused opportunity to gain a three-dimensional understanding of the internal structure of entire salt structures and, by inference, their kinematics.", "For example, by using data from the Groningen Basin and Cleaver Bank High, offshore Netherlands, Van Gent et al. (2011) and Strozyk et al. (2012) mapped the detailed geometry of a highly reflective, thin (40–50 m) layer of claystone-carbonate-anhydrite encased in large (>15 km long), halite-dominated salt diapirs, pillows, and anticlines.", "They documented a range of shortening (in the form of noncylindrical buckle folds) and extensional (boudins) structures within and adjacent to salt anticlines, which they interpreted as forming due to non-plane strain during highly heterogeneous salt flow.", "Fiduk and Rowan (2012) used depth-migrated 3D seismic data from the Santos Basin, offshore Brazil, to constrain the internal structure of walls formed by flow of multilayered Aptian salt, documenting near-isoclinal, recumbent folds and intrasalt thrusts (see also Cobbold et al., 1995; Davison et al., 2012).", "They inferred massive basinward flow of and shortening within the salt.", "Cartwright et al. (2012) inferred the bulk flow profile of salt in a domain of shortening based on 3D seismic analysis of multilayered Messinian evaporites in the eastern Mediterranean.", "Although the salt in this particular example is not diapiric, their study points to further use of seismic reflection data to understand flow kinematics within salt bodies.", "In all these examples, highly reflective layered evaporites acted as internal strain markers within an otherwise acoustically and rheologically homogeneous salt body.", "Despite providing excellent spatial imaging of intrasalt structures, seismic data have drawbacks because positioning of reflections is controlled by uncertain velocity models.", "Furthermore, steeply dipping or highly strained intrasalt strata are typically poorly imaged by conventional seismic reflection data.", "Likewise, some intrasalt units may be too thin to image, despite being acoustically different from the encasing strata.", "Although challenging, understanding large-scale intrasalt structure and kinematics is important for several reasons.", "First, some diapirs are envisaged as repositories for high-level nuclear waste.", "Thus it is critical to understand their internal structure, in particular the location and size of high-permeability structures and stratigraphic units that may hydraulically connect the otherwise impermeable core of the diapir and permeable overburden rocks (Klinge et al., 2007).", "Second, diapirs and their caprocks can host potash deposits (Talbot et al., 2009) or sulphide minerals in Mississippi Valley–type deposits (Light et al., 1987), so there is strong economic incentive to understand their permeability structure.", "Third, drilling wells through thick salt is an engineering challenge because it may contain variably deformed and dismembered interbeds of more-mobile potash evaporites, like sylvite and carnallite, which may flow on drilling timescales.", "Other porous inclusions may be overpressured or underpressured, both of which complicate drilling.", "Anything that can be done to understand and predict intrasalt inclusion geometries mitigates drilling risk.", "Fourth, understanding the internal structure of the salt could help to optimize wellbore trajectories (Weijermars and Jackson et al., 2014).", "Fifth, structural restoration of salt-bearing sedimentary basins is typically focused on retrodeforming the overburden and ignores strain within the salt because the intrasalt structures are too complex or poorly imaged.", "An improved understanding of intrasalt structure should allow us to include intrasalt strain into structural restorations of salt-bearing sedimentary basins (Hossack, 1995; Rowan and Ratliff, 2012).", "This study aims to document the variability in intrasalt structural style in five natural salt diapirs, and to test kinematic and mechanical models for how these structures formed.", "To achieve these aims we use time-migrated 3D seismic reflection and borehole data from the São Paulo Plateau, Santos Basin, offshore Brazil (Fig. 2).", "Despite the inherent shortcomings of seismic data, this area is ideal to investigate the structure and kinematics of diapirs because: (i) detailed mapping of layered and highly reflective evaporites in the autochthonous salt allows us to constrain intrasalt structure within numerous large salt walls; and (ii) the absence of shallow allochthonous salt, or stratigraphic and associated acoustic complexity in the overburden, permits high-quality imaging of the internal structure of the walls.", "Some of the salt structures described here have been investigated by Fiduk and Rowan (2012), using a pre-stack depth-migrated version of the same 3D volume, and by Jackson et al. (2014), using time-migrated 3D seismic and borehole data.", "We greatly add to the data presented in their studies and critically appraise their models by: (i) constructing detailed maps and providing a systematic description of the internal structure of numerous salt walls; (ii) using borehole data to constrain the physical properties, in particular the density, of the autochthonous source layer; and (iii) describing the internal structures of diapirs outside of the domain characterised by complex intrasalt deformation.", "We focus especially on complex structures within a minority of salt walls.", "Such structures have only very rarely been described in detail before (Fiduk and Rowan, 2012; Jackson et al., 2014) and their genesis is enigmatic because we cannot account for them merely by intrasalt shortening Our analysis evaluates previous interpretations of the origin of intrasalt structures on the São Paulo Plateau, and offers a revised explanation.", "Although our kinematic model is based on analysis of the Santos Basin, it may be more applicable to other salt-bearing sedimentary basins.", "A companion paper by Dooley et al. (this volume) uses a series of physical models to explore the mechanical feasibility of our kinematic model.", "The Santos Basin initially formed as a rift basin during the Early Cretaceous as the South Atlantic began to open (Meisling et al., 2001; Modica and Brush, 2004; Karner and Gambôa, 2007; Mohriak et al., 2008; Contreras et al., 2010).", "A series of graben and half-graben were filled by non-marine strata (Picarras and Itapema formations), which are overlain by shallow-marine carbonates (Barra Velha Formation) (Fig. 3).", "During the latter part of the Early Cretaceous (latest Aptian), the number of active upper crustal faults, and the rate of slip on these faults, decreased, and a 2.4–2.6 km thick, post-rift salt-rich succession (Ariri Formation) was deposited (Figs. 2 and 3) (De Freitas, 2006; Davison et al., 2012).", "Margin-scale restorations across the Santos Basin suggest that the salt was fully contained within its original depositional basin (Fig. 2B) and that postdepositional flow caused it to be expelled as salt nappes emplaced on transitional or oceanic crust (Davison et al., 2012).", "During the early Albian, in response to thermally induced subsidence and a eustatic sea-level rise, fully marine conditions became established in the Santos Basin, and a carbonate-dominated succession was deposited (Itanhaem Formation) (Fig. 3).", "Grainstones and oolitic shoals accumulated along the proximal northwestern basin margin; farther basinward to the southeast, beyond the palaeo-shelf edge, a very fine-grained, marl-dominated succession was deposited (Modica and Brush, 2004).", "At this time thermal and isostatic subsidence was focused toward the centre of the embryonic oceanic spreading centre and, during the latest Albian, this subsidence tilted the basin southeastward, creating a thin-skinned array of predominantly seaward-dipping normal faults that broke the Albian carbonate platform into extensional rafts (Demercian et al., 1993; Rouby et al., 1993; Cobbold et al., 1995; Mohriak et al., 1995; Guerra and Underhill, 2012; Quirk et al., 2012).", "Our study area lies downdip of the Albian extensional domain and so is likely to have undergone Albian shortening.", "Shortening may then have continued through to the end of the Cretaceous, but this is debatable.", "A key feature of this debate is the Albian Gap (Demercian et al., 1993; Cobbold et al., 1995; Mohriak et al., 1995; Ge et al., 1997; Modica and Brush, 2004; Gemmer et al., 2005; Davison et al., 2012; Guerra and Underhill, 2012; Quirk et al., 2012).", "Some researchers think the Albian Gap formed mostly by Late Cretaceous gravity-driven extension of the suprasalt cover, in which case the structures in our study area may be part of the Late Cretaceous shortening system that accommodates this extension (e.g., Mohriak et al., 1995; Modica and Brush, 2004; Quirk et al., 2012).", "Others interpret that the Albian Gap formed due to salt expulsion driven by progradation of basin-margin clastic wedges across a pre-existing salt wall (Ge et al., 1997; Gemmer et al., 2005; Krézsek et al., 2007).", "If so, there should be little post-Albian shortening of the overburden in the study area because there was little updip extension to drive it.", "Establishing the origin and timing of intrasalt structures on the São Paulo Plateau can contribute to this debate.", "This study uses a zero-phase processed, time-migrated, 3D seismic reflection data set that covers 20,122 km2 of the São Paulo Plateau in the central Santos Basin (Fig. 2A).", "Inline (east–west) and crossline (north–south) spacing is 18.75 and 25 m, respectively; the vertical sampling interval is 4 ms two-way time (ms TWT) and the data have a record length of 5500 ms TWT.", "The seismic survey is displayed with Society of Economic Geologists (SEG) “normal” polarity, where a downward increase in acoustic impedance is represented by a positive reflection event (white on seismic sections) and a downward decrease in acoustic impedance is represented by a negative reflection event (black on seismic sections) (Brown, 2011).", "The dominant frequency in the Aptian salt is c. 30 Hz and the average interval velocity is 3439 m/s, which is lower than typical for salt (4500–5500 m/s) due to the relative abundance of acoustically slow carnallite layers; together, these values for frequency and velocity yield an intrasalt vertical resolution of c. 29 m. Owing to a similar dominant frequency (c. 31 Hz) and a slower average interval velocity (c. 2015 m/s), the vertical resolution (c. 17 m) in the overburden is slightly higher than in the salt, although this too varies with depth.", "Structural measurements in the salt (e.g., fold amplitude, displacement across intrasalt shear zones) are converted from time (ms TWT) to depth (meters) using the interval velocities defined above.", "Horizontal resolution is taken as twice the seismic line spacing (i.e., 37.5 m in the E–W direction and 50 m in the N–S direction).", "Six boreholes lie within the area covered by the 3D seismic dataset (Fig. 2A).", "These boreholes yield wireline-log (gamma ray, bulk density, sonic travel time), cuttings, and stratigraphic (age) data that constrain the composition and age of the salt and overburden.", "Checkshot data in these wells allow us to tie the stratigraphic data both within the salt in the adjacent minibasins to the seismic reflection data using synthetic seismograms (Fig. 4).", "The top and base of salt are defined by high-amplitude postive and negative reflection events, respectively (TS and BS, Fig. 4).", "The regional distribution and morphology of salt and salt-related structures are defined by a two-way time (TWT) map of the top salt reflection (Fig. 5A; see also Fig. 5B).", "Borehole data and synthetic seismograms also indicate that the Aptian salt has a very distinctive internal stratigraphy that is closely tied to its seismic expression and mechanical properties (Fig. 4); this is described in the following section.", "Borehole data indicate that the salt is halite-rich (typically >80% by volume) (see also Fiduk and Rowan, 2012; Jackson et al., 2014).", "However, on the basis of percentage of nonhalite evaporites (anhydrite, carnallite, tachyhydrite) and their differing densities, the unit is divided into four units (Table 1; Fig. 4) (Jackson et al., 2014).", "A1 is a chaotic-to-weakly stratified, poorly reflective, halite-rich (77–98%) unit, having a bulk density of 2239–2270 kg/m3.", "A2 is a strongly reflective, high-amplitude unit having less halite (67–86%), a bulk density of 2258–2311 kg/m3, and relatively thick (50–235 m), dense (2350–2990 kg/m3) anhydrite interlayers.", "A3 is a poorly reflective unit that has more halite (69–94%) and a bulk density of 2231–2277 kg/m3.", "A4 is a strongly reflective unit having less halite (31–94%) and a bulk density of 2047–2522 kg/m3, but containing some low-density evaporite (carnallite <1800 kg/m3).", "Note that we elect to use a four-fold stratigraphic framework rather than the six-fold framework of Fiduk and Rowan (2012) because: (i) a four-fold sub-division allowed us to extend mapping of intrasalt stratigraphy throughout the basin and within numerous diapirs; and (ii) their nomenclature challenges stratigraphic conventions in that stratigraphic units are numbered from top to bottom (e.g. B1 overlies B2, B2 overlies B3), which is opposite to their age relations.", "Although the four main intrasalt units can be identified across the study area, our borehole data indicate that the present thickness, composition, and, therefore, density stratification of the salt varies (Fig. 6; see also Table 1).", "In the south, boreholes 723C and 709, which are both located on the south-central part of the São Paulo Plateau in the midst of an array of diapirs that contain complex intrasalt structures (see Section 6), indicate that A2–A4 are collectively denser (723C = 2355 kg/m3 and 709 = 2297 kg/m3) than A1 (723C = 2251 kg/m3 and 709 = 2239 kg/m3).", "In contrast, on the northwestern part of the São Paulo Plateau, where diapirs contain relatively simple intrasalt structures (see Section 6), borehole 369A indicates that the salt has a broadly similar density throughout, with A2–A4 (2179 kg/m3) being less dense than A1 (2270 kg/m3).", "This spatial variability in the density stratification of the autochthonous salt is critical to the kinematic model outlined later in this paper (see Section 8.3).", "Because the intrasalt units differ in composition and acoustic properties, their boundaries are defined by regionally mappable reflections (Figs. 4, 6 and 7) that allow us to define the internal structure of a number of salt diapirs (as done by Davison et al., 2012; Fiduk and Rowan, 2012; Jackson et al., 2014).", "Line-by-line (every 18.75–25 m) mapping of intrasalt horizons, in particular the top of A2 (Figs. 4, 7 and 8), allowed us to produce very detailed maps of the internal structure of each diapir (Fig. 9).", "We also mapped several post-Aptian seismic horizons to illustrate overburden structural styles intimately related to the evolution of the salt structures and adjacent minibasins (top Cretaceous = TC, intra-Palaeocene = IP, top Palaeocene = TP; Figs. 3, 7 and 8).", "We first describe the overall distribution and morphology of salt structures on the São Paulo Plateau to provide a semiregional framework for our detailed analysis of intrasalt structure.", "Salt walls predominate on the São Paulo Plateau and salt stocks are rare (Fig. 5).", "A key observation is that the most of the salt walls have relatively simple external morphologies, and are defined by relatively flat crests and relatively smooth, moderately dipping (<20°) flanks (Figs. 5 and 7).", "The relatively simple external form of many of the diapirs masks much greater internal structural complexity, as described in the next section.", "The walls are up to 4.8 km tall, 15 km wide, and 60 km long (Figs. 5A and 7).", "They trend broadly NE-to-NNE in the north and broadly N-to- locally NE in the south (Fig. 5A–B).", "In the north, NW-trending walls link the NE-to-NNE-trending salt walls, resulting in an overall polygonal pattern of salt walls enclosing subcircular minibasins, which together cover much of the central and northern Santos Basin (Fig. 5A–B) (Davison et al., 2012; Guerra and Underhill, 2012; Quirk et al., 2012).", "In the south of the study area, these NW-trending walls are absent and N-trending walls dominate; in this domain occur the complex intrasalt structures that form the focus of this study (Fig. 5A–C).", "There is abundant evidence for mild shortening in suprasalt sediments in some places on the São Paulo Plateau (see also Cobbold et al., 1995).", "For example, relatively closely spaced (<3 km), NE-trending, upright to slightly ESE-verging folds, which have amplitudes of up to 700 m and rounded hinges, are especially common in the northeast margin of the São Paulo Plateau (herein called the Outer São Paulo Fold Belt; Figs. 5B and 8A).", "Northeast-trending buckle folds occur in the southwest, (herein called the Inner São Paulo Fold Belt; Figs. 5B and 8B), where they crosscut N-trending salt walls.", "Locally lower-A1 shows marked, shear zone-induced changes in thickness and the overlying layered evaporites and the overburden are concordantly folded (Fig. 8B).", "NE-striking, salt-cored buckle folds, which are typically underlain by NW-dipping thrusts, also sporadically occur in the central part of the São Paulo Plateau between the variably oriented salt walls (Figs. 5B and 8C).", "Many of these thrusts can be traced into salt walls, where they change strike to become parallel to the margins of the wall (see below).", "Some salt walls are strongly discordant with their overburden and have major cutoffs against flanking strata, thus are characteristic of passive diapirs formed by downbuilding (Fig. 7B) (e.g. Nelson, 1989; Vendeville and Jackson, 1992; Rowan et al., 2003).", "In contrast, other salt walls are only slightly discordant to the onlapping overburden strata and resemble contractional anticlines whose crests have been uplifted and eroded (e.g. Stewart and Coward, 1995).", "Still other diapirs have triangular profiles and a fan of inward-dipping normal faults at their crests, a geometry typical of reactive diapirs initiated by regional extension (e.g. Vendeville and Jackson, 1992; Jackson et al., 2014)", "Highly reflective layered evaporites in the Aptian salt layer (Fig. 4) permit detailed analysis of its internal structure, indicating that the magnitude and style of deformation varies across the São Paulo Plateau (Figs. 5B and 9) (see also Cobbold et al., 1995; Davison et al., 2012; Fiduk and Rowan, 2012; Jackson et al., 2014).", "Five main intrasalt structures are identified: (i) large, upright, symmetrical, internal anticlines that deform A2–4 and are cored by thickened A1 halite (Fig. 10); (ii) near-isoclinal, moderately-inclined-to-recumbent synclines formed in upper A1–4 (Fig. 11); (iii) acoustically transparent, sheetlike bodies overlying complexly deformed walls, attached to narrow, subvertical, acoustically transparent zones linking them downward to autochthonous A1 salt (Fig. 11); (iv) small, upright, symmetrical anticlines in the upper part of A1 that only affect basal parts of salt walls (Fig. 12); and (v) reverse shear zones (Fig. 13).", "In this section we use maps (Fig. 9) and cross sections (Figs. 10–13) to describe these structures, drawing on examples from five of the complex diapirs, which we name after well-known rock ‘n’ roll frontmen (Jimi, Martin, Liam, Freddie, and Axl; Figs. 5B and 9).", "Most salt walls on the São Paulo Plateau, irrespective of their external shape, orientation, or size, contain relatively simple, upright, intrasalt anticlines (Figs. 5B and 7).", "These intrasalt anticlines are cored by thickened lower-A1 and overlain by broadly tabular but folded, layered evaporites (upper-A1–A3 and, where preserved, A4); beneath the adjacent minibasins, lower-A1 is typically very thin (<250 m) or even locally absent where A2 is welded to presalt strata (Fig. 7B).", "The anticline hinge is parallel to the wall long axis, and the fold limbs dip <20°.", "Although generally tabular, upper-A1, A2, and A3 locally display dramatic changes in thickness across the anticlines, which are especially striking on the limbs of some of the high-relief diapirs in the central and northern parts of the central São Paulo Plateau.", "A4 and the upper part of A3 are commonly truncated and are absent at the wall crest across the anticline hinge (Fig. 7).", "Internal anticlines also occur in the domain of complex intrasalt structures on the south-central part of the São Paulo Plateau, but are typically at the low-relief ends of walls (e.g. northern and southern ends of Liam; Figs. 9B and 10B; southern end of Jimi; Figs. 9A and 10B; northern and southern ends of Martin; Fig. 9A; northeastern and southwestern ends of Axl; Fig. 9E) whose culminations are dominated by complex folds and poorly reflective sheetlike bodies (see Sections 6.2 and 6.3).", "Locally, relatively large, upright anticlines occur within the cores of complex walls; these anticlines typically have a moderately inclined, near-isoclinal syncline superposed on the limb facing the wall centre (Fig. 11) (see Section 6.2).", "The cores of the complexly deformed walls are dominated by moderately-inclined-to-recumbent synclines.", "Two fold styles are observed: (i) large (amplitude up to 3 km), relatively continuous (>5 km) folds that either are preferentially developed on one side of the wall or occur in pairs; and (ii) smaller (amplitude up to 1.5 km), isolated folds of limited lateral extent (<5 km).", "The large, near-isoclinal, moderately-inclined-to-recumbent, noncylindrical synclines occur along the southeastern (Freddie, Figs. 9D and 11C; Axl, Fig. 9E) or eastern (e.g., central part of Liam, Figs. 9B and 11F–G) margins of several complex walls.", "These synclines face outward and die out toward the wall margins and continue along strike for up to 20 km.", "Folds change in profile upward from open and rounded in the upper part of A1 to tight and angular in A2.", "Layer A3 commonly forms an undeformed, tabular cap above folded older strata (e.g., Martin, Fig. 11A–B; Freddie, Fig. 11C; Liam, Fig. 11F).", "The major recumbent synclines typically occur on the northwestward-dipping limb of internal anticlines (see Section 6.1) (Fig. 11).", "The axial surfaces of the recumbent synclines are folded across the hinges of the internal anticlines (Fig. 11).", "The northwestern margins of complex walls are either defined by northwestward-dipping panels of undeformed layered evaporites (A2, A3, and, locally, A4; Fig. 11E and G) or open, NW-facing, recumbent-to-slightly-overturned synclines that are deveoped along strike of and locally disrupt the undeformed panels (e.g., Freddie, Fig. 11C).", "In some cases, where the walls narrow at their ends or centres, the syncline on the northwestern wall margin is the same size as that on the southeastern margin (e.g., central part of Martin, Figs. 9A and 11A; southern part of Liam, Figs. 9B and 11F).", "The smaller, moderately-inclined-to-recumbent synclines have limited length (<5 km).", "They trend parallel to and face outward toward the wall margin, and are commonly separated from neighbouring folds by zones of extremely poor reflectivity (Fig. 9A, B and C).", "In some cases their hinges, which may be strongly curved in map-view (e.g., Martin, Fig. 9A), are oblique to the wall axis (e.g., Jimi, Fig. 9C).", "Synclines of this type may overlap in map view (Fig. 9A–C) and be vertically stacked in cross section (Fig. 11B and F), seemingly by horizontal transport in the hanging wall of intrasalt shear zones that also strike parallel to poorly reflective subvertical zones (see Section 6.5).", "Poorly reflective, chaotic, sheetlike bodies dominate the upper parts of the walls and cap the synclines and internal anticlines described above (Figs. 9 and 11).", "The sheetlike bodies are up to 1200 m thick, 5 km wide, and 20 km long and typically fill the entire upper parts of the wall.", "The basal contact between the sheetlike bodies and the underlying folds is subhorizontal or dips in toward the wall centre, climbing to structurally higher levels toward the salt-wall contact (e.g., eastern margin of Liam, Fig. 11F; eastern margin of Martin, Fig. 11A–B; eastern margin of Freddie, Fig. 11C).", "The surface can be diffuse relatively sharp, cross-cutting underlying A2 reflections.", "We interpret that this angular relationship represents highly strained and/or steeply dipping A2 that is poorly imaged immediately below the sheet-fold contact.", "The top of the sheet typically coincides with the top-salt contact (Fig. 11).", "The sheetlike bodies are underlain by and attached to subvertical, weakly reflective zones that connect the sheets downward to autochthonous lower-A1 at the base of the wall (Fig. 11).", "These inclined zones are 0.25–2 km wide, up to 20 km long and 1500 m tall.", "They are almost invariably thrusted, as shown in map view (Fig. 9) and cross section (Fig. 11B, C, E, and F) (Section 6.5).", "Where the weakly reflective zone is thrusted, it dips less steeply (<25°) than where these thrusts are absent, and is typically associated with recumbent folds that overlap in map view (Fig. 9).", "Where a steep zone separates oppositely facing recumbent synclines, one of which is much larger than the other, the zone is located off-centre of the wall's long axis (e.g., Fig. 9A–E; 11C–G).", "However, where multiple subvertical zones are present, this pattern is less obvious, and the zones may show no preferential location with respect to the wall margins (Fig. 9C).", "Upright, symmetrical folds are present near the base of several walls.", "These folds typically trend NW, are up to 5 km long (Fig. 9), have amplitudes of up to 250 m, and have moderately dipping (<20°) limbs (Fig. 12).", "They incorporate layered evaporites of upper-A1 and the lower part of A2 (Fig. 12A) or A1 only (Fig. 12B).", "The folds do not appear to deform the base of the poorly reflective sheetlike bodies that dominate the upper parts of the walls (Fig. 12), although this may reflect poor imaging of the basal sheet contact or modification of the primary stratigraphic relationship by syn- or post-emplacement flow of underlying and overlying evaporites.", "These folds are different from the internal anticlines described in Section 6.1 in that they: (i) typically occur in the wall centres, rather than at the wall lateral margins or ends (Fig. 9); (ii) are smaller in length (<5 km; Fig. 9), amplitude (<250 m), and wavelength (<2.5 km; Fig. 9); and (iii) occur in closely spaced (hinge-to-hinge distance <2.5 km; Figs. 9 and 12) syncline–anticline pairs.", "Reverse-sense shear zones are common in complex walls.", "Most intrasalt shear zones trend N-to-NE, parallel to the long axis of their containing walls (Fig. 9).", "They dip gently to the W or NW (up to 25°), flattening and detaching downward onto the base of salt; the sense of shear is thus top-to-E or -SE, either being wholly contained within A1 (Fig. 13A) or thrusting upper-A1 over layered evaporites of A2 (Figs. 9 and 13B).", "We recognise three types of intrasalt shear zone.", "The first type is up to 5 km long, 1500 m tall, and has up to 2.5 km of displacement.", "These shear zones are relatively closely spaced (<2 km; Fig. 9D) and bound slices of upper-A1 evaporites (Fig. 13A).", "Their upper tips are located within A1 (Fig. 13A) or A2 (Fig. 13B).", "The second type of shear zone occur where intrasalt sheetlike bodies are absent, typically toward the ends of the walls.", "These shear zones are longer (up to 10 km), taller (up to 2200 m), and have more displacement (up to 1150 m) than the first type; they extend up to the A2–A3 contact (e.g. Liam, Fig. 13B).", "These shear zones may offset the limbs of the internal anticlines, although the anticline limbs remain broadly concordant to the salt contact (Fig. 13B).", "The third type of shear zone is somewhat cryptic and occurs within the intrasalt feeders (see below).", "These shear zones are recognised based on the observation that the hinge of one of the feeder-flanking recumbent synclines is elevated up to 700 m above the opposing fold (Fig. 11B and F).", "The close spatial relationship between shear zones and feeders means that many intrasalt shear zones trend highly oblique to shear zones elsewhere within the walls, or to thrusts in flanking minibasins (Fig. 9B, C, and E).", "Examples of obliquity of up to 90° occur at the northern end of Liam (Fig. 9B) and in Jimi (Fig. 9C).", "In contrast, where a wall's long axis and feeder trend NE, such as in Freddie, the intrasalt shear zone also trends NE, parallel to structures in adjacent minibasins (Fig. 9D) and to most other salt-related shortening structures elsewhere on the São Paulo Plateau (e.g., the Inner and Outer São Paulo fold belts; Fig. 9).", "Outside the main walls the thrusts lose displacement and core salt anticlines (eastern end of Fig. 10B; southeastern end of Fig. 11C), and are typically overlain by SE-verging folds across which Upper Cretaceous strata and especially upper Palaeocene strata thin and onlap (fold–thrust pair at southwest end of Fig. 8C).", "Upper Cretaceous strata also change in thickness across folds in the Inner (Fig. 8A) and Outer (Fig. 8B) fold belts, suggesting that structures within these domains are of broadly similar age (Late Cretaceous-to-Palaeocene) to shortening-related structures in the zone of complex diapirs.", "Before inferring the origin of intrasalt structures on the São Paulo Plateau, we must first interpret their kinematics.", "We start with diapirs having relatively simple internal anticlines, which are ubiquitous across the study area, then focus on the more enigmatic, complex diapirs locally developed on the southern margin of the São Paulo Plateau.", "Internal anticlines defined by variably deformed but preserved layering, characterise the majority of salt structures on the São Paulo Plateau and occur in two locations: (i) in simple salt-cored anticlines (Fig. 8; eastern structures on Figs. 10B and 11C) along the entire lengths of simple walls (Fig. 7), and (ii) at the ends of complex walls (Fig. 10A and western structure on Fig. 10B).", "Internal anticlines in salt-cored anticlines are typically cored by intrasalt shear zones.", "These salt-cored anticlines incorporate isopachous but folded Albian strata, are onlapped by Upper Cretaceous-to-Palaeocene strata, and formed in the Late Cretaceous-to-Palaeocene at the same time as the internal anticlines contained within them.", "Internal anticlines in the salt-cored anticlines formed by flow and thickening of mobile lower-A1, which boreholes reveal is halite-rich (average >88%; Table 1), whereas A2–A4 units are broadly isopachous across the fold hinges.", "Internal anticlines at the ends of complex walls, are typically not cored by intrasalt shear zones, and are flanked by Albian strata that may onlap onto or show normal fault-induced structural thinning towards the diapir flanks.", "Stratigraphic relationships adjacent to their host walls, which we will discuss more fully below in Section 8.1, indicate that the internal anticlines formed during the Albian-to-Late Cretaceous.", "Like those outside complex walls, internal anticlines at the complex wall ends formed by inward flow and thickening of lower-A1 mobile halite, in this case expelled from beneath flanking minibasins.", "A2–A4 units are broadly isopachous across the fold hinges, although local thickness changes in A2 and A3 on the anticline limbs may reflect preferential expulsion of halite in these layers as the walls grew (Kupfer, 1968; Wagner and Jackson, 2011; Jackson et al., 2014b).", "These diapirs could have grown reactively or passively, as discussed below in Section 8.1.", "Our interpretation of the origin of the complex intrasalt structures is based on two key features: (i) poorly reflective, chaotic, sheetlike bodies and poorly reflective subvertical zones; and (ii) moderately inclined-to-recumbent synclines.", "Having considered these, we then consider the origin of the upright symmetrical folds and reverse shear zones.", "Based on analysis of depth-migrated 2D seismic reflection data, Davison et al. (2012) interpret that the poorly reflective intrasalt bodies are allochthonous salt sheets and canopies, sourced from A1 and extruded during Aptian salt deposition (see their Fig. 10).", "Likewise Gamboa et al. (2008) also identify sheetlike, A1-sourced bodies capping strongly deformed layered evaporites, attributing these bodies to syndepositional Aptian flow of A1 halite.", "However, they infer that the sheets are not related to Aptian extrusion but to seaward shearing of relatively upright salt diapirs during basinward translation of the overburden linked to Late Cretaceous gravity spreading (Fig. 16.20 in Gamboa et al., 2008).", "In contrast Fiduk and Rowan (2012) suggest that the poorly reflective bodies are recumbent, near-isoclinal folds in which strata were extensively disrupted and therefore poorly imaged.", "They see no evidence for emplacement of allochthonous salt bodies, extrusive or otherwise.", "Because the poorly reflective intrasalt bodies are not penetrated by boreholes, their composition and origin are speculative.", "However, although high strains are eminently capable of disrupting stiff interbeds in an evaporite sequence, we see no reason why these upper poorly reflective areas would be seismically transparent, whereas strata in underlying folds, which are of similar size, shape and attitude, are highly reflective.", "Instead, we infer that the poorly reflective bodies are intrasalt sheets and canopies, based on the following evidence.", "First, the bodies are geometrically and geophysically similar to sheets and canopies described elsewhere from seismic reflection data (e.g., Jackson et al., 1990; Hudec and Jackson, 2006).", "Second, the bodies are physically linked to A1 halite via narrow, subvertical, poorly reflective zones interpreted as feeders (Davison et al., 2012; Jackson et al., 2014).", "Third, the bodies are above overturned flaps, which we relate to breakout of intrasalt allochthons (see Section 7.2.2) by analogy with flaps adjacent to salt sheets described elsewhere, where they are typically confined within overburden.", "Fourth, seismic evidence that lower-A1 was preferentially expelled from beneath minibasins points to flow of this unit as salt structures grew.", "We are thus in broad agreement with Gamboa et al. (2008) and Davison et al. (2012) that the poorly reflective bodies are largely composed of A1 halite.", "Furthermore, we concur with Davison et al. (2012) that these intrasalt bodies consist of allochthonous salt.", "However, as we argue in Section 8.4, we interpret that these sheets and canopies were emplaced during growth of the salt diapirs rather than during salt deposition.", "The near-isoclinal, moderately inclined-to-recumbent synclines are found in all the complex salt walls (Fig. 9).", "These synclines always underlie the allochthonous salt sheets and canopies, and their hinges are flanked by steeper intrasalt feeders (Figs. 9 and 11).", "Thus we interpret these synclines to be overturned flaps related to the rise and breakout of an intrasalt allochthonous sheet of lower-A1.", "These structures are geometrically comparable, if not scale equivalent to, overturned flaps in subsalt strata below allochthonous salt bodies in the Gulf of Mexico (Hudec and Jackson, 2006, 2009), Yemen (Davison et al., 1996), France (Graham et al., 2012), Germany (Schachl, 1987), Mexico (Giles and Lawton, 2002; Rowan et al., 2003), Australia (Kernan et al., 2012; Hearon et al., 2015) and Israel (Alsop et al., 2015), as well as to salt sheets generated in physical models (e.g., Dooley et al., 2009, 2015a).", "The flaps in the Santos Basin differ from these natural and model examples in that they are found within diapiric salt rather than within flanking country rock.", "The upright symmetrical folds have no consistent trend or spatial relationship with the wall orientation or the trend of the internal upright anticlines related to initial diapir inflation.", "Furthermore, the age of these folds is uncertain.", "For these reasons, the origin of the intrasalt upright symmetrical folds is uncertain.", "Possible origins are: (i) simple buoyancy-driven intrasalt diapirism during growth of the larger diapirs containing them; or (ii) buckling in response to regional shortening within the salt.", "In contrast to the upright symmetrical folds, the reverse sense shear zones provide unequivocal evidence for shortening.", "Local continuation of these shear zones into flanking minibasin strata indicates that most but not all of the shortening was accommodated by diapir squeezing.", "We further explore the timing of shear-zone formation and regional shortening in Section 8.", "Before interpreting the origin and kinematics of the complex intrasalt structures, we first discuss the origin of the internal anticlines, which form the dominant intrasalt structures on the São Paulo Plateau.", "Salt flow to form internal anticlines could be due to reactive or passive diapirism, shortening, or a combination of these mechanisms.", "We can assess the relative roles of these mechanisms by considering overburden geometry and thickness, and its seismic–stratigraphic relationship with underlying salt.", "Many of the complex diapirs are overlain by broadly isopachous Albian overburden that drapes the diapir flank and which is deformed by an array of inward-dipping normal faults near the crest (e.g. western diapir in Fig. 10A; western margin of diapir in Fig. 11D; eastern margin of diapir in Fig. 11C; see also Fig. 13A).", "Some of these salt walls have triangular profiles at their low-relief ends (Fig. 10A).", "These geometries are consistant with salt flow triggered by reactive diapirism (Vendeville and Jackson, 1992).", "Reactive diapirism, triggered by broadly dip-parallel (WNW-ESE-to-NW-SE) extension of overburden, could have formed the broadly NE-SW-elongate walls that prevail in the central and eastern Sao Paulo Plataeu (Fig. 5).", "In contrast, some diapirs, particularly those outside the domain of complex diapirs, have Albian (and younger) strata that progressively thin towards and onlap as diapir flanks (Fig. 7B); normal faults are absent.", "These geometries are consistant with passive rather than reactive diapirism (Nelson, 1989; Vendeville and Jackson, 1982; Hudec and Jackson, 2007).", "However, a thorough analysis of triggering mechanisms is beyond the scope of this paper.", "Whatever the trigger, flow of salt formed diapirs and thinned salt beneath flanking minibasins.", "Almost all previous studies infer that, on the distal São Paulo Plateau, salt tectonics in general and intrasalt deformation in particular resulted from regional shortening of seaward-flowing salt during the Late Cretaceous.", "For example, Cobbold et al. (1995) use 2D seismic reflection profiles to argue that the following features are diagnostic of shortening in the Santos Basin: (i) salt tongues in reverse shear zones; (ii) triangular depocentres (in cross section) bound by asymmetrical salt diapirs or basinward-verging salt tongues; (iii) angular and concentric folds in layered evaporites (see A2–4, Fig. 8); and (iv) progressive upward increase in fold wavelength in overburden.", "Furthermore, strain partitioning shows that poorly reflective lower evaporites, equivalent our A1, appear to have flowed into the cores of folds, whereas overlying layered evaporites, equivalent to our A2–4, are broadly isopachous (cf. Figs. 7 and 10), suggesting that salt flow and related deformation occurred after salt deposition (i.e., post-Aptian, in contrast to Gamboa et al., 2008; Davison et al., 2012).", "Guerra and Underhill (2012) use 2D and 3D seismic reflection data to expand the concepts of Cobbold et al. (1995), arguing that intrasalt deformation on the São Paulo Plateau resulted from downdip salt flow and regional shortening caused by thin-skinned gliding and/or spreading within a kinematically linked system they called the “Ilha Grande Gravitational Cell” (Fig. 2A).", "They suggest that the complex pattern of salt structures and minibasins is related to diachronous, gravity-spreading, noncoaxial shortening caused by a switch in sediment input and the direction of sedimentary topographic loading (sensu Hudec et al., 2009).", "Based on their 3D seismic reflection–based study on the southern São Paulo Plateau, Fiduk and Rowan (2012) conclude that intrasalt deformation was primarily caused by basinward flow and bulk shortening of the evaporites, although they recognised it is difficult to determine if this occurred by thin-skinned extension (Cobbold and Szatmari, 1991; Demercian et al., 1993; Cobbold et al., 1995; Guerra and Underhill, 2012), sedimentary topographic loading (Szatmari et al., 1996; Ge et al., 1997; Gemmer et al., 2005), or salt drainage (Davison et al., 2012; Quirk et al., 2012).", "Fiduk and Rowan (2012) suggest that the dominance of noncoaxial deformation patterns within and between structures, may reflect convergent gliding or spreading of the overburden (first inferred by Cobbold and Szatmari, 1991; Demercian et al., 1993), perhaps augmented by a change in the direction of shortening (see also Guerra and Underhill, 2012).", "One or more of these processes could have formed transpressional structures having curvilinear fold shapes, especially if salt flowed in different directions at different rates.", "Davison et al. (2012) and Quirk et al. (2012) also invoke regional shortening as the trigger for intrasalt deformation on the São Paulo Plateau.", "However, unlike the above authors, they interpret that the salt sheets, canopies, and minibasins formed during salt deposition as salt drained seawards before the overburden was deposited.", "Regional intrasalt shortening can indeed explain the growth of several types of structure on the São Paulo Plateau and this is most clearly expressed in the form of Late Cretaceous-to-Palaeocene salt-cored anticlines (e.g. Figs. 5 and 8).", "These contractional structures occur in two areas; at the eastern and western margins of the São Paulo Plateau (i.e. the Inner and Outer fold belts; Fig. 5B), and between complex walls on the south-central part of the plateau.", "Where developed in distinct fold belts, salt anticlines are up to 40 km long and invariably trend NE, normal to the overall south-eastward slope of the margin.", "The style and orientation of salt anticlines in the fold belts are thus entirely consistent with formation as part of a gravity-driven fold belt.", "In contrast, salt anticlines between the complex walls are relatively short (typically <10 km) and display highly variable trends, thus regional SE-directed shortening cannot readily account for the formation of these structures.", "Regional shortening cannot account for the formation of NW-trending salt walls either because: (i) they trend normal to the postulated shortening direction; and (ii) as argued above, these structures are diapiric and initiated during as passive or reactive structures during the Albian or Cenomanian, before witnessing protracted growth through the Late Cretaceous.", "We argue that convergent gravity gliding (Fiduk and Rowan, 2012) is implausible because the structures lie opposite a fairly straight part of the continental margin (Fig. 2A).", "In conclusion, although shortening undoubtedly played a role in the rise of at least some salt walls, the far-field causes, precise timing, and number of folding events is unresolved.", "In the next section we tackle this uncertainty and evaluate: (i) the timing of intrasalt deformation with respect to other structural and stratigraphic events; (ii) the distribution of salt walls that contain complex internal deformation versus those that do not; and (iii) the orientation of salt walls that contain complex internal deformation versus those that do not, and the orientation of structures within complex salt walls.", "Constraining the age of the intrasalt structures with respect to regional structural or stratigraphic events is difficult because most of the structures are contained entirely within salt and lack intrasalt synkinematic strata.", "However, at least two parts of the complex intrasalt assemblage can be dated.", "First, the internal anticlines are inherently the same age as the salt walls, which grew primarily during the Late Cretaceous-to-Palaeocene although, locally, as described above, earlier (Albian) passive growth is recorded by onlap of Albian strata onto their flanks of the walls.", "These anticlines are not part of the complex intrasalt assemblage but are along strike to this assemblage near the ends of the salt walls.", "Allochthonous salt bodies and overturned flaps dominate the high-relief wall centres, and simple inflation-related anticlines are preserved at the lower-relief wall ends (Jackson et al., 2014).", "This link between diapir relief and structural complexity could imply that the simple anticlines formed during early diapir inflation and that the more complex intrasalt anticlines formed later when the diapir had more significant relief.", "In this hypothesis the internal anticlines originally continued along the lengths of the walls, albeit overlain by intrasalt sheets at the wall culminations.", "If so, the complex assemblage would have formed after the internal anticlines, most likely during the Palaeocene.", "An alternative interpretation is that the simple and complex styles are laterally equivalent, rather than different stages of evolution, and thus are of broadly similar age.", "In either case, the complex assemblage of intrasalt structures formed no earlier than the Late Cretaceous.", "A second age criterion is that intrasalt shear zones offset the limbs of the internal anticlines within the diapirs and are thus the same age or younger than the folding (Fig. 13B).", "Where shear zones continue outside the walls into flanking minibasins, overlying growth folds are onlapped by Upper Cretaceous-to-Palaeocene strata, indicating that the intrasalt shear zones and possibly the anticlines, formed during the Late Cretaceous to Palaeocene (Fig. 8) (Jackson et al., 2014).", "The above observations suggest that most salt walls and the shear zones that dissect their internal structures formed during the Late Cretaceous-to-Palaeocene although earlier (Albian) passive growth is locally recorded.", "If these ages are correct, these structures are too young to be the downdip contractional equivalent of the thin-skinned, Albian extensional structures along the basin margin.", "Only if the Albian Gap formed by post-Albian extension could it drive contractional growth of salt walls in our study area (as advocated by Cobbold et al., 1995; Fiduk and Rowan, 2012; Guerra and Underhill, 2012; Quirk et al., 2012).", "Complex structures are developed in only a few diapirs on the São Paulo Plateau.", "Furthermore, these diapirs are located in a relatively small area (Fig. 5B).", "If regional shortening occurred it is unlikely that only a few diapirs, located in the midst of an array of structurally simple diapirs, would be squeezed to a greater degree, although such a scenario could conceivably be explained by inferring special conditions, such as steps in the base of salt or changes in topographic slope during shortening.", "The complex diapirs are located in a domain of relatively low shortening strain, being flanked landward and seaward by the Inner and Outer São Paulo fold belts, where shortening is greater (Fig. 5B).", "Significantly, the shape and dimension of the complex diapirs are similar to diapirs that lack complex internal structures, suggesting they have not been preferentially squeezed (compare diapirs in Fig. 5A–C with those in Figs. 7 and 11) and that shortening alone is not a valid explanation for the complex structures.", "Salt walls characterised by complex internal deformation have trends (e.g., N-to-NE) similar to those having simple internal anticlines (Fig. 5B).", "This suggests that intrasalt deformation was not a function of wall orientation with respect to any postulated shortening direction.", "Regional shortening, driven by broadly SE-directed gliding or spreading, should lead to coaxial deformation and formation of broadly NE-trending salt and intrasalt structures.", "However, intrasalt deformation is strongly noncoaxial, as shown by the presence of near-isoclinal, recumbent folds that have different trends and curved hinges (cf. Fiduk and Rowan, 2012).", "Strongly non-coxial deformation could have been driven by a change in the direction of sediment supply, salt loading and spreading during the early Palaeogene (Modica and Brush, 2004; Guerra and Underhill, 2012), although this does not explain why structures landward of the complex diapirs were “bypassed” and are not internally deformed.", "Although shortening-related squeezing of mature diapirs can lead to high levels of intrasalt strain (e.g., Dooley et al., 2009), passive diapirism in the absence of regional shortening can also result in large-magnitude, three-dimensionally complex strains (Talbot and Jackson, 1987; Jackson and Talbot, 1989; Geluk, 1998; Brun and Mauduit, 2009; Van Gent et al., 2011; Strozyk et al., 2012).", "We thus propose a new hypothesis for the origin of complex intrasalt structures on the São Paulo Plateau, which envisages Rayleigh–Taylor density-driven overturn within the evaporite sequence during diapir growth.", "We discuss this process using the same criteria with which we critiqued the role of shortening in Section 8.2.", "We argued above that Albian thin-skinned extension in the proximal domain occurred before most of the complex intrasalt deformation on the distal part of the São Paulo Plateau indicating that kinematically linked regional shortening of this age cannot have triggered the observed deformation.", "For the Rayleigh–Taylor overturn model outlined below, however, intrasalt deformation need not have coincided with any external deformational events.", "We note that the complex diapirs are located where a density inversion is now present within the source salt layer.", "Borehole data indicate that intrasalt units A2, A3, and A4 are collectively denser than A1, principally because of the presence of relatively thick (50–235 m), dense anhydrite layers in A2 (Fig. 6 and Table 1) (see also Jackson et al., 2014; Dooley et al. this volume).", "Elsewhere on the São Paulo Plateau, this density inversion is not observed.", "This argues that the existence of the complex intrasalt deformation is a function of density structure of the salt layer, rather than a product of some complex, polyphase, far-field deformation events.", "Based on our interpretation of the kinematic and temporal link between the various intrasalt structures, and the spatial relationship of the complex diapirs to the density inversion within the autochthonous salt layer, we propose the following five-stage evolution (Fig. 14) to explain the formation and growth of both the simple and complex diapirs:", "Stage 1—Initial diapir growth was triggered by cover extension (reactive diapirism) or differential sedimentary loading (passive diapirism).", "Irrespective of the cause, initial growth resulted in the formation of an upright internal anticline, which is the fundamental structural style associated with the growth of a salt wall, simply reflecting inward salt flow and wall amplification.", "Lower-A1 halite flowed into the growing structure, with layered evaporites of A2–A4 being folded, stretched and thinned at the diapir crest (Fig. 14B).", "Stage 2—Diapirism continued and, after the diapir had reached a critical height, a series of tensile fractures began to develop in brittle, anhydrite-rich layers (i.e., upper-A1–A3) either at the crest or, as shown in Fig. 11B, on one limb of the internal anticline.", "Eventually, these fractures linked to form a throughgoing network that hydraulically connected lower-A1 halite and halite-rich and potash-rich deposits in A3 and A4.", "Loading of buoyant, mobile lower-A1 halite by denser, more anhydrite-rich, A2–A4 layered evaporites caused the lower-A1 halite to ascend through the fracture network into the upper part of the diapir.", "The position of the fractured breach dictated the feeder location and degree of asymmetry of the overlying sheet.", "For example, where the breach was at the diapir crest, the feeder was located centrally and supplied a symmetrical sheet (e.g. Freddie; Fig. 11C), whereas a breach on the diapir limb was located away from the wall axis and supplied a strongly asymmetrical sheet (e.g. Jimi, Liam and Martin; Figs. 11A and D-G; Fig. 11B).", "Stage 3—Buoyancy drove mobile A1 halite through the dismembered higher levels of evaporites, and an allochthonous salt sheet (where one discrete feeder formed, e.g., Liam) or canopy (having multiple feeders, e.g., Axl, Freddie, Jimi, and Martin) was forcibly intruded into the upper part of the wall; breakout of the lower-A1–sourced sheets above A2 resulted in near-isoclinal folding of upper-A1, A2, and A3 directly beneath the feeder–sheet transition.", "We speculate that sheet emplacement was facilitated by lower-A1 halite forcing aside mobile halite- and potash-rich units in A3 and A4.", "Stage 4—Passive diapirism, ascent of mobile lower-A1 halite, and growth of the allochthonous salt body occurred.", "The flow of lower-A1 halite into the shallow-level allochthonous body depleted the core of the ancestral upright internal anticline, causing it to founder and be left as a relic inside the wall.", "This interpretation implies that the larger, upright, subsheet anticlines were originally connected to the internal anticlines at the wall ends.", "Subsequent deformation linked to sheet emplacement and fold foundering resulted in the ancestral internal anticline hinges having variable trends.", "Furthermore, we interpret that the largely undeformed panels of layered evaporites that extend up to the wall crest are relict limbs of the breached internal anticlines that subsequently foundered towards the wall base.", "Stage 5— Regional shortening resulted in diapir squeezing; this does not preclude earlier episodes of shortening, but it was during our Stage 5 that the imprint of shortening is clearest.", "Upright anticlines in structurally simple diapirs were tightened and, locally, intrasalt shear zones dissected their limbs.", "Owing to the abundance of superposed deformation, the expression of shortening is more cryptic in the centre of complex walls.", "The major fold belts along the seaward periphery of the São Paulo Plateau, generally trend NE, suggesting they formed in response to SE-directed shortening.", "However, the variable trends of the intrasalt shear zones and intracover thrusts, especially within and between closely spaced diapirs in the south-central part of the plateau, suggest causal stresses were locally reoriented and focused onto the diapirs, which were weaker than sediments in flanking minibasins.", "Diapir squeezing was focused on the intrasalt feeders, an observation that has also been made in physical models, where squeezed feeders supply salt to allochthonous salt extruded onto country rock (Dooley et al., 2015a).", "Squeezing of intrasalt feeders may have driven continued inflation of the lower-A1–fed sheet/canopy.", "Our new kinematic model, based on predominantly vertical salt tectonics, augmented by Rayleigh–Taylor density-driven overturn within the salt, can explain why although complex diapirs have trends similar to simple diapirs; their internal structures differ greatly because the former grew in the presence of a density inversion in the source layer.", "The model also explains why noncoaxial deformation is common within diapirs; this is the natural consequence of complex 3D salt flow driven by the fundamental process of passive diapirism augmented by density-driven Rayleigh–Taylor overturn.", "An obvious question is “How much of a density inversion is necessary to drive Rayleigh–Taylor instability?”.", "Because salt has effectively no yield strength, even the slightest density contrast allows it to flow.", "The strength of the stiffer evaporite interbeds, which is related to their thickness and lithology, would impede Rayleigh–Taylor overturn as long as the interbeds remain intact.", "However, as shown by the numerical models of Albertz and Ings (2012), once a stiff layer is breached by boudinage in a rising intrasalt diapir, more-mobile salt can rise unimpeded through the breach.", "Another pertinent question is “to what extent the density inversion recorded by boreholes reflects the original density structure of the autochthonous salt sequence?”.", "Clearly the wide variations in thickness of the evaporite units point to extensive redistribution of the most mobile interbeds.", "More-mobile halite-rich A1 evaporites would be preferentially expelled into the growing salt structures (Kupfer, 1968; Wagner and Jackson, 2011; Albertz and Ings, 2012; Cartwright et al., 2012; Jackson et al., 2014b).", "Expulsion would reduce the density contrast within the sequence, so the Santos density contrast could have been higher in the past, thus making Rayleigh-Taylor overturn more likely in the past than the present, which supports the hypothesis presented here.", "In a companion paper, Dooley et al. (this volume) use physical models to explore the mechanical feasibility of our kinematic model, and to explain the temporal and spatial relationship between intrasalt structures.", "In particular, they illustrate the role that Rayleigh-Taylor density-driven overturn may play in the growth of salt walls having an intrasalt density inversion.", "They further assess the possible contribution that shortening had in forming complex intrasalt structures and how allochthonous salt could have been emplaced within salt walls, highlighting the role of intrasalt rheology.", "3D seismic reflection and borehole data from the São Paulo Plateau, Santos Basin, offshore Brazil, documents the variability in intrasalt structural style in salt walls.", "Detailed mapping of layered evaporites in the upper part of an autochthonous salt layer allowed us to provide a detailed, systematic description of the internal structure of numerous salt walls, leading to the following conclusions.", "Simple, upright, internal anticlines are the most common intrasalt structure on the São Paulo Plateau, forming in response to the inward flow, thickening and rise of salt within diapirs.", "Salt was principally sourced from halite-rich evaporites in the basal part of the autochthonous layer (lower-A1).", "On a relatively small part of the south-central margin of the São Paulo Plateau, several diapirs contain a range of complex and spectacularly well-imaged intrasalt structures, including (i) poorly reflective, chaotic, sheetlike bodies in the top of the diapirs, which are underlain by and physically linked to poorly reflective, subvertical zones; (ii) moderately inclined-to-recumbent synclines below the sheetlike bodies, which are flanked by the subvertical zones; these folds face outward toward and trend parallel to the salt-wall contact; (iii) small upright folds at the base of the walls; and (iv) reverse shear zones.", "Simple internal anticlines also occur in this area, but only at the lateral, low-relief ends of the walls, passing along strike into more complex structural styles where wall relief is greatest.", "We interpret that these structures document three main processes: (i) rise of lower mobile halite (lower-A1) through an arched and structurally thinned roof of slightly denser layered evaporites (lower A1–A4), and emplacement of an intrasalt sheet or canopy (poorly reflective horizontal sheetlike bodies) fed by single or multiple feeders (poorly reflective subvertical zones); (ii) folding and formation of overturned synclinal flaps in the form of moderately inclined-to-recumbent synclines kinematically linked to salt breakout, and emplacement of intrasalt allochthonous bodies; and (iii) diapir squeezing, forming reverse shear zones and potentially small upright folds.", "Complex diapirs are restricted to an area that borehole data indicate is characterised by a density inversion within the autochthonous salt (upper, anhydrite-rich layered evaporites of lower-A1–A4 are collectively denser than lower, more halite-rich evaporites of lower-A1).", "Outside this area, across most of the São Paulo Plateau, there is no density inversion, and diapirs contain only simple internal anticlines.", "Most diapirs initially rose via growth of internal anticlines, but locally, where a density inversion existed in the autochthonous salt, Rayleigh-Taylor–driven density overturn occurred within the growing diapir.", "During this overturn, less-dense evaporites rose into the diapir crest and formed allochthonous bodies and breakout-related flaps.", "This conclusion is based on: (i) the along–strike transition from simple to complex structural styles, which appears to be linked to diapir relief and thus maturity; and (ii) the spatial relationship between complex diapirs and inverted density in the autochthonous salt", "Intrasalt and intra-minibasin reverse shear zones and buckle folds record late-stage regional shortening on the São Paulo Plateau.", "However, we conclude that shortening alone is unable to account for the timing, distribution, and style of most of the complex intrasalt structures.", "Because much of the intrasalt folding occurred during passive diapir rise, complex intrasalt structures may thus not be diagnostic of regional shortening and should be examined with caution when being used constrain structural restorations in salt basins.", "We have shown that 3D seismic reflection data provide far greater spatial sampling of salt bodies than do mines or typical field exposures and have revealed, arguably for the first time, the internal kinematics of a suite of large salt walls.", "Our kinematic model is based an analysis of salt walls in the Santos Basin, but it may be more widely applicable to other salt-bearing sedimentary basins.", "The internal structures documented here may be discovered elsewhere, and similar studies using 3D seismic reflection data could be profitably applied in other areas of thick, layered evaporites (e.g., Red Sea, Kwanza Basin, Central North Sea, Barents Sea).", "Uninterpreted versions of seismic profiles shown in (a) Fig. 7; (b) Fig. 8; (c) Fig. 10; (d) Figs. 11A–C; (e) Figs. 11D–G; (f) Fig. 12; and (g) Fig. 13.", "Line drawings illustrating intrasalt deformation; these are based on depth-converted, VE = 1:1 versions of seismic profiles shown in: (a) Fig. 7; (b) Fig. 8; (c) Fig. 10; (d) Figs. 11A–C; (e) Figs. 11D–G; (f) Fig. 12; and (g) Fig. 13.", "Depth conversion was based on the following interval velocities: (i) seawater = 1500 m s−1; (ii) overburden = 3000 m s−1; salt = 4500 m s−1." ]	[ "3D seismic data document internal structure of salt diapirs, offshore Brazil.", "Most diapirs characterised by simple upright anticlines.", "Some diapirs contain complex internal structures.", "Complex diapirs occur where a density inversion exists in autochthonous salt layer.", "Rayleigh–Taylor density-driven overturn appears a key driver for diapir growth." ]
Cyclophosphamide pharmacokinetics and pharmacogenetics in children with B-cell non-Hodgkin's lymphoma	Introduction Variation in cyclophosphamide pharmacokinetics and metabolism has been highlighted as a factor that may impact on clinical outcome in various tumour types. The current study in children with B-cell non-Hodgkin's lymphoma (NHL) was designed to corroborate previous findings in a large prospective study incorporating genotype for common polymorphisms known to influence cyclophosphamide pharmacology. Methods A total of 644 plasma samples collected over a 5 year period, from 49 B-cell NHL patients a&18 years receiving cyclophosphamide (250 mg/m), were used to characterise a population pharmacokinetic model. Polymorphisms in genes including CYP2B6 and CYP2C19 were analysed. Results A two-compartment model provided the best fit of the population analysis. The mean cyclophosphamide clearance value following dose 1 was significantly lower than following dose 5 (1.83 1.07 versus 3.68 1.43 L/h/m, respectively; mean standard deviation from empirical Bayes estimates; P < 0.001). The presence of at least one CYP2B6∗6 variant allele was associated with a lower cyclophosphamide clearance following both dose 1 (1.54 0.11 L/h/m versus 2.20 0.31 L/h/m, P = 0.033) and dose 5 (3.12 0.17 L/h/m versus 4.35 0.37 L/h/m, P = 0.0028), as compared to homozygous wild-type patients. No pharmacokinetic parameters investigated were shown to have a significant influence on progression free survival. Conclusion The results do not support previous findings of a link between cyclophosphamide pharmacokinetics or metabolism and disease recurrence in childhood B-cell NHL. While CYP2B6 genotype was shown to influence pharmacokinetics, there was no clear impact on clinical outcome.	[ "BIOC", "MEDI" ]	[ "B-cell NHL", "Chemotherapy", "Cyclophosphamide", "Paediatrics", "Pharmacogenetics", "Pharmacokinetics" ]	[ "1306", "2730" ]	[ "The oxazaphosphorine alkylating agent cyclophosphamide is used across a wide range of tumour types in childhood cancer [1,2].", "In order for cyclophosphamide to exert its antitumour activity, the prodrug requires metabolic activation by hepatic cytochrome P-450 (CYP) enzymes to generate active alkylating species [3,4].", "Key enzymes involved in the initial metabolic step to form 4-hydroxycyclophosphamide, include CYP2B6, CYP2C19 and CYP3A4, expression of which can vary markedly between individuals [5].", "The active metabolite 4-hydroxycyclophosphamide exists in equilibrium with its tautomeric form, aldophosphamide, with these metabolites transported to tumour cells via the systemic circulation [6].", "Further spontaneous breakdown is required to form the active DNA-crosslinking metabolite, phosphoramide mustard, with the release of the urotoxic metabolite, acrolein [7].", "A number of inactivating metabolic pathways may also play a role in determining cyclophosphamide efficacy, leading to the formation of 4-ketocyclophosphamide (KetoCP), dechloroethylcyclophosphamide (DCCP) and carboxyphosphamide (CXCP) [8].", "The overall metabolism of cyclophosphamide is therefore a complex process, involving numerous enzymes which may vary in expression and activity in cancer patients.", "Variation in the metabolism of cyclophosphamide between individuals has been highlighted as a factor that may impact on clinical outcome, in terms of both response and toxicity, in tumour types including breast cancer in adults and non-Hodgkin's lymphoma (NHL) in children.", "A study carried out in the United Kingdom (UK) by Yule et al., previously indicated that lower clearance of cyclophosphamide to its active metabolites was associated with an increased risk of disease recurrence in paediatric B-cell NHL patients.", "In addition, likelihood of disease recurrence was positively related to a higher formation of inactive metabolites following cyclophosphamide administration [9].", "A number of enzymes involved in the metabolism of cyclophosphamide, including CYPs, UGT and GST enzymes, may exhibit variable expression and activity between patients which could impact on cyclophosphamide metabolism and/or clinical response and toxicity [10].", "Of particular note, CYP2B6 and CYP2C19 genotype have previously been shown to influence cyclophosphamide pharmacokinetics and activation, in terms of half-life of the parent drug, in breast cancer patients [11,12].", "The current study in children with B-cell NHL was designed to corroborate the findings of Yule et al. [9], in a larger prospective study incorporating genotyping for the common polymorphisms in genes known to influence the pharmacology of cyclophosphamide.", "While high cure rates are commonly seen in B-cell NHL with the use of cyclophosphamide-containing regimens [13], it is important to investigate factors that influence response rates and incidence of toxicity to further improve outcome in childhood cancer.", "Patients 18 years or younger, receiving cyclophosphamide as part of their standard clinical treatment for B-cell NHL, were eligible to participate in the trial.", "The study was approved by the UK Trent Multicentre Research Ethics Committee and registered through the appropriate clinical trial registries (PK 2005 02 – REC 04/MRE04/68; CTA: 17136/0243/001; EUDRACT: 2004-003731-31) prior to patient recruitment.", "Participating centres obtained written informed consent, either from patients or parents as appropriate, for all patients entered onto the study.", "Patients were required to have central venous access, in the form of double lumen central venous catheters, in order to participate in this pharmacokinetic study.", "Baseline toxicity data prior to cyclophosphamide treatment, including baseline haemoglobin, white blood cell and platelet counts, were obtained from patients' notes and details of concomitant medications prior to and/or in combination with cyclophosphamide were recorded.", "Additional patient characteristics and clinical parameters including glomerular filtration rate, serum creatinine, alanine transaminase (ALT) and bilirubin measurements were also collected for post-study analysis.", "Cyclophosphamide (250 mg/m2) was administered as a 15 min infusion twice daily on days 2, 3 and 4 of treatment (six doses in total) as part of the COPADM regimen.", "This consisted of cyclophosphamide (1.5 g/m2 total dose as described, with hydration continued at 3000 ml/m2/day until 12 h after the final dose), vincristine (2.0 mg/m2; bolus intravenous infusion on day 1), prednisolone (60 mg/m2/day, days 1–7), doxorubicin (60 mg/m2, 1–6 h infusion on day 2), high dose methotrexate (3 g/m2, intravenous infusion over 3 h on day 1), folinic acid (15 mg/m2 orally every 6 h, beginning at 24 h from the start of methotrexate as required) and intrathecal methotrexate/hydrocortisone (8–15 mg on days 2 and 6).", "Toxicity following cyclophosphamide treatment was assessed by the National Cancer Institute Common Toxicity Criteria version 2.0.", "Progression free survival data were obtained from 6 monthly follow-up visits to the centre where treatment was undertaken.", "Blood samples (2.5 ml) for pharmacokinetic analysis were obtained from a central line prior to administration of the first dose of cyclophosphamide on day 2, at the end of infusion and at 1, 2, 4, 6 and 12 h after the start of infusion.", "Additional samples were obtained prior to administration of the first dose of cyclophosphamide on day 4 of treatment (dose 5), at the end of infusion and at 1, 2, 4, 6 and 12 h after the start of infusion of dose 5.", "All samples were taken from a different lumen from that used for drug administration following a standardised procedure.", "Plasma was separated from whole blood samples by centrifugation (1200g, 4 °C, 10 min) and stored at −20 °C prior to analysis.", "Samples were sent by overnight courier, on dry ice and in an insulated container, to the Northern Institute for Cancer Research, Newcastle University.", "Concentrations of cyclophosphamide and its stable inactivated metabolites, KetoCP, DCCP and CXCP were measured in plasma using a validated LC/MS method as previously described [14].", "Cyclophosphamide was obtained from Sigma (Poole, Dorset).", "The inactive metabolites and the internal standard deuterated cyclophosphamide (D4CP) were obtained from IIT (University of Bielefeld, Germany).", "The assay had a limit of quantification of 0.5 μg/ml for cyclophosphamide and 0.05 μg/ml for the metabolites and exhibited within- and between-run coefficients of variation and bias below 15%.", "QC samples for each analyte were included in each assay.", "Standard curves were linear between 0.5–10 μg/ml for cyclophosphamide and 0.05–1 μg/ml for CXCP, DCCP and KetoCP with r2 values ≥ 0.99.", "Samples containing concentrations of cyclophosphamide or metabolites above the linear range were diluted with blank plasma.", "A population pharmacokinetic model for cyclophosphamide was developed using nonlinear mixed effects modelling (NONMEM version 7.2), based on the enhancement of a model previously published by our group [14].", "The first order conditional estimation method with η/ɛ interaction was used, together with ADVAN1/TRANS2 or ADVAN3/TRANS4 as appropriate.", "A composite error model was most appropriate to describe within-subject error.", "An additive error model, on the logarithmic scale, was used for inter-individual variability in pharmacokinetic parameters.", "As cyclophosphamide concentrations were available from days 2 and 4 (doses 1 and 5) for each patient, additional error terms were included to account for inter-occasion variation (IOV) on clearance (CL) and volume of distribution in the central compartment (V1).", "The exact timing of the second dose administered on day 2 and of the doses administered on day 3 were not recorded.", "Given this unknown dosing history, to allow for non-zero cyclophosphamide concentrations prior to the start of the first cyclophosphamide infusion on day 4, a rate-controlled steady-state ‘infusion’ into the central compartment was assumed (terminating at the start of the current infusion).", "This has the effect of initialising all compartments with an appropriate amount of drug.", "The rate of the ‘infusion’ was allowed to vary across the population [15].", "Allometric scaling was used for all population pharmacokinetic parameters; the approach taken to this scaling was the same as that used in a previously published analysis [16].", "Changes in NONMEM objective function value (OFV), and examination of residual plots guided model structure development.", "Empirical Bayes estimates of pharmacokinetic parameters including CL, V1 and cyclophosphamide area under the plasma concentration-time curve (AUC) were obtained from the final population model.", "The covariates body weight, age, gender, ALT, bilirubin and creatinine, alongside genetic variation including CYP2B66 genotype were assessed for their relationship with CL.", "Plasma concentrations of the inactive metabolites CXCP, DCCP and KetoCP were determined on day 2 (dose 1) and day 4 (dose 5) of cyclophosphamide treatment.", "Calculation of metabolite AUCs from time 0–6 h was carried out using the trapezoidal rule on day 2 (dose 1) and day 4 (dose 5).", "Genomic DNA was obtained from whole blood samples using Qiagen QIAamp® DNA Blood Maxi kits according to the manufacturer's instructions.", "DNA purity and concentration were measured using a NanoDrop ND-1000 (Thermo Scientific, Rockford, United States of America [USA]) and stored at −20 °C prior to pharmacogenetic analysis.", "Genotyping for SNPs CYP2B65 1459C > T (rs3211371), CYP2B66 785A > G (rs2279343) & 516G > A (rs3745274), CYP2C192 681G > A (rs4244285), CYP2C1917-806C > T (rs12248560), GSTP12 313A > G (rs1695), CAR 540C > T (rs2307424) and PXR-25385C > T (rs3814055) were performed using TaqMan® probes and an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, California, USA) according to manufacturer's instructions.", "Allelic discrimination was performed using sequence detection software (Applied Biosystems).", "For the analysis of pharmacogenetic data, overall differences between groups were assessed with the Mann–Whitney and Kruskal–Wallis tests using GraphPad Prism version 5.0 software (GraphPad Software, Inc., San Diego, CA, USA).", "Analysis of linkage disequilibrium was performed using Fisher's exact test (two-sided) for general contingency tables with SPSS version 15.0 software (SPSS Inc., Chicago, IL, USA).", "Time to disease progression was calculated as the delay between the first day of cyclophosphamide treatment and the first observation of disease progression or death.", "Hazard ratios for disease progression were estimated for individual covariates using the univariate Cox proportional hazards regression model approach, with each model fitted separately.", "Potential prognostic factors were tested as continuous variables.", "Statistical significance was given for P values < 0.05.", "A total of 49 patients receiving cyclophosphamide as part of their standard clinical treatment for B-cell NHL were entered onto the study over a 5 year period.", "Patients were recruited from nine UK centres.", "The study population had a median age of 11.7 years (range 3.5–18.7) and included 42 male and seven female patients.", "Patient characteristics including ethnicity, age, sex and body weight are provided in Table 1.", "A population pharmacokinetic analysis using data collected from 48 patients on day 2 (dose 1) of cyclophosphamide treatment and 46 patients on day 4 (dose 5) of treatment was performed using NONMEM.", "Pharmacokinetic data were available following dose 1 and dose 5 from 45 patients, with a total of 644 plasma samples available for analysis.", "The best fit of the population analysis was obtained with a two compartment model including random effects on CL, V1, Q and V2 allowing for correlation between CL and V1.", "The model included IOV for CL and V1.", "Allometric scaling was used to allow for differences in body size; population parameters are therefore scaled to a standard body surface area of 1.4 m2.", "Mean (coefficient of variation) parameters were CL 2.2 (37%) L/h/1.4 m2; V1 18.4 (25%) L/1.4 m2; Q 5.2 (69%) L/h/1.4 m2 and V2 21.8 (19%) L/1.4 m2.", "Estimates of IOV were CL 18% and V1 21%.", "Estimates of the composite intra-subject error model were 0.1 μg/ml and 14% for the additive and multiplicative components respectively.", "There was a significant increase in CL on day 4 (OFV change of 116); on average CL increased by a factor of 2.1.", "No change was apparent for V1.", "The covariates body weight, age, gender, ALT and bilirubin were not observed to have a significant effect on cyclophosphamide pharmacokinetics.", "However, homozygous wild-type CYP2B6 patients were shown to have on average a 34% higher cyclophosphamide CL than patients with at least one variant allele (OFV change 7.7).", "Creatinine also had a relationship to CL (OFV change 10.6); as creatinine increased, CL tended to decrease.", "Table 2 provides a summary of empirical Bayes estimates of cyclophosphamide pharmacokinetic parameters obtained from the final population model following dose 1 and dose 5.", "The pharmacokinetic parameter estimates obtained were in general agreement with values previously reported in the literature in paediatric patient populations.", "Cyclophosphamide Cmax values were observed between 15 min and 120 min following dose 1, ranging from 9.8–114.6 μg/ml and between 15 min and 60 min following dose 5, ranging from 8.53–53.7 μg/ml.", "The mean cyclophosphamide CL following dose 5 was significantly greater than that following dose 1 (3.68 ± 1.43 versus 1.83 ± 1.07 L/h/m2, respectively; p < 0.001).", "This shift in cyclophosphamide CL between doses 1 and 5 resulted in a significantly lower cyclophosphamide AUC following dose 5 (10.3 ± 5.3 mg/ml.", "min for dose 1 versus 4.7 ± 1.7 mg/ml.", "min for dose 5, p < 0.001).", "The volume of distribution in the central compartment (V1) was comparable following doses 1 and 5, with values of 17.3 ± 8.2 and 17.4 ± 7.9 observed, respectively.", "Cyclophosphamide metabolites CXCP, DCCP and KetoCP could be quantified throughout the defined pharmacokinetic sampling period in all patients studied.", "A summary of cyclophosphamide metabolite AUC0–6h values observed is provided in Table 3, with marked inter-patient variability observed for all metabolites.", "Significantly greater AUC0–6h values were seen following dose 5 of cyclophosphamide treatment, as compared to dose 1, for all metabolites measured (CXCP – 198.9 ± 137.9 μg/ml.", "min for dose 5 versus 103.7 ± 60.9 μg/ml min for dose 1, p < 0.001; DCCP – 105.6 ± 60.9 μg/ml min versus 76.7 ± 49.6 μg/ml min, p = 0.008; KetoCP – 153.4 ± 61.3 μg/ml min versus 63.6 ± 27.5 μg/ml min, p < 0.001).", "The impact of pharmacogenetic variation on cyclophosphamide pharmacokinetics was investigated in all 49 patients studied, for whom both pharmacogenetic and pharmacokinetic data were available.", "Seven variant alleles of putative relevance for cyclophosphamide disposition were analysed.", "The allele frequencies for CYP2B65, CYP2B66, CYP2C192, CYP2C1917, GSTP12, CAR 540C > T and PXR-25385C > T were in accordance with those observed previously in Caucasian populations and were consistent with Hardy-Weinberg equilibrium.", "For the 49 patients studied, the number of individuals with each individual CYP genotype studied were as follows: 1/1 (40), 1/5 (7) and 5/5 (2) for CYP2B65; 1/1 (21), 1/6 (22) and 6/6 (6) for CYP2B66; 1/1 (34), 1/2 (12) and 2/2 (3) for CYP2C192 and 1/1 (37), 1/17 (10) and 17/17 (2) for CYP2C1917.", "Supplemental Table A.1 shows the individual genotype data and associated cyclophosphamide CL values for all patients studied.", "Relationships between cyclophosphamide CL following doses 1 and 5 of treatment and CXCP, DCCP and KetoCP AUC values and the studied genetic variants were investigated.", "A significant effect of CYP2B66 on cyclophosphamide CL was observed.", "The presence of at least one variant allele was associated with a lower cyclophosphamide CL following both dose 1 (1.54 ± 0.11 L/h/m2 versus 2.20 ± 0.31 L/h/m2, p = 0.033) and dose 5 (3.12 ± 0.17 L/h/m2 versus 4.35 ± 0.37 L/h/m2, p = 0.0028) of treatment, as compared to homozygous wild-type CYP2B6 patients.", "It should be noted that within the CYP2B61/1 patient group, there are a number of patients who are carriers of other CYP2B6 variant alleles and similarly, within the CYP2B66 heterozygote group there are several patients who may be carriers of the CYP2B67 variant allele (see Supplemental Table A.1 for individual patient genotype data).", "We have not attempted to further stratify the patients into additional CYP2B6 groups based on CYP2B6 genotype due to the limited number of patients involved.", "No other statistically significant influence on cyclophosphamide CL was found for any of the genetic variants investigated, including the previously reported effect of CYP2C1917, although there was a trend towards a lower cyclophosphamide CL in patients with at least one CYP2C1917 variant allele following both dose 1 (1.49 ± 0.19 L/h/m2 versus 1.94 ± 0.19 L/h/m2, p = 0.22) and dose 5 (3.12 ± 0.36 L/h/m2 versus 3.86 ± 0.25 L/h/m2, p = 0.13), as compared to homozygous wild-type CYP2C19 patients.", "Nor was there any effect of genetic variation on CXCP, DCCP or KetoCP AUC values observed following doses 1 or 5 of treatment.", "Relationships between genotype for CYP2B66, CYP2C1917 and CYP2C192 and cyclophosphamide CL following doses 1 and 5 of treatment are shown in Fig. 1.", "Of the patients studied, 38/49 (78%) were alive with no disease at follow-up.", "The median time of follow-up was 6 years, with a range of 1–9 years.", "Of the remaining patients, two (4%) were alive with disease progression and seven (14%) had died following disease relapse or due to treatment-related deaths.", "The remaining two patients were lost to follow-up.", "In the Cox proportional hazards regression model, cyclophosphamide CL values following doses 1 or 5 had no significant influence on progression free survival.", "Cyclophosphamide metabolite AUC0–6h values for patients alive with no disease and those who relapsed are listed in Table 3.", "DCCP AUC0–6h determined following dose 1 had a negative prognostic effect on progression free survival (p = 0.05).", "However, this result should be treated with caution, bearing in mind the borderline significance and the fact that a total of eight variables were investigated.", "DCCP AUC0–6h determined following dose 5 had no prognostic effect on progression free survival (p = 0.26).", "Fig. 2 shows Kaplan–Meier curves of progression free survival according to (A) cyclophosphamide CL following dose 1, (B) cyclophosphamide CL following dose 5, (C) DCCP AUC0–6h following dose 1 and (D) DCCP AUC0–6h following dose 5 of treatment, with patients stratified above and below the median values for each parameter.", "No other pharmacokinetic parameters investigated were shown to influence progression free survival.", "Similarly, there was no effect of genetic variation on clinical outcome for any of the genetic variants studied.", "Mean DCCP AUC0–6h values following doses 1 and 5 were 74% and 38% higher, respectively, in patients who relapsed as compared to patients who were alive with no disease at long-term follow-up (dose 1 − 122.0 ± 92.2 μg/ml min versus 70.1 ± 37.6 μg/ml min; dose 5 − 138.7 ± 86.8 μg/ml min versus 100.4 ± 55.6 μg/ml min).", "However, due to the level of inter-patient variability exhibited and the small number of patients in the relapsed group, these differences were not statistically significant.", "The metabolic elimination of cyclophosphamide has previously been identified as a factor which may influence exposure of both tumour and host tissues to active species and thus affect clinical outcome [5,8–12].", "Many of these studies have been in adult cancer patients across a number of different tumour types.", "One study in patients with childhood B-cell NHL found that cyclophosphamide CL and the extent of formation of inactive metabolites influenced the likelihood of disease relapse [9].", "The current study was designed to corroborate these results in a larger prospective study, and to incorporate an investigation of genetic variants thought to influence the pharmacology of cyclophosphamide.", "This allowed us to address the hypothesis that pharmacogenetic variability, mediated by an effect on cyclophosphamide metabolism and pharmacokinetics, impacts on response to treatment.", "A total of 49 children with B-cell NHL were recruited at nine UK centres over a 5 year period, with combined information generated on cyclophosphamide pharmacokinetics, metabolism, pharmacogenetics and clinical outcome.", "The length of time taken to complete the current study highlights the challenges faced when carrying out clinical pharmacology studies in a paediatric oncology setting, requiring the collection of serial blood samples from patients with a particular tumour type, receiving specific chemotherapeutics on a defined treatment protocol.", "The main aim of the study was to investigate the direct influence of pharmacogenetics on the pharmacokinetics and metabolism of cyclophosphamide, based on existing pharmacokinetic and clinical response data in a comparable patient population.", "In contrast to the Yule study, no correlation was observed between cyclophosphamide clearance following either dose 1 or dose 5 of treatment and patient outcome, with no difference in clearance observed in patients alive and disease-free at long-term follow-up, as compared to patients who had experienced disease relapse.", "While cyclophosphamide pharmacokinetics were shown to be influenced by CYP2B66 genotype, with the presence of at least one variant allele associated with a lower cyclophosphamide CL, the variations in cyclophosphamide CL observed had no apparent bearing on clinical response.", "No other effect of pharmacogenetic variation on cyclophosphamide pharmacokinetics or metabolite formation was observed.", "Previous studies have reported modest effects of both CYP2B6 and CYP2C19 genotype on the pharmacokinetics of anticancer drugs, including cyclophosphamide, with the impact being both dose dependent and potentially influenced by disease status [5,11,12,17–19].", "The extent of cyclophosphamide metabolism in the current study was comparable to data from previous publications in children, with significant increases in metabolite formation observed over several days of treatment due to the induction of CYP enzymes [14,20].", "Although DCCP AUC0–6h following dose 1 of treatment was associated with worse progression free survival, the level of significance and number of tests carried out warn against drawing firm conclusions from this result.", "Similarly, although DCCP AUC0–6h values following doses 1 and 5 of treatment were higher in patients who relapsed as compared to patients who were alive with no disease at long-term follow-up, these differences were not statistically significant.", "No other relationships between the extent of cyclophosphamide metabolism and clinical outcome were observed.", "Again, these data contrast with the previous findings of Yule et al. in a comparable patient population [9].", "Although a more sensitive LCMS assay was used in the current study, with a ten-fold lower limit of quantification for cyclophosphamide metabolites, this should not have influenced the ability to detect an underlying difference in metabolite levels between relapse and disease-free patients.", "Other disparities between the current study and the Yule et al. publication include differences in cyclophosphamide dose and patient gender and a high percentage of Burkitt's lymphoma patients in the current study.", "A lower dose of cyclophosphamide was administered to patients in the current study (250 mg/m2 versus 1000 mg/m2) and a lower percentage of females were included as compared to the previous study (14% versus 42%, respectively).", "Cyclophosphamide dose and gender have previously been reported to influence both pharmacokinetics and metabolism of the drug and therefore may explain the differences in findings between the two studies [20,21].", "In particular, the association between cyclophosphamide pharmacokinetics and clinical outcome in breast cancer patients was in the setting of high-dose chemotherapy (6000 mg/m2) [22].", "Based on the potential for saturation of metabolism at an increased dose of cyclophosphamide, it is certainly possible that the more frequent administration of lower drug doses in the current study may explain our findings.", "Beyond these factors the patient populations were comparable in terms of age ranges and other patient characteristics.", "In terms of data analysis the current study included the more robust use of a univariate Cox proportional hazards regression model to assess the impact of cyclophosphamide pharmacokinetic parameters and metabolite levels on clinical outcome, as opposed to the direct comparison of median values between relapse and remission patients in the previous study.", "The current study involved quantification of cyclophosphamide alongside its inactive metabolites, predominantly in an attempt to replicate the data generated by Yule et al. [9] and recognising the difficulty of accurately quantifying the unstable metabolites in a multi-centre study.", "While other investigators have suggested that CXCP (also known as CEPM) may be a marker of cyclophosphamide bioactivation [23–25], in the current study there was no link between CXCP plasma concentrations and clinical outcome.", "Interestingly, there were proportionally larger increases observed in CXCP and ketoCP AUC0–6h values between cyclophosphamide dose 1 and dose 5, as compared to the increase in DCCP AUC0–6h.", "Cyclophosphamide auto-induction models have previously been studied in some detail by Huitema et al. [26,27].", "In summary, the findings of the current study do not support a link between cyclophosphamide metabolism and recurrence of disease in childhood B-cell NHL with the cyclophosphamide dosing regimen described.", "While the influence of CYP2B6 genotype on cyclophosphamide pharmacokinetics was confirmed, there was no clear impact of pharmacogenetic variation on clinical outcome.", "This work was supported in part by Cancer Research UK, the North of England Children's Cancer Research Fund and the Experimental Cancer Medicine Centre Network.", "No funding bodies played a role in the study design, the collection, analysis or interpretation of data, the writing of the report or the decision to submit the article for publication.", "The authors have no financial relationships relevant to the work contained in this article to disclose and no other conflicts of interest to disclose." ]	[ "The influence of cyclophosphamide clinical pharmacology on childhood cancer outcome has been investigated", "The presence of at least one CYP2B6*6 variant allele was associated with a lower rate of cyclophosphamide clearance", "Pharmacokinetic parameters investigated were not shown to have a marked influence on clinical outcome", "Findings do not support a link between cyclophosphamide metabolism and disease recurrence in B-cell non-Hodgkin's lymphoma" ]
Using epigenetic networks for the analysis of movement associated with levodopa therapy for Parkinson's disease	Levodopa is a drug that is commonly used to treat movement disorders associated with Parkinson's disease. Its dosage requires careful monitoring, since the required amount changes over time, and excess dosage can lead to muscle spasms known as levodopa-induced dyskinesia. In this work, we investigate the potential for using epiNet, a novel artificial gene regulatory network, as a classifier for monitoring accelerometry time series data collected from patients undergoing levodopa therapy. We also consider how dynamical analysis of epiNet classifiers and their transitions between different states can highlight clinically useful information which is not available through more conventional data mining techniques. The results show that epiNet is capable of discriminating between different movement patterns which are indicative of either insufficient or excessive levodopa.	[ "BIOC", "MATH" ]	[ "Artificial gene regulatory networks", "Classification", "Epigenetics", "Parkinson's disease", "epiNet" ]	[ "1300", "2604", "2611", "2613" ]	[ "Since the beginnings of computer science, there have been many successful attempts at capturing biological models within a computational framework.", "This field, known as bio-inspired computation, has given rise to algorithms which have been shown to out-perform humans on real world tasks, for instance in object recognition (He et al., 2015).", "Indeed, many of the computational algorithms that are now state of the art are built upon bio-inspired principles (Oquab et al., 2014; Hinton et al., 2012; Yang et al., 2013; Zhou et al., 2011).", "The design of bio-inspired algorithms falls on a spectrum.", "At one end of this spectrum are attempts to capture as much detail from the biological system as possible, to best promote the possibility of emergent behaviour.", "At the other are intentionally minimalist approaches, using pared-back models that capture only an abstract representation of the biological system.", "Each of these approaches has advantages, but often the simpler models are functionally complex and capable of capturing real-world biological dynamics (Bull, 2013; Wang et al., 2012).", "In this work we use a novel bio-inspired architecture, termed epiNet, which is modelled upon the interactions between genetic and epigenetic processes within biological cells (Turner et al., 2015).", "Computational modelling of genetic processes, in the form of artificial gene regulatory networks, is nothing new (Lones, 2016).", "However, the inclusion of epigenetic elements allows for a connectionist architecture with a dynamical topological morphology – that is, the ability for a computational network to autonomously partition itself and select partitions based upon environmental or internal state.", "This serves two primary advantages.", "First, it supports task specialisation, where a partition assigned to a specific environmental or internal context is only used in that scenario.", "Secondly, with minimal analysis it provides a method of characterising a network's behaviour from the ground up, by mapping the functionality of the individual partitions and building up an image of the system's transitions between these partitions.", "This helps automate the process of model validation.", "Because of this, we propose that the properties of epiNet lend themselves well to the classification and analysis of real world time series data, where the underlying generative model is often poorly understood.", "We describe the application of epiNet to the particular problem of understanding and classifying the movements associated with the neurodegenerative disorder Parkinson's disease when a patient is undergoing treatment with the dopamine replacement drug levodopa.", "Incorrect dosage of levodopa can have severe manifestations, notably the involuntary and violent muscle spasms known as levodopa-induced dyskinesia (LID).", "Correct dosage involves finding a balancing point between the removal of Parkinsonian symptoms and the onset of side-effects, providing a challenge to both patients and clinicians alike.", "In this work, we demonstrate the potential of epiNet by analysing the topological changes and transitions between dynamical states within the model when classifying movement data collected during levodopa therapy.", "We show that it is possible to provide clinically relevant information about LID, the underlying data and the processes underpinning why at any given point epiNet has made a certain decision.", "Epigenetics refers to mechanisms which result in changes in gene expression without altering the underlying DNA (Bird, 2007; Turner, 2001).", "In Fig. 1, a general eukaryotic arrangement of DNA is illustrated.", "Within the cell nucleus, DNA is wrapped around a histone octamer over 1.67 turns.", "This combination of DNA and histones is referred to as chromatin, one of the major epigenetic structures.", "Chromatin has two prominent biological roles: compressing the size of a DNA strand, and controlling physical access to the DNA.", "The dynamically varying structure of chromatin allows DNA to move relative to it, allowing the cellular machinery (e.g. polymerase, transcription factors) to access it.", "Controlling this movement means controlling which genes are actively being transcribed at any given moment.", "This idea is central to the model used within this paper, where there exists a ‘bank’ of genes (the genome) which is inactive by default.", "Then, chromatin reorganises itself relative to the DNA in order to change which genes are accessible to the cellular machinery at any given time.", "Additionally, there are other epigenetic marks.", "One of the most pervasive is DNA methylation, where a methyl group is added to either the cytosine and adenine nucleotides within DNA.", "In a similar vein to chromatin modifications, methylations typically prevent transcription by physically inhibiting the cellular machinery's ability to straddle the DNA, preventing processes such as transcription (Bird, 2007; Turner, 2001).", "There are also other epigenetic mechanisms, such as micro RNAs, prions and SRNAs.", "The model used in this work (previously described in Turner et al. (2015)), builds upon a range of previous work from more statically derived genetic and epigenetic functionality (representing genetic networks and static epigenetic marks such as DNA methylation) (Lones et al., 2013; Turner et al., 2014; Reil, 1999; Bull, 2014) to more dynamic models which take inspiration from chromatin modifications (Turner et al., 2013, 2015, 2016).", "Indeed, the low-level element of epiNet, the artificial gene model, is derived from (Lones et al., 2010), and remains unchanged.", "This artificial gene is a computational element that takes inputs and processes them using a function, producing a single transformed output.", "In this paper, we use a sigmoid function, meaning that these low-level elements are similar to perceptrons in artificial neural networks.", "This function can be parameterised during evolution, allowing the gradient of the function and offset to change for each individual gene, allowing for functions ranging from the typical sigmoid to an approximate step function (Fig. 2).", "Many of these genes are linked together to form an artificial gene regulatory network, in which certain genes are mapped to external inputs and outputs (Lones et al., 2010).", "We have explored several approaches to building epigenetic structures into, or on top of, existing artificial gene regulatory networks (Turner et al., 2013, 2014, 2015).", "In these models, genes are generally always active unless made inactive by an epigenetic analogue.", "Additionally, the epigenetic analogues are static and fixed in place and control a small part of the network.", "With epiNet, by comparison, genes are inactive until turned on by an epigenetic analogue.", "The epigenetic analogues are then regulated by genes, causing them to move around the genome, switching on and off different parts of the network over the course of time.", "The main structure in this model is a genetic structure, consisting of a number of genes (30–100) which exist on a 1-dimensional linear scale (Fig. 3) between [0,1].", "These genes are static and are not directly executed.", "Execution of the genes occurs when genes are copied from the genetic structure to the protein network.", "The protein network functions in a similar way to the networks in (Lones et al., 2010), where it is the structure which directly interacts with an external environment (task) and is executable.", "However, which genes are copied from the genetic structure to the protein network is controlled by the epigenetic molecule.", "The epigenetic molecule(s) within epiNet straddles the genome, existing in the same 1-dimensional space.", "Each epigenetic molecule has a position in this 1-dimensional space and a size (Fig. 1).", "The size is fixed, but the position is a variable.", "At each time step, whichever genes exist within the space occupied by the epigenetic molecule are then copied from the genome to the protein network.", "At each time step, the epigenetic molecule takes selected inputs from the protein network, and processes them using a sigmoid function (identical to the genes) and this output then becomes the epigenetic molecules new position.", "Hence, the position of the epigenetic molecule on the genome is the product of the structure and state of the protein network.", "Upon initialisation, epiNet will contain 3 epigenetic molecules, however multiple epigenetic molecules can be incorporated to the network throughout the optimisation process.", "After each execution the protein networks expression values are mapped back to the genome.", "This serves to give the protein network and genome a relative memory of its previous state.", "Following execution, all the proteins within the protein network are removed and will become repopulated according to the position of the epigenetic molecule on the next step.", "An external task can interface with epiNet by modifying protein expression within the protein network.", "In this work, epiNets are initialised with 40 genes and 3 epigenetic molecules; however, new components may be generated and removed allowing the modification of their parameters throughout the course of evolution.", "Parkinson's disease (PD) is a chronic neurodegenerative disorder which is characterised by progressively impaired motor control due to loss of dopaminergic neurons in the brain (Nalls et al., 2014).", "Although the underlying cause of Parkinson's is unknown, it has been linked to various genetic and environmental factors (Impellizzeri et al., 2015; Lolekha et al., 2010).", "Currently approximately 1% of people over 60 suffer from PD; however, with a globally ageing population, it is estimated that this will increase significantly (Lill et al., 2012).", "Clinical diagnostic accuracy for PD is relatively low and research studies have shown that approximately 8–25% of cases are misdiagnosed (Schrag et al., 2002; Hughes et al., 1992).", "This is in part due to the overlap of symptoms associated with a range of other diseases including progressive supranuclear palsy, multiple system atrophy, corticobasal syndrome, and vascular Parkinsonism.", "One of the most successful treatments for the motor symptoms of Parkinson's is a dopamine-replacement drug, levodopa.", "When using levodopa, there is an issue of fine tuning a patient's medication to their exact requirements: insufficient dosage results in ineffective treatment, excess dosage can lead to levodopa-induced dyskinesia (LID), which may manifest as violent muscle spasms.", "Clinicians fine-tune a patient's medication regimens based on their own ratings of the patient's symptoms and on patient-rated treatment response.", "These metrics may be insensitive to small but important effects and additionally, these metrics correlate only weakly with each other (Fahn et al., 2004).", "Moreover, it is often difficult to separate the symptoms of Parkinson's from LID.", "Hence, a more personalised computational approach is desirable, where dosage guidance can be inferred from models trained using real patient data.", "The objectives of this work are:", "To understand the properties of epiNet as a predictive modelling tool for the analysis of levodopa-induced dyskinesia", "To understand how the analysis of epiNet's topological morphology when classifying the data can provide useful clinically relevant information about levodopa-induced dyskinesia", "To achieve these objectives, we applied epiNet to the classification of real world medical data derived from accelerometers worn by Parkinson's patients undergoing a levodopa based medication regime.", "The data was acquired from 25 patients who had confirmed symptomatic Parkinson's disease and were being treated with levodopa therapy.", "The patients wore small lightweight sensing devices with integrated accelerometers and gyroscopes to monitor their movements at a sample rate of 100 Hz.", "These devices each stored 6 channels of information, corresponding to acceleration in the three translational and three rotational axes.", "The patients were unconstrained yet asked to complete various movement tests throughout a 2 h window at non-specific intervals.", "Whilst doing this, the patients were recorded using an infrared video camera.", "Trained clinicians then used these recordings to grade the data, using the Unified Dyskinesia Rating Scale (UDysRS) to mark up periods of LID.", "The data was cross-correlated between clinicians to ensure accuracy over the grading of the patients.", "The patients were rated on a scale of 0–4, where 0 corresponds to the patient displaying no signs of dyskinesia, and 4 corresponds to severe dyskinesia.", "Table 1 summarises the number of instances of each grade of dyskinesia within the data set.", "Evolutionary algorithms (EAs) are population-based metaheuristics which carry out optimisation procedures motivated by biological evolution.", "Evolution is the process of adapting an individual or population to environmental dynamics over successive generations via the stochastic breeding of population members.", "Evolutionary algorithms model this principle by using the data of a system or model as a genome, and by crossing over and mutating solutions within a population according to an objective measure of their ability to solve a problem (known as their fitness).", "Over successive generations, the individuals’ fitness will improve as the population learns to solve a particular task.", "EAs are relatively flexible in how they represent and evaluate solutions.", "Their appropriateness for optimising epiNets is also suggested by the role of biological evolution in designing biological genetic regulatory systems.", "For these reasons, we used them to optimise both the architecture and parameters of the epiNet model in order to solve the problem of predicting the degree of dyskinesia in segments of accelerometry time series data.", "We used the multi-objective evolutionary algorithm NSGA-II (Deb et al., 2002) with a population size of 400 and an optimisation period of 250 generations.", "We used tournament selection of size 4.", "Point mutation was used for all components of the genome and epigenome with a probability of 0.05.", "The crossover operator exchanged 30% of the data between individuals, and the crossover rate is 0.5.", "These parameters were selected as previous exploratory experimentation has shown that these allow for the evolution of complex behaviours which were not able to be learned by other algorithms within identical time frames (Turner, 2013; Turner et al., 2015).", "We use a multi-objective fitness function in this work.", "The first objective is to correctly classify each time step within a data segment, with one point awarded for each correct classification.", "The second aggregates the points awarded for a data segment; if over 50% of the time points are correct, that data segment is considered to be correctly classified and a point is awarded.", "This allows the system to handle variable-sized data segments (204–16,001 time steps) without biasing search towards classifiers that perform well on only the shorter or longer samples, which tends to happen when only one of these objectives is used.", "The complete data set contained 9672 labelled sequences.", "To make the optimisation process computationally tractable, we uniformly sub-sampled (without replacement) 1000 instances for use as a training set.", "The remaining samples were used for testing, allowing a fairly robust estimate of a classifier's generality.", "The data was split into 2 classes of dyskinesia severity: high and low.", "High indicates an LID rating of either 2, 3 or 4.", "These correspond to clinically significant levels of dyskinesia that require a change in levadopa dosage.", "Low indicates an LID rating of 0 or 1, i.e. no, or very mild, dyskinesia.", "The training data was split accordingly; the low class comprising 500 data segments, with 250 rated 0 and 250 rated 1, the high class comprising 500 data segments with 166 rated 2, 167 rated 3, and 167 rated 4.", "Each data segment comprises 6 times series, corresponding to changes of acceleration over time in the three translational and rotational axes.", "At each time step, this multivariate data is fed into an epiNet classifier as a group of 6 values, each copied into the activation levels of input genes.", "After the epiNet has processed the input, a single output is read from the expression level of an output gene.", "An output value below 0.5 is interpreted as indicating low dyskinesia; a value equal to or greater than 0.5 indicates high dyskinesia.", "We carried out 20 independent runs, during which each epiNet classifier was evaluated on its ability to correctly predict the labels of each time step and each data segment within the training set.", "Following these 20 runs, all the final generation classifiers were re-evaluated on the test set.", "Fig. 4 shows the performance of these controllers on both the training and test sets, with each point showing the per-step and per-segment accuracy of a single controller.", "On the training set, classifiers were found with accuracies of ∼80%.", "When re-evaluated on the test set, accuracies dropped to around ∼70%; however, most showed an ability to generalise.", "Fig. 5 summarises the distributions for both objectives.", "It is notable that test set accuracies were significantly higher for whole data segments (more important from a clinical viewpoint) rather than individual time steps.", "This is unsurprising, given the larger amount of historical time series data present in an entire data segment, and the relative stochasticity of movements at a single time step.", "However, it is also evident from Fig. 4 that the majority of the classifiers are fairly balanced in their responses at the per-step and per-segment level, with most clustered along the diagonal.", "The results show that trained instances of the epiNet model are capable of classifying movements associated with levodopa therapy.", "We next carried out post-hoc analysis of the dynamical behaviour of the epiNet classifiers, in order to gain some insight into how they achieve this.", "Figs. 6 and 7 show the dynamical response of a single evolved epiNet classifier whilst processing two different acceleration time series data segments.", "First of all, it is evident that the dynamics of the network are adaptive, with the activation levels of the genes and the positions of the epigenetic analogues undergoing significant changes during the course of classification.", "This change in dynamics is caused by the autonomous topological changes of the network as a result of changing dynamics in the environmental input data.", "Analysis of a sample of the evolved classifiers suggests that this behaviour is typical, with <5% of the instances not undergoing topological change whilst processing the input data, and these topologically-invariant solutions having generally lower fitness values.", "This suggests that topological change is a useful mechanism, and it seems likely that topological changes are driven by, and hence are a signal of, dynamical changes in the patient's movements.", "For the network analysed in Figs. 6 and 7, some genes are almost always active (approximately 3–5), some genes are never active, and others are intermittently active.", "Additionally, the size of the expressed network varies significantly over time; sometimes no genes are active, sometimes upwards of 15 are active.", "It can also be noticed (particularly in Fig. 7) that some changes in the activity of the genes do not lead to changes in the overall dynamics of the network.", "For example, at time steps 220–800, gene 31 is constantly being activated and deactivated, yet this causes no discernible difference in the network's output.", "The evolutionary algorithm was given the freedom to evolve the architecture of the epiNets within bounds.", "The initial parameters for the network were 40 genes and 3 epigenetic molecules and most networks maintained a similar architecture throughout optimisation.", "The number of genes varied little, with the largest solutions containing 52 genes, the smallest containing 30.", "There appeared to be no discernible difference between the performance of networks based on the number of genes alone.", "Most functional solutions contained 3 epigenetic molecules.", "However, a significant number contained only 2, but these otherwise functioned similarly to the networks containing 3: suggesting that useful behaviour can be achieved with less molecules, but with some impairment to evolvability.", "Networks which contained 1 or 4 epigenetic molecules had poor functionality, with neither of them resulting is a solution in the top 20% of the population.", "A possibility, as seen is previous work is that too many epigenetic molecules leads to a greater likelihood of interference between regulatory regions.", "The data used to train the networks contains 6 input channels and a single output.", "EpiNet allows these inputs to be mapped onto genes multiple times (i.e. one input gets mapped onto multiple genes); it also allows particular inputs to be ignored by the network (Fig. 8).", "To understand the way in which epiNet classifiers use the inputs, we analysed the best evolved solutions.", "A pervasive trend was for 3 of the 6 inputs to be used significantly more than the others, although none of the high fitness networks used a permanently static mapping.", "Inputs 2, 3 and 6 were a common choice; however, 4 and 5 were also used widely in other networks.", "It was also common for input 1 (acceleration in the x-plane) to be completely ignored by the networks.", "In many solutions, single inputs were mapped onto multiple genes (mostly 2, sometimes 3 or 4), which were then excited in tandem.", "Hence, a particular input could drive multiple regulatory circuits.", "This can be interpreted as an indication of the relative importance of the input variable.", "However, over the course of execution, not all inputs remained in use.", "This can be seen as a context sensitive switch, with input signals driving topological change that causes the network to focus on movement in particular axes.", "For instance, for over 75% of the networks’ execution, half of the inputs are not used.", "For further analysis, we looked at creating a minimum working example (MWE) of an evolved solution, assuming that evolved epiNets would be architecturally more complex than they needed to be in order to solve the problem.", "This was done by iteratively removing a gene from the network and then re-evaluating it.", "If the performance worsened, the gene was replaced; if not, it was removed permanently.", "The epiNet architecture is particularly robust to deleterious mutations as its genes are not all interconnected during execution, and it is therefore unlikely that all of the dynamical regimes of the network would be affected by a single gene deletion.", "We found that removing genes had a less damaging effect to the networks’ performance then removing epigenetic molecules, which almost always adversely affected performance.", "The networks we considered initially started with 40 genes, and in general could be reduced to around 5 genes (Fig. 9).", "Although this had no effect on functionality, the networks in this state were more sensitive to perturbations; because of this increased sensitivity, it is probable that they lacked the ability to move through the search space in as timely a manner as the original networks, as small perturbations would be likely to dramatically alter behaviour.", "Because these networks were artificially created by removing genes, it is difficult to fully assert what the networks structure would be if they were evolved to be as small as possible.", "However, in previous work (Turner et al., 2015) it was shown that doing such, removed the robustness of the networks during execution, but the networks were still able to find solutions to complex tasks.", "Hence it is likely that artificially altering network structure has different effects than evolving the networks to do so.", "The functioning of an MWE version of the network analysed in Fig. 9 is depicted in Fig. 10, showing the activity of each gene over a transitional period.", "This network was capable of classifying both time steps and data segments with over 69% accuracy.", "It can be seen that genes 1 and 5 initially oscillate.", "Whilst in this dynamical phase, the network is completely inactive for 50% of the time.", "Once the transition occurs, genes 3 and 4 become permanently active.", "This switching behaviour was common in the MWEs, and in general the networks appeared to have two stable attractors corresponding to movements in the high and low dyskinetic classes.", "The transition between attractors was also, in most cases, permanent, suggesting that once a significant dyskinetic signal was detected, the network remained in the high dyskinesia state, increasing the likelihood of classifying the whole segment as exhibiting dyskinesia.", "For most MWEs, it was also evident that the transition between attractors was governed by a single input.", "Although other inputs might be mapped to genes after this transition, the permanent change in attractor meant that they were not being used.", "This suggests that it is possible to train effective classifiers using only a single channel of the accelerometry data.", "This is significant, as it could considerably reduce the training time of the networks.", "It also means that patients could be fitted with simpler sensors that consume less energy and are therefore less bulky.", "Furthermore, the MWE equivalents of the evolved classifiers are quite small.", "In principle, this means they could be implemented in a relatively small digital (or potentially analogue) circuit deployed within a sensor platform.", "This could provide a single-device solution to monitoring dyskinesia in Parkinson's patients.", "In this paper we have shown that epiNet is able to classify levodopa-induced dyskinesia within patients suffering from Parkinson's disease.", "We have also shown how analysis of an epiNet's structure can give us insight into both how and why classification decisions have been made.", "In particular we showed that post-processing of the networks could often reduce the number of genes in the network from 40 to around 5 without altering functionality.", "This also showed that the networks could make decisions based on a single accelerometer input, significantly increasing the efficiency of execution.", "This opens up the opportunity for small, wearable diagnostic tools to be implemented using a hardware model of the MWEs.", "The results are promising, however, there were specific caveats associated with the work in this paper.", "The first is that the combination of population based evolutionary algorithms and epiNet meant that run times were relatively high.", "This limited the scope of the networks ability to learn, but conversely, also allowed for a comprehensive method of testing because a large amount of data was not used in training.", "It would have also been beneficial to allow for a larger population size and more generations to gather a better understanding of epiNet's potential.", "A further possibility to improve this is to migrate the software to a GPU implementation as a significant part of the processing within the networks is floating point arithmetic, hence, the performance increase should be substantial.", "Within this work there has not been a direct focus on objective performance, but a focus on the underlying dynamics of the networks when classifying high frequency movement data.", "Because of the novel dynamical properties of the network, we feel that direct comparison with other techniques with differing dynamical functionality would at present be of limited value as variations in performance could be attributed to the parameters of the respective techniques.", "The work in this paper is to be used as an underpinning of future research in which a robust analysis of epiNets performance will be carried out in reference to other models, such as those in Lones et al. (2014b).", "Within this work we had to reclassify the data into either a high or low levodopa-induced dyskinesia rating.", "In addition, there was a class imbalance where there was an order of magnitude more data available data sets with no levodopa-induced dyskinesia, compared to those who had the most pronounced symptoms.", "Hence, it might be possible to look at a class weighting which is dependent on classification outcomes (and how incorrect classifications may effect patients) and the relative performance of the networks.", "Indeed, it may also be beneficial to look at re-balancing the classes by omitting certain classifications within the data (to only focus on the most severe symptoms) as seen in Lones et al. (2014a).", "Overall, the results in this paper have shown that epiNet has significant potential as a classification tool, and that the analysis of the network structure and dynamics can provide clinically relevant information about the decisions made by epiNet which are important both for patient outcome and model validation." ]	[]
Loss of Parkin contributes to mitochondrial turnover and dopaminergic neuronal loss in aged mice	Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by the loss of nigrostriatal dopamine neurons. PARK2 mutations cause early-onset Parkinson's disease (EO-PD). PARK2 encodes an E3 ubiquitin ligase, Parkin. Extensive in vitro studies and cell line characterization have shown that Parkin is required for mitophagy, but the physiological pathology and context of the pathway remain unknown. In general, monogenic Parkin knockout mice do not accurately reflect human PD symptoms and exhibit no signs of dopaminergic (DA) neurodegeneration. To assess the critical role of Parkin-mediated mitophagy in DA neurons, we characterized Parkin knockout mice over a long period of time. At the age of 110 weeks, Parkin knockout mice exhibited locomotor impairments, including hindlimb defects and neuronal loss. In their DA neurons, fragmented mitochondria with abnormal internal structures accumulated. The age-related motor dysfunction and damaged mitochondria pathology in Parkin-deficient mice suggest that impairment of mitochondrial clearance may underlie the pathology of PD.	[ "NEUR" ]	[ "Aging", "Dopaminergic neuron", "Mitochondria", "Parkin", "Parkinson's disease" ]	[ "2808" ]	[ "Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by the loss of nigrostriatal dopamine neurons and the formation of intracellular Lewy bodies (LBs), which consist primarily of α-synuclein (Spillantini et al., 1997).", "Interactions between genetic predisposition and environmental factors are likely to be primarily responsible for mitochondrial dysfunction and oxidative damage, which result in oligomerization and aggregation of synuclein; however, the underlying molecular mechanisms remain poorly understood.", "The vast majority of PD is sporadic, and inherited familial forms of the disease accounting for only 5% of all cases (Dauer and Przedborski, 2003).", "The identification of PD-related genes and risk factors has implicated several pathways in PD etiology, with accumulating evidence suggesting a link between dysfunctional mitochondrial clearance and PD pathogenesis.", "In PARK2-linked PD, intrinsically disordered mutant Parkin initiates PD pathology (Kitada et al., 1998).", "Since the discovery of Parkin, the protein has received special interest because it has ubiquitin–protein ligase activity.", "Early studies assumed that proteolytic dysfunction causes substrate accumulation, resulting in massive loss of dopaminergic (DA) neurons (Chung et al., 2001; Shimura et al., 2000).", "Subsequently, several lines of Parkin-null mice were analyzed, but the causative mechanisms underlying early-onset PD (EO-PD) remain largely unknown, in part because most substrates of Parkin reported thus far remain unchanged in the disease state.", "Furthermore, loss of DA neurons is not observed in Parkin-deficient mice (Goldberg et al., 2003; Periquet et al., 2005; Sato et al., 2006).", "Given that Parkin is associated with mitochondrial degradation in vitro (Matsuda et al., 2010; Narendra et al., 2008), it is likely that a dysfunction of mitophagy (i.e., autophagy of the mitochondria) contributes to the neurodegenerative process.", "Macroautophagy (hereafter, autophagy) is a highly conserved bulk protein degradation pathway in eukaryotes.", "Cytoplasmic proteins and organelles, including mitochondria, are engulfed within autophagosomes; these bodies eventually fuse with the lysosome, where they are degraded along with their cargo (Levine and Klionsky, 2004).", "Insights regarding the functions of PINK1 and Parkin (Pickrell and Youle, 2015), which shed light on the mitochondria, have also enhanced our understanding of EO-PD pathogenesis.", "Specifically, experiments in which cultured cells were treated with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), which causes mitochondrial depolarization, revealed that the PINK1/parkin pathway is associated with mitochondrial quality control.", "Several lines of evidence indicate that damaged mitochondria are predominantly degraded by mitophagy in vitro.", "In addition, Parkin mutations implicate induction of mitophagy in PD, and suggest that accumulation of damaged mitochondria contributes to DA neurodegeneration.", "However, the phenotypes of mice harboring Parkin deletion have not been fully examined.", "To elucidate the critical role of PINK1/Parkin-mediated mitophagy in DA neurons, we characterized the age-related pathological and motor phenotypes in Parkin knockout mice.", "A targeting vector was constructed using 1.5- and 7-kb DNA fragments as the 5′ and 3′ homologous sequences, respectively.", "A negative selection cassette, DTA, which encodes the diphtheria toxin, was also included.", "The linearized targeting vector was transfected into TT2 ES cells.", "After selection in G418, clones were screened for homologous recombination by Southern blotting.", "Using the 5′ external probe and probe specific for neo sequence, we confirmed that the clones carried the desired homologous recombination.", "ES cells derived from these clones were injected into C57BL/6 J embryos.", "Chimeric offspring were crossed with C57BL/6 J mice to obtain germline transmission, which was confirmed by Southern analysis with the 5′ probe.", "Heterozygous mice were then interbred to obtain homozygous knockout and wild-type control mice.", "Mice were subsequently genotyped by PCR using primers specific for the wild type and the targeted allele(Sato et al., 2006).", "All animals were kept in a pathogen- and odor-free environment, which was maintained under a 12 h light/dark cycle at ambient temperature.", "Procedures were approved by the Animal Experimental Committee of the Juntendo University Graduate School of Medicine, and performed in accordance with the guidelines of NIH and the Juntendo University Graduate School of Medicine.", "Locomotor behavior was assessed in mice from 100 to 125 weeks of age.", "Accelerating rotarod tests were performed on a rotarod machine with automatic timers and falling sensors (MK-660D, Muromachi Kikai, Japan).", "Male Parkin knockout mice and wild-type mice (n = 10 for each genotype) were placed on a 3-cm diameter rotating rod covered with rubber, and rotation was accelerated from 3 to 40 rpm over 5 min.", "Fall latency was recorded, and the first fall latency of the third trial was used for analysis.", "The runway test was performed using a narrow, horizontally fixed beam (3 cm wide, 80 cm long, held at a height of 40 cm from the table).", "The animal was placed at one end of the beam and urged to move toward the opposite end, where an escape platform was located.", "Each genotype mice were randomly selected and behavioral tests were conducted in a blinded fashion.", "A combination anesthetic (Kawai et al., 2011) was prepared with 0.3 mg/kg of medetomidine hydrochloride (DO-0003; Nippon Zenyaku Kogyo, Japan), 4.0 mg/kg of midazolam (001541582; Astellas Pharma, Japan), and 5.0 mg/kg of butorphanol (DO-0023; Meiji Seika Pharma, Japan).", "The anesthetics were administered to mice by intraperitoneal injection.", "Mice were perfused with 4% paraformaldehyde (PFA), and their brains were immersion-fixed at 4 °C for 36 h.", "The fixed samples were cryoprotected with 20% sucrose and sliced on a freezing microtome to obtain 40-μm-thick floating sections.", "For immunohistochemistry of TH, sections were incubated with anti-TH antibody (657,012; Calbiochem, Germany) and visualized with DAB (Diaminobenzidine), which generates brown precipitates.", "The sections were observed on a VS120 (Olympus, Japan).", "The counting of dopaminergic neurons was done with unbiased stereology.", "For double immunofluorescence staining for cytochrome c (green) and TH (red), floating sections were incubated with anti–cytochrome c antibody (ab90529; Abcam, UK) and anti-TH (clone TH-16; Sigma-Aldrich, USA) antibody.", "The sections were then incubated with anti–guinea pig IgG conjugated with Alexa Fluor 488 and anti-rabbit IgG conjugated with Alexa Fluor 546.", "Fluorescence signals were captured on an FV3000 confocal microscope (Olympus).", "Using these fluorescence data, the number and area of green stained mitochondria within red stained dopaminergic neurons were measured using “cellsens” analysis software (Olympus, Japan).", "For stereological quantification, three areas (VTA: ventral tegmental area; SNcc: center area of substantia nigra pars compacta; SNcl: lateral area of substantia nigra pars compacta) were selected.", "Every other 40-μm section of serial coronal brain slices for each genotype were stained for DAB.", "Quantification was performed with design-based stereology system (Stereo-Investigator version 2019, MBF Bioscience, Williston, VT, USA).", "The sampling parameters were set up according to the software guide to achieve the coefficient of error ranged between 0.07 and 0.09 using the Gundersen test, normally with a counting frame size 30 × 30 μm, optical dissector an average of 10 sampling sites per section.", "For conventional electron microscopy, mice were fixed by cardiac perfusion with 2.5% glutaraldehyde in 0.1 mol/L PB (pH 7.2).", "Brain slices were embedded in epoxy resin, and ultrathin sections (70 nm thick) were cut and observed on an HT7700 electron microscope (Hitachi, Japan).", "Using these electron microscopy data, the number and area of mitochondria within dopaminergic neurons were manually measured using “cellsens” analysis software (Olympus, Japan).", "Dorsal striata from 120-week-old mice were dissected, quickly frozen on dry ice, and then homogenized with 0.5 mL of 0.2 M perchloric acid containing 100 μM EDTA per 100 mg wet tissue.", "Samples were centrifuged at 20,000 ×g for 15 min at 4 °C, and the supernatant was collected and analyzed by HPLC.", "Statistical significance was determined by Student's t-test (STATVIEW; SAS Institute).", "Data are expressed as means ± S.E. P < .05 was considered significant.", "Previously, we generated mice deficient in exon 2 of Parkin (Parkin knockout mice) and characterized them in the early part of life.", "When young, the mice exhibited very little phenotypes other than downregulation of DA release and upregulation of D1 and D2 dopamine receptors in the striatum.", "However, the abundance of DA neurons was not decreased in 48-week-old mice (Sato et al., 2006).", "Hence, we observed Parkin knockout mice over longer period of time.", "Parkin knockout mice were viable at birth, indistinguishable in appearance from their littermates, and had a normal survival rate.", "Although these mice have not yet been observed over their entire lifespan, they began to show motor behavioral deficits at 110 weeks.", "These clinical abnormalities could be observed by the runway (Fig. 1A a, b, and c; Video S1, Parkin KO; Video S2, Parkin WT), footprint (Fig. 1B), and rotarod tests (Fig. 1C).", "In the runway test, wild-type mice exhibited well-coordinated movement and almost no slips of the forepaw or hindpaw from the beam; by contrast, Parkin knockout mice could hardly move on the beam, and frequently slipped.", "In particular, the hindpaws of Parkin knockout mice often slipped off the beam (Fig. 1A b and c).", "Next, we conducted the footprint test, because a short stride is a characteristic of PD, including patients with PARK2 mutations.", "Parkin knockout mice had a shorter foot range than wild-type mice (Fig. 1B).", "Furthermore, in the accelerating rotarod test, fall latency was reduced in Parkin knockout mice (Fig. 1C).", "There were no effects on weight (Supplementary. Fig. S2.", "A) and lifespan (Supplementary. Fig. S2.", "B).", "Gait disturbance progressed, and by the terminal stage (beyond 120 weeks), the majority of affected mice could hardly move.", "Parkin knockout mice were viable at birth, indistinguishable in appearance from their littermates, and had a normal survival rate.", "Although these mice have not yet been observed over their entire lifespan, they began to show motor behavioral deficits at 110 weeks.", "These clinical abnormalities could be observed by the runway (Fig. 1A a, b, and c; Video S1, Parkin KO; Video S2, Parkin WT), footprint (Fig. 1B), and rotarod tests (Fig. 1C).", "In the runway test, wild-type mice exhibited well-coordinated movement and almost no slips of the forepaw or hindpaw from the beam; by contrast, Parkin knockout mice could hardly move on the beam, and frequently slipped.", "In particular, the hindpaws of Parkin knockout mice often slipped off the beam (Fig. 1A b and c).", "Next, we conducted the footprint test, because a short stride is a characteristic of PD, including patients with PARK2 mutations.", "Parkin knockout mice had a shorter foot range than wild-type mice (Fig. 1B).", "Furthermore, in the accelerating rotarod test, fall latency was reduced in Parkin knockout mice (Fig. 1C).", "There were no effects on weight (Supplementary.", "Fig. S2.", "A) and lifespan (Supplementary.", "Fig. S2.", "B).", "Gait disturbance progressed, and by the terminal stage (beyond 120 weeks), the majority of affected mice could hardly move.", "Accumulating knowledge regarding PINK1 and Parkin, both of which are associated with mitochondria, has increased our understanding of the proteins' cellular functions.", "The PINK1/Parkin pathway is associated with mitochondrial quality control, but it remains unclear how Parkin influences mitochondrial function or structure in vivo.", "To evaluate its in vivo function, we analyzed mitochondria in DA neurons in 110-week-old Parkin-deficient mice, which exhibit locomotor impairments.", "To identify DA neuron, we performed immunofluorescence staining for tyrosine hydroxylase (TH) and cytochrome C (mitochondria inner membrane protein).", "Specifically, after fixation with 4% paraformaldehyde (PFA), 6-μm paraffin-embedded tissue sections were cut and stained for TH and cytochrome c. For immunofluorescence, secondary antibodies were coupled to Alexa fluorophores (Invitrogen).", "In Parkin knockout mice (Fig. 2A d–f), small mitochondria accumulated in the substantia nigra (SN) to a greater extent than in wild-type mice (Fig. 2A a–c).", "These mitochondria were observed not only in the cytosol, but also outside the cell bodies.", "We speculated that the phenomenon would also be observed in axon and dendrites.", "To evaluate mitochondria in DA neurons, we analyzed mitochondrial area in the TH-positive cell bodies.", "In Parkin knockout DA neurons, mean mitochondria area was smaller than in wild-type mice (Fig. 2B).", "Next, we investigated the percentage of the cytosolic area occupied by mitochondria.", "DA cells in Parkin knockout mice trended toward a higher ratio, but did reach significant difference from wild type mice (Fig. 2C).", "Together, these observations suggest that mitochondrial fragmentation might be facilitated in the DA neurons of aged mice.", "We supposed that damaged mitochondria were fragmented and the number of mitochondria increased.", "To further characterize these mitochondria, we performed ultrastructural analysis in DA neurons of 110-week-old mice (Fig. 3A a–c, wild-type control; Fig. 3A d–f, Parkin knockout mouse sample 1; Fig. 3A g–i, Parkin knockout mouse sample 2).", "We observed small, round, fragmented mitochondria in Parkin-deficient DA neurons (Fig. 3A d–i), but not in controls (Parkin wild-type) (Fig. 3A a–c).", "Precise quantification revealed that mitochondria area was reduced (Fig. 3B left panel) and the number of fragmented mitochondria per unit of cytosolic area was elevated (Fig. 3B right panel) in Parkin-deficient DA neurons.", "In order for damaged mitochondria to be degraded by autophagy, they must be segregated by fission (Twig et al., 2008).", "If Parkin-mediated mitophagy might contribute to mitochondria degradation systems, our in vivo results are reasonable.", "Next, we observed mitochondrial microstructure in Parkin knockout mice.", "Electron microscopy revealed that the fragmented mitochondria had normal outer membrane structure (Fig. 3A c inset), but displayed irregular inner structure, i.e., the normal structures of matrix and cristae were broken.", "This observation indicates that damaged mitochondria accumulate due to abrogation of Parkin-mediated mitophagy.", "Finally, we confirmed the presence of Lewy bodies, but we could not find them in Parkin knockout mice.", "Although the molecular components required for mitophagy have been identified through extensive in vitro work, the physiological pathology and context of these pathways remains largely unknown (Rodger et al., 2018).", "Many genetically modified mice have been developed as PD models, but most of them do not exhibit neuronal loss.", "To assess the contribution of the accumulation of damaged mitochondria, we compared the number of TH neurons between aged Parkin knockout and control mice.", "As demonstrated in the rotarod test (Fig. 1C), Parkin knockout mice exhibited locomotor dysfunction at ages above 110 weeks.", "We sacrificed these mice (Parkin knockout, n = 5; wild type, n = 5) and counted TH neurons in three sections (VTA: ventral tegmental area; SNcc: center area of substantia nigra pars compacta; SNcl: lateral area of substantia nigra pars compacta) (Fig. 4).", "Parkin knockout mice had fewer TH neurons than wild-type control mice.", "The reduction in TH cell number was most prominent in SNcc (Fig. 4B).", "We conducted stereological quantification of the area of cell bodies, but we did not observe a difference between Parkin WT (mean 374.48 ± 10.1 μm2) and Parkin KO (mean 312.85 ± 33.6 μm2).", "Furthermore, we conducted stereological quantification of striatal DA fibers (Supplementary. Fig. S1.", "B).", "Parkin KO mice demonstrate significant loss of dopamine fibers in the striatum.", "The possibility that the behavioral changes are due to Parkin deficiency in other parts of the brain must be elucidated.", "Then, we conducted further pathological examination in cortex, cerebellum and striatum.", "But, we found no obvious pathological changes including Lewy pathology (Supplementary. Fig. S1).", "Although the molecular components required for mitophagy have been identified through extensive in vitro work, the physiological pathology and context of these pathways remains largely unknown (Rodger et al., 2018).", "Many genetically modified mice have been developed as PD models, but most of them do not exhibit neuronal loss.", "To assess the contribution of the accumulation of damaged mitochondria, we compared the number of TH neurons between aged Parkin knockout and control mice.", "As demonstrated in the rotarod test (Fig. 1C), Parkin knockout mice exhibited locomotor dysfunction at ages above 110 weeks.", "We sacrificed these mice (Parkin knockout, n = 5; wild type, n = 5) and counted TH neurons in three sections (VTA: ventral tegmental area; SNcc: center area of substantia nigra pars compacta; SNcl: lateral area of substantia nigra pars compacta) (Fig. 4).", "Parkin knockout mice had fewer TH neurons than wild-type control mice.", "The reduction in TH cell number was most prominent in SNcc (Fig. 4B).", "We conducted stereological quantification of the area of cell bodies, but we did not observe a difference between Parkin WT (mean 374.48 ± 10.1 μm2) and Parkin KO (mean 312.85 ± 33.6 μm2).", "Furthermore, we conducted stereological quantification of striatal DA fibers (Supplementary.", "Fig. S1.", "B).", "Parkin KO mice demonstrate significant loss of dopamine fibers in the striatum.", "The possibility that the behavioral changes are due to Parkin deficiency in other parts of the brain must be elucidated.", "Then, we conducted further pathological examination in cortex, cerebellum and striatum.", "But, we found no obvious pathological changes including Lewy pathology (Supplementary.", "Fig. S1).", "No neuronal loss was observed in young mice, which did not exhibit motor dysfunction (data not shown).", "Consistent with our results, a reduction in the abundance of TH neurons also occurs in PD, primarily in the SNcc.", "Loss of DA neurons may contribute to the motor impairment observed in the late stages of disease.", "In addition, we tested DA physiology in these 120-week-old Parkin knockout mice by neurochemical analysis of the dorsal striata.", "High-performance liquid chromatography (HPLC) revealed a reduction in striatal DA levels in Parkin knockout mice relative to control mice (Fig. 4C).", "Thus, DA content is affected by loss of DA neurons.", "Intriguingly, the significant decrease in dopamine is not accompanied by concomitant reductions in the dopamine catabolites DOPAC and HVA.", "Currently, we have not clarified the reason, but abnormal mitochondrial accumulations may induce a partially modified DA metabolism (Thiffault et al., 2000).", "Great progress has been made toward understanding the pathogenesis of familial PD, mainly due to the discovery of PINK1/Parkin-mediated mitophagy.", "However, little is understood about the in vivo functions underlying PD pathogenesis.", "In this study, we carried out detailed observations of old Parkin-deficient mice, which exhibit motor dysfunction and loss of DA neurons in association with aging.", "In general, the phenotypes of Parkin knockout mice do not accurately reflect the symptoms of PD (Goldberg et al., 2003; Periquet et al., 2005; Sato et al., 2006).", "In 2006, we performed the first examination of the status of DA metabolism in the brain, focusing on DA release and DA receptors using in vivo autoradiography.", "In vivo autoradiography using [11C] raclopride revealed a clear decrease in endogenous DA release after methamphetamine challenge in Parkin-deficient mice.", "Furthermore, Parkin deficiency was associated with considerable upregulation of dopamine D1 and D2 receptor binding in the striatum, as well as elevated DA levels in the midbrain.", "These results suggested that DA neurons behave abnormally prior to their death; this event may be the initial change underlying the development of EO-PD (Sato et al., 2006).", "Parkin knockout mice tend to anticipate phenotypes including DA neuronal loss (Pickrell et al., 2015; Pinto et al., 2018), but monogenic Parkin knockout mice do not accurately reflect human PD symptom and show no signs of DA neurodegeneration.", "The most parsimonious explanation for the lack of DA neurodegeneration in genetic PD models is the existence of a compensatory mechanism resulting from adaptive changes during development, making it hard to observe the degenerative phenotype over the lifespan of the mice.", "To identify the phenotypes associated with Parkin deficiency, we observed motor and pathological phenotypes in mice older than 100 weeks.", "Interestingly, PD model mice began to exhibit a motor phenotype (Fig. 1C) and neuronal loss (Fig. 4B) around the age of 110 weeks.", "In 2018, in an effort to create a PD model, we generated DA neuron–specific autophagy-deficient mice (Atg7F/F:TH-Cre), and we observed the same motor dysfunction and neurodegeneration phenotypes in these animals.", "Specifically, Atg7F/F:TH-Cre mice began to exhibit impairment in motor coordination tasks around 100 weeks and apparent motor behavioral deficits around 110 weeks.", "In contrast to control mice (Atg7F/F), which exhibited well-coordinated movement and almost no slips of the forepaw or hindpaw from the beam, Atg7F/F:TH-Cre mice could hardly move on the beam and slipped frequently.", "In particular, the hindpaws of Atg7F/F:TH-Cre mice often slipped off the beam.", "Furthermore, in the accelerating rotarod test, fall latency was reduced in Atg7F/F:TH-Cre mice.", "Moreover, aged Atg7F/F:TH-Cre mice exhibited synuclein accumulation in neurites and/or cell bodies, as well as loss of DA neurons (Sato et al., 2018).", "These features may represent a common motor phenotype in PD models.", "Long-term disruption of autophagy results in widespread synuclein accumulation in DA neurons.", "In a previous study, Friedman et al. (Friedman et al., 2012) reported that DA axons developed synuclein aggregates at the age of 20 months, and suggested that autophagy is involved in synuclein homeostasis in mice.", "The protein catabolic pathways that regulate synuclein degradation have been discussed by multiple authors (Ebrahimi-Fakhari et al., 2011; Rideout et al., 2004; Webb et al., 2003).", "Additionally, autophagy plays essential roles in the clearance of organelles, including mitochondria (Twig et al., 2008).", "Many mutations at the PARK2 locus induce autosomal recessive Parkinsonism (Hattori et al., 1998; Lucking et al., 1998), as do mutations of the PINK1 (PTEN-induced kinase 1) gene at the PARK6 (Valente et al., 2004).", "PINK1 contains an N-terminal mitochondrial targeting signal and a highly conserved serine/threonine kinase domain, in which many missense and nonsense mutations have been reported.", "The activity of PINK1 kinase is crucial for the phosphorylation of ubiquitin (Kane et al., 2014) (Kazlauskaite et al., 2014) (Koyano et al., 2014) and Parkin (Shiba-Fukushima et al., 2012), which in turn are required for mitochondrial maintenance.", "Loss of PINK1/Parkin function may increase the cell's vulnerability to various stresses.", "In Drosophila models, PINK1 and parkin ensure stable mitochondrial function.", "Parkin-null mutants exhibit severe mitochondrial pathology in association with reduced lifespan, apoptosis, and muscle degeneration.", "The PINK1 mutant phenotype can be rescued by overexpression of Parkin, suggesting that Parkin and PINK1 act in a common pathway to maintain mitochondrial integrity.", "PINK1 loss-of-function decreases mitochondrial membrane potential, and the PINK1/parkin pathway is associated with mitochondrial elimination.", "To date, the exact mechanism leading to mitochondrial autophagy has not been fully elucidated (Pickrell and Youle, 2015).", "Based on previous findings, we carefully monitored Parkin-deficient mice and observed accumulation of fragmented mitochondria (Fig. 2B, 3B) in the DA neurons and livers of aged mice (data not shown).", "In Parkin knockout mice, small mitochondria accumulated in DA neurons (Fig. 2A d–f).", "Interestingly, these mitochondria were observed not only within the cytosol, but also outside the cell.", "Mean mitochondrial area was smaller in Parkin-deficient DA neurons than in wild-type mice (Fig. 2B).", "To further characterize these mitochondria, we conducted ultrastructural analysis.", "In Parkin knockout mice, DA neurons were filled with small, round mitochondria (Fig. 3); mean mitochondria area was smaller than in wild-type mice, and the number of mitochondria per unit of cytosolic area as elevated (Fig. 3B).", "Careful observation of the internal structure of mitochondria in Parkin-deficient mice revealed that the matrix and cristae structure were broken (Fig. 3A).", "In aged Parkin-deficient DA neurons, fragmented and damaged mitochondria accumulated.", "As to how Parkin deficiency is linked to mitophagy and fission/fusion, recent studies of the substrates of mitochondrial ubiquitin ligases including MULAN, MARCH5, and Parkin, identify them as potential critical mediators that govern selective fusion and mitophagy.", "Parkin was shown to translocate selectively to energetically impaired mitochondria.", "Studies in Drosophila have shown that upon recruitment to depolarized mitochondria, Parkin ubiquitinates Mfn (Ziviani et al., 2010).", "And the knockout of Parkin leads to an accumulation of Mfn and to mitochondrial elongation (Poole et al., 2010).", "These results in Drosophila suggest that in the absence of Parkin, damaged mitochondria are rescued by fusion instead of being removed by mitophagy.", "This compromises mitochondrial quality maintenance.", "It should be mentioned that the effects of manipulations on Parkin had an ambiguous effect on mitochondrial morphology between Drosophila and mouse.", "In our report, we observed that Parkin KO led to mitochondria fragmentation in Parkin-deficient mice.", "Other reports on Parkin KO mice support the observation of fragmented mitochondrial phenotypes and demonstrate no Mfn accumulation (Stevens et al., 2015) (Pinto et al., 2018).", "The reasons for these conflicting reports remain to be fully elucidated, but, species-specific differences may be a contributing factor.", "Considering mitochondria degradation in cultured cells, depolarized mitochondria were clustered with p62 (Narendra et al., 2010) (Okatsu et al., 2010).", "Specifically, depolarized mitochondria are ubiquitylated by Parkin, recognized by p62 (Bartlett et al., 2011), transported via microtubules to the cytosol, and degraded by autophagy (Komatsu et al., 2007).", "Loss of Parkin may inhibit physiological clustering of depolarized mitochondria and microtubule transport to the cytosol.", "An electron microscopic analysis by Gai et al. revealed that many mitochondria are concentrated in the early forms of Lewy bodies (LBs) (Gai et al., 2000), and Bedford et al. also reported deposits of mitochondria in early LBs in PD patients (Bedford et al., 2008).", "We speculate that parkin-mediated clustering of p62 may be a key process involved in LB formation.", "Consistent with this, LBs are usually absent in EO-PD caused by Parkin mutations (Mori et al., 1998).", "In general, Parkin KO mice show a rare phenotype, namely, that they do not demonstrate DA neuronal loss and movement dysfunction.", "Previously, we have reported no loss of neurons in Parkin exon 2 deletion mutant mice at ages up to 12 months (48 weeks) (Sato et al., 2006) and Perez and Palmiter claimed no loss of neurons at ages up to 22 months (88 weeks) (Perez and Palmiter, 2005).", "Specifically, Parkin KO mice did not recapitulate central signs of EO-PD.", "But in our investigation, the long-term observation enabled us to find dopaminergic neuronal loss at 120 weeks.", "Previous reports indicated that Parkin-deficient mice display inconsistent phenotypes (Itier et al., 2003), (Goldberg et al., 2003), (Von Coelln et al., 2004), (Palacino et al., 2004).", "One of these reasons is differences in the genetic background between the mouse strains tested.", "Thus, we performed further examinations in mice with the same genetic background.", "Intriguingly, our Parkin KO mice are B6;129 genetic background, the same as in Perez's study.", "Our results demonstrated that Parkin exon 2 deletion mutant mice exhibited age-dependent DA neuronal loss at 120 weeks.", "In addition to the genetic background, the type of Parkin mutation could explain the phenotypic differences in various Parkin KO mice, because these finding could be due to the loss of Parkin function or expression of a mutant Parkin.", "Although the phenotypes of Parkin-deficient mice could be modified by genetic background or the mutation, carefully controlled studies are required to rule out the possibility that these phenotypes are not the result of Parkin deletions and instead reflect artifacts due to several technical confounding factors of gene targeting in mice (Gerlai, 1996), (Gerlai, 2001).", "Further long-term observation beyond 110 weeks will be needed to identify Parkin KO phenotypes.", "Identifying the reasons, why highly aged Parkin KO mice demonstrate Parkinsonism, could provide us with important clues to elucidate the pathogenesis of PD.", "For example, an analysis of PARIS (ZNF746) levels using a conditional knockout of Parkin in adult animals may reveal to progressive loss of dopaminergic neurons in a PARIS-dependent manner (Shin et al., 2011).", "In our investigation, the lack of Parkin leads to an increase in the small size of mitochondria, which occupy the same or greater proportion of cytosolic area of DA neurons in aged mice.", "Stevens et al. reported that conditional knockout of Parkin in the adult mice ventral midbrain leads to a decrease in mitochondria size and number with dopaminergic neuronal loss (Stevens et al., 2015).", "On the other hand, Pinto et al. showed that Parkin-deficient mice, where mitochondrial DNA undergoes double-strand breaks in the dopaminergic neurons only, exhibit a small but significant decrease in the number of mitochondria in the dopaminergic axons only (Pinto et al., 2018).", "Fragmented small mitochondria are commonly observed in Parkin KO mice.", "In general, damaged mitochondria are fragmented.", "Considering the pathophysiology of Parkin loss, it was assumed that the accumulation of damaged mitochondria might be derived from defects in Parkin-mediated mitophagy.", "However, given the formation of damaged mitochondria, other factors remain to be elucidated.", "Currently, mutant mtDNA levels and levels of proteins associated with mitochondrial dynamics, such as Mfn2, Opa1, Drp1, and a repressor of PGC-1αcalled PARIS, may contribute.", "The age-related motor dysfunction and pathology we observed in Parkin-deficient mice suggests that impairment of mitochondrial clearance may underlie PD pathology.", "Our PD model mice exhibited accumulation of damaged mitochondria and neuronal loss.", "Furthermore, DA levels were affected by DA neuronal loss (Fig. 4B).", "These PD models will provide insight into mitochondria-associated PD pathology, and will be crucial for the development of novel therapeutic targets.", "The following are the supplementary data related to this article.", "A. Histological analyses of cortex (Cx), cerebellum (Cl) and striatum (St) in a 120-week-old Parkin knockout (n = 5) and wild-type (n = 5) mice.", "Paraffin sections were stained with hematoxylin–eosin (HE).", "None of neuronal loss and morphological changes including Lewy pathology was observed.", "Scale bars: 200 μm.", "B. Stereological quantification of TH fibers in the striatum with a 120-week-old Parkin knockout (n=5) and wild-type (n=5) mice Data are means ± SE; ** P < 0.01 (Student’s t-test).", "A. Body weight of aged Parkin knockout and wild-type mice at 100 weeks, 110 weeks and 120 weeks.", "Data are means ± SE (Parkin knockout mice, n = 20; wild mice, n = 20) N.S.; Parkin knockout and wild mice denote not significant.", "B. Kaplan–Meier analysis of survival of wild and Parkin knockout mice.", "The age at death of two groups of 105-week-old animals (Parkin knockout mice, n = 20; wild mice, n = 20).", "The survival curves for wild type and Parkin knockout mice are not different by Log rank test.", "Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2019.104717.", "This work was supported by a KAKENHI Grant-in-Aid for Scientific Research from JSPS.", "Authors' contributions SS, NH designed the study, and wrote the initial draft of the manuscript.", "SN, TF contributed to analysis and interpretation of data, and assisted in the preparation of the manuscript.", "All other authors have contributed to data collection and interpretation, and critically reviewed the manuscript.", "All authors approved the final version of the manuscript, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved." ]	[ "Aged Parkin knockout mice exhibit motor dysfunction and dopaminergic neuronal loss.", "Mitochondrial fragmentations are facilitated in the Parkin KO dopaminergic neurons.", "In the Parkin knockout dopaminergic neurons, damaged mitochondria are accumulated." ]
Validation of the blocked force method for various boundary conditions for automotive source characterization	Vibro-acoustic source characterization is an essential task in vehicle development to enable prediction of receiver response. For structure-borne noise, the interface forces in multiple degrees of freedom due to internal loads are often quantified for root cause analyses in a single system assembly, as in transfer path analysis (TPA). However, for a reliable prognosis of the acoustic performance of a known component such as a motor or pump, a receiver-independent source characterization is required, and the method of acquiring blocked forces from in-situ measurements has been shown to be a preferred technique for such purposes. The benefits of the method are the characterization of the intrinsic properties of the source and the possibilities of measuring the component attached to receivers with varying dynamic properties. There is to date a limited number of validation cases where blocked forces from in-situ measurements are acquired for automotive source-receiver assemblies. In this study the blocked forces of a vacuum pump in nine degrees of freedom were determined when connected to a bracket whose boundary conditions were modified in order to achieve four assemblies with different source/receiver dynamic properties. The results show that the blocked forces are transferable, i.e. the receiver response in one assembly was predicted in a wide frequency range by combining source-receiver transfer functions of that assembly with blocked forces estimated in another assembly. Furthermore, an in-situ blocked force TPA was applied to a double-isolated complete vehicle source-receiver case of an electric rear axle drive with interior compartment sound pressure as target. The reconstructed magnetic tonal harmonics agreed with the measured target response in the frequency range 50-500 Hz, which further motivates the use of the blocked force principles for TPA and source requirements specifications.	[ "PHYS" ]	[ "Blocked force", "Source characterization", "Transfer path analysis" ]	[ "3102" ]	[ "In automotive development, tools and methods for structure borne noise diagnosis and prognosis are essential.", "The structure-borne receiver response (e.g. sound pressure, vibration velocity) due to a vibrating source is caused by its internal forces and the transfer functions relating source and receiver.", "The internal forces are often difficult to model or measure, so instead, equivalent operational interface forces are typically used.", "In most situations, direct load determination by force transducers mounted at the connection interfaces is practically difficult.", "Indirect determination of loads in multiple degrees of freedom are often performed by inverse methods which are widely used in transfer path analysis (TPA), a diagnosis tool which enables separation of partial path contribution from the total sum.", "Each partial contribution can further be broken down into source strength (force) and system sensitivity (transfer functions), providing useful underlying information about a noise issue.", "Comparing the reconstructed response resulting from the partial contributions of all accounted transfer paths with a measured response is a common method for judging the quality of a classical TPA-model.", "One major disadvantage associated with classical TPA based upon inverse force synthesis is that the source needs to be mechanically decoupled from the receiving structure when measuring transfer functions between the interface and receiver.", "In many cases, this operation is time-consuming and can induce errors related to the decoupling stage.", "Also, and very important, is that the interface forces derived from classical TPA are not valid for predicting the response when the same source is assembled to a dynamically different receiver.", "For such a prognosis, an independent source characterization is required.", "Elliot and Moorhouse et al. [1–5] have in recent years illuminated the blocked force as being beneficial for intrinsic source characterization.", "The blocked force is analogous to the open-loop voltage in electrical networks and can be estimated in-situ from e.g. operational velocity and mobility.", "This approach is often more practically realizable compared to direct measurements of the blocked force which requires blocking of all interface degrees of freedom (DoF).", "In [1], the blocked force was validated in-situ for two source–receiver assemblies consisting of beams with two rigid connections, and with force excitation in one single DoF.", "It was also shown that the interface forces varied while the blocked forces remained the same for the two different assemblies.", "In [2], the in-situ method for acquiring blocked forces was experimentally carried out on wind turbine prototype assemblies with different receiver poles.", "The validation of the independent source properties were in the two studies above mentioned performed through the following steps: first, the blocked forces for one source–receiver assembly is derived.", "Then, those forces are applied to a dynamically different receiver and the velocity response is calculated.", "Finally, the reconstructed velocity on the second receiver is compared to the directly measured response on the second receiver.", "This is an elegant way of verifying that the blocked force estimates are unaltered instead of comparing them directly especially in the case of multiple interface DoF.", "There are apparently major potential benefits associated with the blocked force methods.", "Besides the advantages of reducing the time effort and avoiding potential errors thanks to the possibility of working in the source/receiver coupled state, the outcome of a TPA can be further utilized for making reliable prognoses.", "For instance, the same blocked forces can be re-used in calculations where the receiver is modified/improved.", "Another advantage is that the blocked forces of a source may be determined for receivers with various boundary conditions.", "The alternatives to in-situ conditions (for automotive applications meaning a prototype or production vehicle) as in the case of TPA, are quasi-free boundary conditions (the free velocity approach is standardized in ISO 9611:1996) [3] or in a test bed or other fixture with no particular stiffness requirements.", "Finally, the blocked forces can also be obtained directly by force transducers if the receiver structure can be made perfectly rigid.", "There are only a limited number of case studies on more complex assemblies available to date.", "One exception is [6], where a blocked force tensor for a rigidly mounted oil pump connected to a truck engine proved to be successful.", "Also, Elliott et al. [5] showed that TPA based upon blocked forces obtained from in-situ measurements provided at least as good results as the classical inverse force synthesis method for structure-borne road noise, with the added value of reducing the time effort by 50%.", "In this work, we wish to highlight practical aspects related to in-situ measurements of blocked forces for automotive engineering applications.", "The objective of the paper was to investigate the following:", "By what accuracy can the response for one source/receiver assembly be reconstructed from blocked forces obtained from measurements of the same source but being attached to other receivers, yielding dynamically different assemblies?", "By what accuracy can the structure borne sound from an electric motor transmitted into the cabin of a car be estimated by contribution analysis based on blocked forces obtained with the in-situ method?", "The results provide information on the expected accuracy of the in-situ blocked force method when used in automotive applications.", "First the findings from measurements on a vacuum pump for brake pressure attached in three connection points to a rig-mounted bracket, which boundary conditions were altered, are presented.", "The operational excitation included both lateral as well as in plane forces acting on the receiver.", "Secondly, a blocked force TPA case-study concerning electromagnetically induced structure-borne tonal vibration and noise from a double-isolated electric rear axle drive (ERAD) installed in a passenger car is described.", "Limitations and applicability related to the two cases are discussed.", "In many structure-borne noise applications related to automotive NVH, the problem to be solved can be illustrated by the sketch shown in Fig. 2.1.", "The source A is connected to a receiver B, either rigidly or through vibration isolators, therefore forming the coupled assembly C.", "The source is an operational dynamic load at a pre-defined interface location.", "In general it is difficult to perform direct measurements of the operational dynamic load.", "Therefore a number of different methods are available for quantifying the excitation.", "The most common methods are:", "Complex stiffness method [9,10].", "Inverse Inertance method [11,12].", "Transfer path analysis in combination with hybrid analysis is also becoming a tool for making NVH prognosis [13].", "The fundamental principle is to be able to predict the operational load with analytical models or from a test rig and to be able to combine the operational load with transfer functions to predict the future vehicle response.", "Many of the components used in car development projects today are off-the-shelf parts, e.g. they already exist in current vehicle production.", "Using the component’s source characteristic, i.e. blocked force, together with a finite element model of the complete vehicle, a prediction of the vehicle responses could be done early in the vehicle development phase, before physical prototypes of the vehicle exist.", "For validation of the blocked forces from in-situ measurements, a pump for providing brake vacuum mounted to a bracket was used.", "In its real environment, the bracket is integrated to the car body.", "For the experiments, the pump and bracket was instead mounted in a laboratory rig (see Fig. 3.1).", "The bracket was equipped with seven triaxial accelerometers where six of them acted as indicators for the indirect determination of the blocked forces and one as target response (the Z-direction of the target accelerometer is presented in the result plots).", "There was no particular strategy for deciding upon the accelerometer positions aside from having them remote from the interface points and also spread out over the receiver.", "For the frequency response function (FRF) measurements, impact hammer excitation was executed at the three connection points in the three translational directions X, Y and Z. Transfer mobilities to all accelerometers were calculated with the H1 estimator.", "The frequency resolution was set to 1.6 Hz and the number of averages was seven.", "It was assumed that the distance between the origin of the internal pump forces and the connection points would provide a lever effect so that the influence of moments acting on the receiver would be small.", "Therefore, rotational DoF were not measured in order to simplify the experimental procedure.", "The vacuum pump was driven by an external power supply in steady state mode during 30 s for each run.", "The X-direction channel of the accelerometer circled in Fig. 3.1 acted as phase reference for calculation of operational spectra.", "The procedure for the operational measurements and FRF tests was repeated in the same fashion for four types of assembly configurations.", "For two of the assemblies, soft rubber bushings were used at the interface between the pump and the bracket while the remaining two configurations employed bolts as connectors.", "The boundary condition between the bracket and the frame work was either fixed or free.", "Fig. 3.2 illustrates the difference between the fixed and free case.", "The four assembly cases are from now referenced as “bushed-fixed”, “bushed-free”, “bolted-fixed” and “bolted-free”.", "To verify that the four assemblies were dynamically significantly different, the summed FRF for each assembly was plotted (see Fig. 3.3) for providing a general and global comparison.", "The question addressed is how well an operational response due to a source in one assembly can be reconstructed from blocked forces estimated from the same source but mounted to a different receiver.", "Blocked forces were estimated for each of the four assemblies.", "A relative threshold of 1% was used for the truncation of each of the four mobility matrices, constraining the condition number to not exceed 100, in order to omit the least significant singular values for removing measurement noise or redundant information.", "First, the measured operational target response on the bolted/fixed assembly was compared to the reconstructed response (the summed products of each blocked force and the respective source-target transfer function as in Eq. (2.6)) from the same assembly.", "The differences are very small throughout the entire frequency range (compare the (iii) and (ii) in Fig. 3.4).", "This indicates that the quality of the measurements was satisfying (e.g. positioning of sensors, signal-to-noise ratios, impact excitations).", "Fig. 3.4 also includes the response reconstructed by forces obtained from FRF and operational measurements of the bolted/free assembly (blue dotted curve).", "For frequencies between approximately 430 and 600 Hz, the fit is again typically within 3 dB.", "From Fig. 3.3 it is apparent that the two assemblies behave rather similarly dynamically in that frequency range.", "In other frequency ranges such as 120–140 Hz and 180–200 Hz, there are deviations between the two reconstructions of about 10 dB.", "Again from Fig. 3.3, it is evident that the bolted/free assembly has an anti-resonant behavior in that range, which is likely to cause errors in the matrix inversion process.", "This will be further discussed in Section 5.", "The most important frequencies regarding perception are however those where the peaks in the spectrum are located.", "The difference is typically within 3 dB for most of the peaks corresponding to the pump’s 10 most prominent orders (see bottom plot, Fig. 3.4).", "The blocked forces estimated from the two bolted assemblies are plotted for each interface DoF in Fig. 3.5.", "The plots indicate that the global trends for the estimated forces between the two conditions are similar but local differences can be significant.", "The same comparison was done for the two bushed cases and the result is depicted in Fig. 3.6.", "Here, the bushings were chosen to be part of the receiver in order to not modify the source between the bolted and bushed cases.", "The vibration response is now reduced by the insertion loss due to the bushings (note the different y-axis scale between Figs. 3.4 and 3.6).", "Again, the sum of partial contributions agrees almost fully with the measured response for the bushed/fixed assembly (compare (iii) and (ii) in Fig. 3.6).", "The velocity reconstructed by forces estimated from the bushed/free assembly and FRFs to target measured at the bushed/fixed assembly deviates from the measured response a little more (blue bars bottom plot, Fig. 3.6) compared to the previous case in Fig. 3.4.", "This is not very surprising since soft rubber bushings between the source and receiver make the FRF measurements more challenging.", "The pump was also measured in quasi-free conditions (hanging in elastic bands) as an alternative experimental method to derive the blocked forces.", "In this configuration, the blocked forces were obtained from only three triaxial accelerometers, positioned at the attachment points both during FRF tests and operational tests.", "If the reconstructed velocity from those blocked forces is added to the curves in Fig. 3.4, several interesting observations can be made.", "From Fig. 3.7a, it is clear that the response reconstructed from free velocity measurements of blocked forces deviates substantially from the response reconstructed from the bolted assemblies.", "On the other hand, it fits rather well to the response reconstructed from the two bushed cases (see Fig. 3.7b).", "This indicates a change in source activity between the quasi-free and the bolted conditions that has not been accounted for when measuring the sets of transfer mobilities on the receiver side.", "Hence it seems more likely to generally achieve better results when conducting blocked force measurements in-situ compared to utilizing the quasi-free approach.", "The blocked forces estimated from the quasi-free measurements are compared to estimates from the bushed cases in Appendix A for Point 1.", "The predictions related to these experiments were limited in the sense that the mobility matrices of the source, the mounts and receiver alone were never measured.", "Instead, the left hand expression of Eq. (2.3) were measured directly in order to short-cut the in reality often needed sub structuring process.", "Again, sub structuring itself is very challenging and practical explorations are left out of the scope in this work.", "This section deals with the findings from a blocked force TPA on a complete passenger car.", "The purpose of the tests was to investigate whether the interior sound pressure due to structure borne noise from an electric rear axle drive could be reconstructed successfully.", "In comparison to the vacuum pump case, this is an example of a double isolated suspension system (see Fig. 4.1) and the response target is interior sound pressure at outer ear positions in front and rear seats rather than bracket vibrations.", "Three rotational-speed-dependent components (orders) originating from the electro-magnetic forces are prominent at take-off from stand still up to about 20 km/h.", "For higher speeds, these harmonic orders are masked by the tire/road interaction noise.", "Fig. 4.2 displays the interior sound pressure (co-driver’s outer ear position) for this specific driving condition.", "As can be seen in Fig. 4.2, the three orders have poor signal-to-noise (SNR) ratio in several frequency bands.", "Having strongly coherent response with respect to the exciting force is crucial for the quality of the reconstructed response, which will be shown.", "The strategy for obtaining the blocked forces in the three translational directions on the source side of the ERAD bushings and acquiring the necessary data for performing the blocked force TPA can be summarized in the two following measurement steps:", "FRF measurements with force excitation by impact hammer at the four locations marked with red crosses in Fig. 4.1, acceleration measured at the eight locations marked by green dots and target interior compartment sound pressure at the four locations marked by stars.", "The latter quantity is often referred to as a noise transfer function (NTF).", "Operational measurements where acceleration and sound pressure are recorded, at the same locations as during the FRF measurements.", "Generally for obtaining blocked forces from in-situ measurements it is faster to measure all the transfer functions direct, meaning force input at the blocked force points in contrast to the reciprocal alternative.", "Thus, the accelerometers do not need to be moved between operational and FRF measurements.", "The accelerometer indicator positions were spread out over the primary substructure of the receiver – the subframe, in order to achieve as clean data as possible for estimating blocked forces.", "During the FRF measurements, the vehicle was lifted (in order to access the impact points) at the wheels in order to get the correct loading of the wheel suspension.", "Transfer mobilities to all accelerometers were calculated with the H1 estimator.", "The frequency resolution was set to 1 Hz and the number of averages was three.", "Again, the moments and angular movements were not measured due to practical reasons.", "The operational tests were run on a road with a moderate upward slope in order to increase the loading of the electric drive.", "The spectra were obtained by tracking the 10th, 30th and 90th orders with order bandwidth of 1, 3 and 9 respectively.", "The X-direction of one of the indicator accelerometers acted as phase reference.", "Although the entire sweep up to 1500 rpm was approximately 30 s long, the 90th order is present only up to about 340 rpm corresponding to an acquisition time less than 6 s.", "Fig. 4.3 displays the measured and reconstructed interior sound pressure response for the 30th order together with the average NTF coherence.", "Appendix A also provides figures for the 10th and 90th order.", "It is important to point out which factors that can influence the agreement between the measured and reconstructed acoustic response; the coherence of the NTF (which was not always satisfying), the influence of two persons in the front seats during the operational measurements but not during the FRF tests, the temperature difference between the two on-road operational measurements and the FRF tests.", "Therefore, for the follow-up analysis, one of the subframe accelerometers was acting as target response.", "For this case the identified factors which had impact on the reconstructed interior sound pressure were less influential.", "The measured subframe acceleration and the reconstructed acceleration are displayed in Fig. 4.4.", "Clearly, the fit is significantly improved compared to the interior sound pressure case.", "As always when it comes to measurements, high quality signals are essential in order to achieve good results.", "For blocked force TPA, a general recommendation is to put effort in achieving a high coherence in the FRF measurements and good signal to noise ratio in the operational data.", "From the two case studies on the vacuum pump and ERAD, the in-situ blocked force method proved to be an attractive method for determining vibro-acoustic source activity.", "Performing a conventional TPA on an electric powertrain (see e.g. Anderson et al. [14]) implicates high complexity with respect to the installation.", "Drive shafts, engine mounts, electric harness and cooling hoses need to be separated from the source which makes the process more protracted.", "An electric powertrain is an example of a source being subject to both airborne and structure borne noise which will be carried over from one hybrid car model to new hybrid car models.", "Hence, performing a blocked force TPA will in addition to the root cause analysis exemplified in the previous section also facilitate prediction possibilities.", "The vacuum pump experiments show that as long as high quality in the measurement data (e.g. signal-to-noise ratio, coherence) is obtained, and no modification to the source activity is made, the response in a different source–receiver assembly can be reconstructed.", "It is not fully clear which factors that were mainly responsible for the discrepancies between the measured and the mixed reconstructed response.", "However, the non-ideal repeatability between the operations of the pump is likely to have had some influence.", "Special caution should be made regarding modified source activity.", "It is important to understand if the underlying mechanisms of the internal forces are affected by changes in the receiver, e.g. by loading.", "For example, cases can exist where the internal force generation is unaffected by changes on the receiver but where instead the reaction forces at the interface are altered.", "Such a case could be when a very flexible source is rigidly connected to a rig that is exposed to stress in such a manner that the transfer between the locations of the internal force and interface reaction force are altered.", "Hence, a general recommendation when measuring the blocked forces is to strive for a test environment which is similar to the real in-situ environment.", "Blocked force characterization from in-situ measurements is also to be preferred since it is more likely that the loading of the source is more accurate compared to when the source is free-free or mounted to a test bed.", "Numerical errors are often associated with the matrix inversion.", "Such errors may impact the magnitude of the blocked force.", "Attention should be made to truncation and regularization techniques for improving the potentially ill-conditioned nature of the mobility matrix which is to be inverted.", "Accurate positioning and sufficient number of indicator sensors will however reduce these potential uncertainties [15], but are usually difficult to point out prior to the test.", "Extending the method from the frequency domain to the time domain would presumably reduce the influence of numerical errors.", "Also, as pointed out in [16], the time domain approach also accounts for non-linear receiver sub-structures.", "Apart from the fact that the process of performing a transfer path/contribution analysis using the in-situ blocked force method is faster compared to the conventional method, it is not prone to the potential errors associated with mounting/disconnecting the source from the receiver.", "In a recent study on a washing machine on a wood floor [17], it was concluded that the structure-borne sound prediction from the coupled state led to more accurate levels compared to the prediction from independently measured components.", "In some cases, such as for ships and wind turbines [18,2], it might not even be an option to separate the source from the receiver due to practical implications.", "Finally, for component or system target setting of tolerable interface force levels for a future assembly, the blocked forces are essentially the only ones that are reliable due to their independent characterization.", "There are though major challenges associated with both a correct determination of blocked forces as well as using them in a finite element based sub-structure model that needs further exploration.", "This paper has investigated the in-situ blocked force method further in the context of automotive vibro-acoustic sources.", "Experiments were made on a vacuum pump measured in nine degrees of freedom and fixed to a bracket that was modified in order to achieve different dynamical properties.", "The results showed that the forces derived from one source–receiver assembly could be used to reconstruct the response when the same source was mounted to a different receiver.", "Moments and angular movements were disregarded but seemed to have small (bolted case) or minimal (bushed case) impact on the reconstructed response.", "Further, the approach was extended to a transfer path analysis case where the interior compartment sound pressure was reconstructed from transfer functions and blocked forces of an electric motor drive estimated in-situ.", "For obtaining best possible reconstructed response it is necessary to obtain as good quality signal data as possible (coherence, signal to noise ratios).", "Caution should also be made not to modify the source activity severely by the installation if the blocked forces are obtained from rig measurements.", "The findings justify the use of the in-situ blocked force method as an appropriate and time-saving approach both for source characterization and troubleshooting for NVH engineers." ]	[]
Analytical data of synthesized deuterated isopropyl myristate and data about the influence of IPM/IPMdeut on the thermodynamics and morphology of 2D Stratum Corneum models	The data in this article shows the effect of isopropyl myristate (IPM) on a 2D Stratum Corneum lipid model. In the first part, the analytical characterization of the synthesized deuterated isopropyl myristate is given. Then a BAM image of the pure Stratum Corneum model used is shown and a dataset of surface-pressure – area isotherms considering various ratios of deuterated and non-deuterated IPM and the Stratum Corneum model mixture is provided. Assuming that after the plateau in the isotherm the area per molecule corresponds only to the Stratum Corneum model (squeezing out of IPM), the value of the area will correspond to the percentage of these lipids in the mixture when considering the pure SC model. The comparison of the real and the calculated areas per molecule is also done.	[ "MULT" ]	[]	[ "1000" ]	[ "The analytical characterization of the newly synthetized and purified deuterated isopropyl myristate was performed by HPLC, HRMS and NMR as shown in Figs. 1–4.", "The Stratum Corneum (SC) is the outermost layer of our skin and the main problem when addressing topical/transdermal delivery drugs [1].", "Langmuir monolayers serve as simple biological models to study the interaction between molecules, as for instance the effect of a penetration enhancer in the SC [2].", "As a SC lipid model a mixture of ceramide [AP], stearic acid and cholesterol in a molar ratio of [1:1:0.7] was used.", "Fig. 5 shows Brewster angle microscopy images from this model at different lateral pressures.", "Ceramide [AP] presents diastereomers: the D-form, which occurs naturally in our skin, and the L-form, which exists only if synthetized.", "Therefore, a SC model with only the D-form of ceramide [AP] in a mixture with the other two components - which will be called D-SC - is a better model of the human skin lipids.", "Fig. 6 shows the surface pressure – area isotherms of isopropyl myristate, pure D-SC monolayer and the mixture of the two in different ratios.", "For comparison and validation reasons the systems with non-deuterated isopropyl myristate (on the left) are compared with the deuterated ones (on the right), and the area per molecule (obtained by averaging the molecular weight of the molecules present in the mixtures) in such isotherms at selected surface pressures is presented in Table 1.", "In Table 2 the comparison of such areas regarding the calculated area corresponding to the ratio of D-SC present in the mixture is shown.", "The surface-pressure area isotherms were also measured for the SC model with the racemic mixture of ceramide [AP] (isotherms presented in [3]).", "Table 3 shows the area per molecule taken from the isotherm of the different systems, while Table 4 shows the comparison of such areas regarding the calculated area corresponding to the ratio of SC present in the mixture.", "Analytical characterization of the newly synthetized and purified deuterated Isopropyl myristate", "For the HPLC measurement, a stock solution of IPMdeut in CH3CN with the concentration of 1.5 µg/mL was prepared.", "As mobile phase, a mixture of CH3CN:MeOH:H2O 36;54:10 (v:v:v) was used.", "The flow rate was 1.2 mL/min with a flow gradient of 100.0 mL/min² and a pressure of 80 bar.", "The chromatogram was obtained at 265 nm.", "For the measurements, a stock solution of IPMdeut in CHCl3:MeOH 60:40 (V:V) with the concentration of 1.0 µg/mL was prepared.", "The data was collected at 1.3 kV for HR-MS and at 5.0 kV for ESI-MS.", "For the NMR measurement, 10 µg IPMdeut were diluted in 3 mL CDCl3.", "The spectrum was obtained at 27 °C.", "Stock solutions of each lipid were prepared in CHCl3 (SA, CHOL and IPM or IPMdeut) or in a CHCl3:MeOH mixture [7:3] (w/w) (CER[AP]).", "Lipid concentrations were in the range of 1 mM.", "All mixtures were prepared by mixing the respective amount of stock solutions in the desired ratio to a final concentration around 1 mM.", "To promote mixing, the solution was gently heated, followed by stirring and vortexing for three times.", "The solutions were spread onto the subphase using a 100 μL Hammilton micro-syringe.", "10–15 min were given for the evaporation of the solvent.", "To mimic the skin surface conditions, a pH 5.5 buffer solution (adjusted with 1 mM HCl) containing 150 mM NaCl and 1 mM EDTA was used as subphase.", "Brewster Angle Microscopy (BAM)", "A BAM2plus from NanoFilm Technologie (Göttingen, Germany) equipped with a miniature film balance from NIMA Technology (Coventry, UK) was used to image the morphology of the monolayer.", "The microscope has a frequency-doubled Nd-YAG laser (532 nm, ~50 mW), a polarizer, an analyzer, and a CCD camera.", "When p-polarized light is directed onto the pure air/water interface at the Brewster angle (~53.1°), zero reflectivity is observed.", "When a monolayer is added, the light starts to be reflected because of the different refractive index of the surface layer.", "The reflected light is registered by the CCD camera after passing the analyzer.", "BAM images of 355×470 µm2 were digitally recorded during compression of the monolayer with a resolution of approx. 2 µm.", "The pressure-area isotherms were recorded on a computer interfaced home-made Langmuir trough.", "The trough is equipped with a Wilhelmy balance using a glass plate.", "The compression speed was 3 Å2 molecule-1 min-1.", "The temperature was kept constant at (21±0.1) °C.", "Isotherms were measured three times on individually prepared samples to check for reproducibility.", "The isotherms were plotted in Origin 9 and the area per molecule was read directly from the graph at the surface-pressure considered.", "The calculated area corresponding to the ratio of SC model present in the mixture was obtained by multiplying the area obtained in the isotherm by the percentage of SC in the mixture.", "For example, at 35 mN/m the area per molecule of the SC model is 30 Å2.", "Considering the mixture with 90 mol% SC, the calculated area which corresponds to the SC is 27 Å2." ]	[]
ESX1-dependent fractalkine mediates chemotaxis and Mycobacterium tuberculosis infection in humans	Mycobacterium tuberculosis-induced cellular aggregation is essential for granuloma formation and may assist establishment and early spread of M. tuberculosis infection. The M. tuberculosis ESX1 mutant, which has a non-functional type VII secretion system, induced significantly less production of the host macrophage-derived chemokine fractalkine (CX3CL1). Upon infection of human macrophages ESX1-dependent fractalkine production mediated selective recruitment of CD11b+ monocytic cells and increased infection of neighbouring cells consistent with early local spread of infection. Fractalkine levels were raised in vivo at tuberculous disease sites in humans and were significantly associated with increased CD11b+ monocytic cellular recruitment and extent of granulomatous disease. These findings suggest a novel fractalkine-dependent ESX1-mediated mechanism in early tuberculous disease pathogenesis in humans. Modulation of M. tuberculosis-mediated fractalkine induction may represent a potential treatment option in the future, perhaps allowing us to switch off a key mechanism required by the pathogen to spread between cells. © 2014 Elsevier Ltd. All rights reserved.	[ "IMMU", "MEDI" ]	[ "ESX-1", "Fractalkine", "Infection", "Mycobacterium", "Tuberculosis" ]	[ "2403", "2404", "2725", "2726" ]	[ "Mycobacterium tuberculosis (Mtb) remains the leading cause of bacterial mortality world-wide [1].", "Little is known about the earliest events in human tuberculous infection during which the inhaled bacillus must spread from permissive cell to cell and establish its infective niche.", "Rapid availability of niche host cells early on (i.e. during the first 24–48 h) is essential for early establishment of infection and subsequent bacillary dissemination.", "The percentage of macrophages, representing the primary niche of the tubercle bacillus, in the human lungs is relatively low (ranging from 3 to 19%) with approximately 1 macrophage per 9 ml lung volume [2].", "As such, a chemotactic call from the infected “Trojan horse” aggregating more of these naive cells could increase niche availability, cellular infection and bacterial dissemination.", "It is increasingly recognised that chemotaxis-driven aggregation of macrophages is essential for establishment of the infectious niche and early bacillary spread in the Mycobacterium marinum-infected zebrafish [3].", "This pioneering study reported how early granuloma formation (“macrophage infiltration and aggregation”) in the zebrafish resulted in increased bacterial growth and spread.", "Intriguingly, M. marinum's ability to establish infection and dissemination through early cellular aggregation was dependent on region of difference 1 (RD1), the most strongly virulence-associated genomic region of Mtb.", "Notably, the reduced cellular infiltration observed in transparent zebrafish embryos infected with the M. marinum RD1 mutant was due to reduced ESAT-6-mediated production of matrix metalloproteinase 9 (MMP-9) by epithelial cells which neighbour the infected macrophages [4].", "The role of RD1, and hence ESX1, in early cellular aggregation and local spread of infection has not hitherto been tested in M. tuberculosis in humans.", "Similarly to mutants of M. marinum, Mtb mutants without RD1 are highly attenuated yet unlike the zebrafish pathogen they have no defect in growth [5,6] and thus represent a pure pathology-deficient phenotype or pat mutant [7].", "Mtb region of difference 1 (RD1) represents the primary attenuating deletion of the vaccine strain Bacillus Calmette Guerin (BCG) [8] and spans a 9 gene region from Rv3871-Rv3879, central to which are the highly antigenic molecules early secreted antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10).", "Recently, RD1 was determined to be part of a novel type VII secretion system called ESX1 which is critical for virulence and was recently determined to be required for the survival of tubercle bacilli in the primate lung [9].", "A number of virulence mechanisms have been attributed to ESX1 including phagosomal maturation arrest [10], cytosolic bacterial translocation [11] and host cell necrosis in cells 72 h post-infection [5,12,13], although it is unknown which of these is the most important in human tuberculosis in vivo.", "The early virulence mechanisms utilised by the tubercle bacillus to obtain and set up its infective niche are likely pivotal in determining the outcome of M. tuberculosis exposure and infection and therefore important to elucidate.", "We hypothesised that ESX1 may play a similarly crucial role in early monocyte recruitment and bacterial dissemination in humans to that observed in the zebrafish.", "Using an M. tuberculosis mutant and human cells we assessed the role of ESX1 in M. tuberculosis-induced chemokine-mediated cellular aggregation and bacillary dissemination.", "All samples were taken with approval from St. Mary's REC for 5 years (REC Number: 07/H0712/85) and with written informed consent.", "All animal work was carried out in accordance with the UK Animal (Scientific Procedures) Act 1986 and was approved by the animal use ethical committee of Oxford University.", "Mtb H37Rv was grown in Category III conditions on Middlebrook 7H9 (Becton Dickinson) medium plus 10% Middlebrook OADC (oleic acid, albumin, dextrose and catalase; Becton Dickinson) supplement, 0.5% glycerol (Sigma Aldrich) and 0.05% Tween 80 (Sigma Aldrich) at 37 °C at 100 rpm until they reached mid log phase when aliquots were frozen for use in 10% glycerol and plated out for colony forming units (CFU) on Middlebrook 7H10 agar plates plus 10% OADC and 0.5% glycerol at 37 °C for 2–3 weeks.", "Human THP-1 cells (a monocyte-derived cell line; American Type Culture Collection: ATCC), human A549 alveolar epithelial cells, murine J774 macrophage cells (ATCC), primary human alveolar epithelial cells (ATCC) or primary MDMs (isolated as in [5]) were infected at a multiplicity of infection (M.O.I) of 10:1, as described in detail in5.", "THP-1 cells were pre-treated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich) for 18 h to stimulate adherence prior to infection or activation.", "In brief, cells were seeded in 12-well plates, or chamber slides (VWR) to determine successful intracellular infection, at 5 × 105 cells/ml, and, after infection with bacilli for 2 h, any extracellular bacilli were washed off with warm RPMI and the media replaced.", "For supernatant analysis, the supernatant was removed and filtered using an ultra-free centrifugal filter device 0.22u (2 ml; Millipore) prior to removal from the CL3 suite.", "The cells were harvested for counting or flow cytometry by scraping.", "Trypan blue (Sigma) was used to determine viability, except in the flow cytometry where a viability stain was used (see below).", "To plate for extracellular CFUs a dilution series was prepared and plated out as above and for intracellular bacilli 0.1% Triton X-100 was used to lyse the cells prior to diluting and plating out.", "The anti-human CX3CL1/fractalkine antibody (R and D systems) was added at 1 μg/ml, the recombinant full length human CX3CL1/fractalkine (R and D systems) at 100 ng/ml, lipopolysaccharide at 10 ng/ml and piceatannol at 50 μM (Sigma Aldrich) for the stated time.", "For direct observation of the infected monolayers, coverslips were added to the well prior to the MDMs and staining was carried out using the TB cold stain kit (BDH).", "Washed cells were stained with aqua LIVE⁄DEAD® Fixable Dead Cell Stain Kit, (Invitrogen), CD3-Pe-Cy7 (e-bioscience), CD11b-APC-Cy7 (BD bioscience) and FITC anti-human CX3CR1 (Cambridge Bioscience) at 1:100 dilutions for 30 min.", "Unstained controls were used to set voltages, single-stained controls for compensation and fluorescence minus one (FMO) controls for gate setting during analysis.", "The samples were run on a Becton Dickinson LSR Fortessa and analysed using FlowJo 9.3 (©TreeStar,Inc).", "Events were gated on live singlets then analysed for cell surface marker expression.", "Cells infected with Mtb H37Rv-FITC were stained with aqua LIVE⁄DEAD® Fixable Dead Cell Stain Kit (Invitrogen) and then acquired on Becton Dickinson LSR Fortessa before analysis with FlowJo 9.3.", "The murine lung flow cytometry panel was composed of annexin-V/propidium iodide (AbCam), CD11b-APC-Cy7.7 (Becton Dickinson) and CD3-Pacblue (Cambridge Bioscience).", "For the single stained controls anti-rat/hamster murine beads were used (Invitrogen) and an unstained and FMO controls were also carried out.", "The samples were run on a Beckman Coulter CyAn ADP and live singlet cells were gated and analysed using Flowjo 9.3.", "Balb/C mice were infected intra-nasally with 300 CFU Mtb H37Rv or Mtb H37Rv ΔESX1.", "Mice (n3 per time point plus 1 naive uninfected control) were sacrificed at days 1, 3, 7, 14 and 28 post-infection.", "At each time point serum samples were taken and the lungs were resuspended in 2 mls of DMEM medium (Sigma Aldrich) plus 1.4 mg/ml collagenase (Sigma Aldrich) and 60 μg/ml DNase I (Sigma Aldrich).", "Following 45 min incubation at 37 °C the lungs were homogenised and the samples were centrifuged.", "The supernatant was saved as lung lysate and the lymphocytes were isolated from the cell pellet using Lympholyte M (CedarLane) and then processed for flow cytometry.", "At day 1 and day 28 post-infection one lung per mouse was homogenised and plated out for CFU as noted above in Mtb growth.", "MDMs, matured for 6 days at 5 × 105 cells per well, were infected with Mtb as above and 5 × 105 PBMCs added in 12-well millicell hanging culture inserts with 5μ filters, which would allow both monocytes and lymphocytes to pass through (Merck Millipore).", "Following 24 h infection (∼maximal fractalkine production) the inserts were removed and the cells were harvested by centrifuging the supernatant and by scraping for adherent cells prior to counting in a haemocytometer and flow cytometry staining.", "The 12-plex human cytokine array MPXHCYTO-60K-12 (Merck Millipore), human fractalkine single-plex MPXHCYTO-60K-01 (Merck Millipore), murine single-plex MPXMCYP2-73K-01(Merck Millipore) and matrix metalloproteinase panel 1 HMMP1-55K-03 (Merck Millipore) were carried out as per the manufacturer's instructions.", "LDH release was quantified at specified time points by using the cytotoxicity detection kit (Roche Diagnostics), according to the manufacturer's instructions.", "The maximum and background controls used were 0.1% Triton X-100-treated cells (maximum) and uninfected cells (background).", "Values were calculated by using the following equation: % LDH release: (sample-background)/(maximum-background) × 100.", "The necrosis positive control was 0.1% Triton X-100 (Sigma).", "BAL samples were obtained from the Tuberculosis Service at St Marys hospital, transferred to CL3 facilities and following passage through a 70μ cell strainer (VWR) and centrifugation the cells were pelleted and frozen in foetal calf serum plus 10% DMSO (Sigma Aldrich) at −80 °C until use in flow cytometry.", "The supernatant or BAL filtrate was filtered through a 0.2μ filter and stored at −20 °C until use.", "BAL samples were also acquired from healthy and asthmatic individuals too.", "We previously constructed a Mtb H37Rv mutant in the RD1 region Rv3871-Rv3879 which has a defective ESX1 secretion system [5].", "We infected the adherent THP-1 cells at a multiplicity of infection (M.O.I) of 10:1 with the wild-type and mutant strains and took supernatants at 24 h and 48 h post-infection (since the H37Rv ESX1 mutant exhibits a defect in cellular necrosis at 72 h post infection [5]).", "Along with the mutant and wild-type, infection was also carried out with a complemented mutant [5] and controls included uninfected and LPS-treated adherent human cells.", "Secretion of all of the innate cytokines measured in response to infection with Mtb was similar to that with infection with the Mtb ΔESX1 mutant (Figure 1a–f), with the exception of the chemokine fractalkine (Figure 2a, P = 0.041).", "Therefore, uniquely among the panel of chemokines and cytokines evaluated, fractalkine induction at 24 h post-infection in Mtb-infected THP-1 cells is ESX1-mediated.", "This difference was also observed at 48 h post-infection (data not shown) but in both cases was only observed in resting macrophages, the suggested preferred interstitial niche of the tubercle bacillus [14] and was not seen in cells activated by the Th1 cytokine interferon-γ (IFN-γ; Figure 2b).", "We also determined that fractalkine production was ESX1-dependent in primary human MDMs (Figure 2c, P = 0.040).", "In contrast, there was no production of fractalkine noted in mruine macrophages.", "The ESX1 region of Mtb is also associated with increased cellular necrosis and cellular egress at 72 h post-infection [5] so we assessed whether fractalkine plays a role in cellular necrosis at this time point using an anti-fractalkine monoclonal antibody, recombinant fractalkine and Triton-X 100 induction of necrosis as a positive control.", "Infection with Mtb H37Rv resulted in the induction of necrosis similar to the Triton-X 100-treated positive control but this was not reduced by the addition of anti-fractalkine (Figure 2d).", "Moreover, the addition of fractalkine itself did not increase necrosis over the basal uninfected level confirming that fractalkine does not induce macrophage necrosis in this system.", "Elucidation of the fundamental role of M. marinum-induced ESX-1-dependent MMP-9 secretion in zebrafish embryos [4] after we generated the above data prompted us to investigate ESX1-dependence of MMP-9 secretion in our human system.", "We assessed production of MMP-9 in THP-1 cells, both with and without IFN-γ, and in human MDMs and found no difference between Mtb H37Rv and the Mtb ΔESX1 mutant.", "This was also the case with cells activated with conditioned media from infected macrophages (Figure 3a–c).", "Although we observed substantial production of MMP9 in human alveolar epithelial cells (A549 cell line; Figure 3d) there was no ESX-1 mediated difference.", "The zebrafish immune system differs substantially from the human, so we studied several other MMPs (1, 2, 7 and 10) to see if an alternative MMP might be acting in the place of the zebrafish MMP9 and there was no observable ESX1-mediated difference in the MMPs 1, 2 7 or 10 (Supplementary Figure 1).", "Fractalkine's primary function is as a chemoattractant to both monocytic and lymphocytic cells.", "Accordingly we set up a modified transwell assay (see Figure 4a) in order to test for Mtb-induced ESX1-dependent cellular migration.", "Direct observational counting in a haemocytometer on blinded samples revealed a reduction in cellular migration in the Mtb ΔESX1-infected transwell experiment in all three healthy donors.", "This difference was significantly different to wild-type-infected cells and was restored by complementation of the ESX1 mutation (Figure 4b).", "The cytoplasmic tyrosine kinase Syk is required for monocyte/macrophage chemotaxis to fractalkine [15] and addition of the Syk inhibitor picetannol resulted in a similar migratory reduction to that observed with Mtb ΔESX1 confirming that Syk-dependent ESX1-mediated fractalkine production results in cellular migration towards Mtb-infected cells.", "We then determined the nature of these migratory cells using flow cytometry.", "We stained for dead cells using a live/dead discrimination kit then stained with the fluorochrome-conjugated antibodies, CD3-Pe-Cy7 and CD11b-APC-Cy7.", "CD3 is a pan-T cell marker and CD11b is an integrin also known as Mac-1 often used as a pan monocyte-macrophage marker and used in murine studies for macrophage depletion [16].", "While the CD3+ cell number exhibited no difference (Figure 4c), the number of CD11b+ cells was significantly reduced in the Mtb ΔESX1-infected transwell assay when compared to the Mtb wild-type-infected cells (Figure 4d).", "A similar reduction was seen in CD11b+ cell migration when the wild-type infected cells were treated with an anti-fractalkine antibody or the Syk inhibitor picetannol, but not with an anti-TNF antibody (data not shown).", "Thus, Mtb ESX1-mediated fractalkine production results in enhanced recruitment of CD11b+ monocytic cells, the niche for the tubercle bacillus, but not in CD3+ T cells, many subtypes of which are implicated in protection against tuberculosis.", "The Mtb ESX1 mutant does not exhibit a growth defect in human cells or in vivo in the murine model [5] and this was also determined to be the case in the transwell system by plating out colony forming units (CFU) from 0, 24, 48 and 72 h post-infection plus and minus anti-fractalkine antibody (data not shown).", "However, we hypothesised that the increased recruitment of niche cells for the bacilli would result in an increase in the percentage infection rate of the cells early after infection.", "Therefore, fluorescently labelled wild-type bacteria (FITC-labelled Mtb H37Rv) were used to infect MDMs in the modified transwell experiment with and without anti-fractalkine antibody and the numbers of infected cells determined using flow cytometry in the FITC channel.", "The percentage infected cells were significantly reduced upon treatment with anti-fractalkine, which reduces CD11b+ monocytic cellular migration (Figure 5a, P < 0.05).", "Direct observation, again blinded, of the infected monolayers was also carried out following Ziehl-Neelsen staining.", "This confirmed the above finding, showing reduced infection levels in the anti-fractalkine treated wells 72 h post-infection (Figure 5b).", "This also enabled us to count the number of bacilli per infected cell (Figure 5c) and there were significantly higher (P = 0.026) numbers of intracellular bacteria associated with the anti-fractalkine treated cells (mean = 35) than with the control (mean = 12) at this time point.", "Representative fields are shown in Figure 5d–g, where 5f and g show the anti-fractalkine treated wells and 5d and e the untreated wells.", "Blocking fractalkine thus results in higher numbers of bacilli per cell and fewer infected cells, indicating that fractalkine-mediated monocyte influx early in infection aids in bacillary spread.", "ESX1-mediated fractalkine production from infected macrophages thus results in increased influx and infection of monocytic cells and promotes bacillary dissemination.", "We sought to determine the involvement of fractalkine in granulomatous disease, employing sarcoidosis as an example of a non-TB disease associated with the formation of granulomas in the lung.", "Therefore, we obtained bronchoalveolar lavage (BAL) fluid from patients with TB (n13), sarcoidosis (n3), asthma (n3) and normal healthy (n3) controls, filtered the supernatants and measured fractalkine levels using a multiplex cytokine assay.", "The BAL fluid from TB patients contained significantly higher levels of fractalkine than the inflammatory but non-granulomatous lung disease asthma (P = 0.001) and the healthy controls (P = 0.004, Figure 6a).", "Sarcoidosis also exhibited significantly increased levels of fractalkine compared to asthma (P = 0.026) and healthy controls (0.028) suggesting an association with granulomatous disease.", "In 5 patients with active cavitatory pulmonary tuberculosis with consolidation, we enumerated the number of CD11b+ and CD3+ cells by flow-cytometry in BAL and correlated these with fractalkine levels (Figure 6b) in filtrates of the same BAL samples.", "The levels of fractalkine at the site of disease clearly correlated with the number of CD11b+ cells (Figure 6c, P = 0.021), but not CD3+ cells (Figure 6d, P = 0.383), thus suggesting that increased fractalkine in the lung is significantly associated with increased monocytic cell numbers and niche expansion.", "This increase in available niche cells would be expected to result in increased cellular infection and egress, potentially associated with more disseminated disease in the lung.", "A major limitation of the murine model of infection with Mtb is the lack of granuloma formation.", "We intra-nasally infected Balb/C mice with the Mtb ΔESX1 mutant and wild-type strains.", "No ESX1-mediated differential fractalkine (generally called lymphotactin in the mouse with structural differences to the human homologue [17]) production was observed in the lung lysates (as had been seen previously in murine macrophages in vitro) and no differential infiltration of CD11b+ or CD3+ cells was observed between the mutant and wild-type at days 1, 3, 7, 14, 21 and 28 (data not shown).", "In addition, there was no difference noted in colony forming units as previously determined [5].", "The early events in TB infection are likely critical to determining whether the outcome favours the host or the pathogen.", "Following inhalation the bacilli must infect and then replicate within its primary niche cell; the macrophage.", "At this early stage the absence of further permissive niche cells will halt the spread of infection and conversely the influx of niche cells would enable the infection to spread.", "Here we show that the tubercle bacillus utilises the very cell-type evolved to destroy invading pathogens in order to establish and locally disseminate infection by signalling sibling-cells and increasing its permissive niche.", "Our findings suggest that the Mtb-mediated influx of uninfected niche cells allows the bacteria to infect new host cells, disseminate and to set up a foothold of infection within the human lung.", "Elegant studies in the M. marinum-infected zebrafish model of infection describe how this early aggregation of macrophages, termed a granuloma, is utilised by the bacilli to facilitate bacterial growth and the spread of infection [3].", "However, although the M. marinum-zebrafish model has fundamentally advanced our understanding of early Mtb infection, zebrafish do not have lungs and so whether or not ESX1-dependent cellular aggregation and infectious dissemination occurs in the lung is unknown.", "In addition, zebrafish embryos lack a fractalkine homologue and also lack T and B cells [18], so whether preferential recruitment of macrophages also occurs in models with both lymphoid and myeloid cells in humans needs to be determined.", "Using a non-growth deficient Mtb ESX1 mutant, unlike the M. marinum mutant, here we provide the first evidence of ESX1-dependent specific fractalkine-mediated chemotaxis of monocytic cells to infected macrophages in the presence of lymphocytes.", "We further demonstrate a relationship between monocyte infiltration and fractalkine levels in tuberculous human lung.", "The chemokine fractalkine (CX3CL1) exists in a class of its own containing a unique C-X3-C motif and can exist in both membrane-bound and soluble forms, the latter of which has potent monocyte and T cell chemoattractant properties, higher than the chemokine RANTES [19].", "Fractalkine is secreted by many cell types including macrophages and can exist as a membrane bound molecule only in endothelial cells due to the presence of a mucin-like stalk, which is cleaved from the soluble form [20].", "Although fractalkine has been determined to be present at the site of disease in patients with pulmonary tuberculosis [21,22] its role in human tuberculosis was hitherto unknown.", "The cognate receptor for fractalkine, CX3CR1, is necessary for migration and not adhesion and shown to be expressed by the majority of monocytes, NK cells and about 10–15% of T cells [23].", "Interestingly, CX3CR1 is a requisite for rapid tissue invasion of resident monocytes at the site of infection in mice peritoneally infected with Listeria monocytogenes [24].", "We identified ESX-1-dependent induction of the chemokine fractalkine from host macrophages.", "A modified trans-well migration assay revealed ESX-1-mediated FKN-dependent migration of CD11b+ (a pan-macrophage marker) cells, with no significant effect upon migration of CD3+ (pan-T cell marker) cells at this sample size.", "The recruitment of uninfected macrophages by fractalkine by the Mtb-infected macrophage resulted in productive infection, increasing the proportion of infected cells and thus bacillary dispersal and dissemination.", "The addition of anti-fractalkine resulted in fewer infected cells with higher bacterial loads.", "We next assessed the role of fractalkine in vivo.", "In our murine experiments there was no fractalkine production and no ESX1-mediated difference in cellular recruitment of CD11b+ or CD3+ cells into the murine lung, suggesting this artificial model system which lacks granuloma formation and which, unlike the zebrafish-M.", "marinum model, is not a natural host/pathogen pair, is substantially different to human infection.", "In vivo in the human lung, fractalkine levels were elevated in BAL from TB patients compared to healthy controls and asthmatic controls and were moreover strongly correlated with the extent of monocytic cell infiltration.", "Fractalkine levels were also elevated in BAL from sarcoidosis patients, suggesting an association of fractalkine with pathogenesis of granulomatous disease, consistent with earlier observations in TB, sarcoidosis and Wegeners granulomatosis [22,25].", "The role of fractalkine in granulomatous disease clearly requires further investigation and, in the case of TB, it would be of interest to investigate ESX-1-mediated fractalkine-induction in a human in vitro mycobacterial granuloma model similar to that of Birkness et al. where the involvement of the cytokines IFN-γ and TNF-α was elegantly dissected [26].", "Fractalkine itself acts as a potent polariser of the Th1 immune response and its role in the adaptive immune response to Mtb remains to be fully elucidated.", "Increased fractalkine has also been associated with factors which increase the risk of the development of TB disease and of disease reactivation including age [27] and HIV infection [28].", "Further work is now required to correlate fractalkine levels with bacillary burden and patient outcomes including risk of disease progression and response to therapy.", "The type VII secretion system ESX1 system has also been implicated in cellular necrosis leading to increased cellular egress later in infection [5] and fractalkine could represent an early stage recruiting permissive cells prior to their release by necrosis.", "Indeed, abrogating the effect of fractalkine resulted in reduced CD11b+ cellular recruitment and reduced percentage infection, without affecting cellular necrosis.", "The ESX1-secreted protein ESAT-6 was responsible for the MMP9 production from neighbouring epithelial cells in M. marinum-infected zebrafish embryos [5,29].", "The complex secretory co-dependence of this system makes the exact role of each of the ESX1-secreted proteins difficult to dissect and this work is currently ongoing.", "In conclusion, Mtb ESX1-fractalkine-mediated CD11b+ cellular migration may play a role in establishment of a foothold in initial infection, maintenance and spread of bacilli through niche expansion.", "Our data moreover suggest that the fractalkine/fractalkine receptor axis could represent an attractive target for novel immunomodulatory interventions for treatment or prevention of tuberculosis infection.", "A human fractalkine analogue which inhibits CD11b+ chemotaxis in human MDM's has recently been described [30] and our findings support the clinical evaluation of this as a potential novel strategy to reduce the spread of the tubercle bacilli in vivo.", "All samples were taken with approval from St. Mary's REC for 5 years (REC Number: 07/H0712/85) and with written informed consent.", "All animal workwas carried out in accordance with the UK Animal (Scientific Procedures) Act 1986 and was approved by the animal use ethical committee of Oxford University.", "This work was funded by the Wellcome Trust.", "S. M. H-W.", "and A.L. designed the experiments and wrote the paper.", "S. M .", "H-W carried out the experiments with the exception of the mouse experiments which were carried out by E.T., O.M.K, L.G., L.P and A.S. provided the BAL samples and D. C. and K. P. helped with their analysis.", "S. M. H-W., T. H. And W. R. J Jnr created the Mtb ESX1 mutant.", "The authors declare no competing financial interests." ]	[]
Determination of unresolved heteronuclear scalar coupling constants by J(up)-HSQMBC	Long-range heteronuclear scalar coupling constants provide important structural information, which is necessary for obtaining stereospecific assignment or dihedral angle information. The measurement of small proton-carbon splittings is particularly difficult due to the low natural abundance of carbon-13 and the presence of homonuclear couplings of similar size. Here we present a real-time J-upscaled HSQMBC, which allows the measurement of heteronuclear coupling constants even if they are hidden in the signal linewidth of a regular spectrum.	[ "BIOC", "PHYS" ]	[ "Band-selective decoupling", "HMBC", "Homonuclear decoupling", "J-upscaling", "NMR spectroscopy", "Structure analysis" ]	[ "1303", "1304", "3104", "3106" ]	[ "NMR spectroscopy is one of the most powerful and frequently used tools for the analysis of small to medium sized molecules.", "Chemical shift, as well as scalar coupling information, play an important role for structural and conformational investigations.", "Long-range J-couplings are used to establish through-bond connectivities.", "They are necessary for example for the stereospecific assignment of carbon resonances attached to olefinic double bonds.", "Additionally, three-bond coupling constants provide information about dihedral angles through the Karplus relation [1].", "However, the extraction of accurate and/or small coupling constants is often complicated, especially when they are smaller than the digital or spectral resolution.", "For proton–carbon coupling constants, the situation is made even more difficult by the low natural abundance of 13C.", "While large one bond proton–carbon coupling constants (1JHC) can be determined easily from 13C satellites, heteronuclear correlations exceeding one bond, with J values of ∼0–15 Hz [2] are typically much more difficult to be measured [3–7].", "The situation is further complicated by the similar magnitude of proton–proton and proton–carbon coupling constants.", "Such nJHC (n > 1) can be measured for example by E.COSY type experiments [8,9], like the HETLOC (heteronuclear long-range couplings) [10–12].", "Although the HETLOC and its modifications provide a relatively sensitive technique for the measurement of small heteronuclear couplings in TOCSY-like spectra, there are several shortcomings connected with it.", "Firstly, correlations are limited to 1H–1H spin systems and the intensity of the cross peaks are dependent on magnetization transfers over this homonuclear correlations.", "But the more severe problem is spectral overlap in both dimensions, which is due to the limited spectral range of hydrogen.", "For these reasons, experiments used for the detection and measurement of long-range proton–carbon couplings are mainly based on the HMBC experiment [13].", "The determination of heteronuclear long-range couplings by direct inspection of HMBC peaks is basically impossible due to the evolution of homonuclear couplings during the pulse-sequence, combined with the anti-phase appearance of H–C couplings.", "Due to these complications, heteronuclear long-range coupling constants can only be obtained from HMBC spectra by line-shape fitting [14–17], which is made easier by avoiding proton–proton scalar coupling evolution [18–26] or by using the IPAP approach [27].", "Heteronuclear long-range coupling constants can also be obtained from the indirect dimension of J-resolved HMBC spectra [28–30] or by recording series of HMBCs with different coupling evolution delays and analysis of signal intensities as a function of this delay – the quantitative HMBC [31,32].", "One major problem of HMBC spectra is the limited resolution in the proton dimension.", "Pure shift NMR techniques by homonuclear broadband decoupling, have been described as a way to enhance the resolution of proton spectra [33–37].", "Pure shift versions of HMBC spectra have been described using homonuclear broadband decoupling through J-resolved spectroscopy [38], pseudo 2D slice-selective (Zangger–Sterk) decoupling [39] or very recently by PSYCHE-decoupling [40].", "These spectra are all essentially pseudo 3D experiments, since an additional decoupling dimension is required.", "Pure shift experiments typically suffer from severe sensitivity losses, which limit their application for carbon-correlated experiments at natural abundance.", "Real-time BIRD-decoupling [41], which offers the unique feature to enhance both the resolution and sensitivity is possible only for HSQC [42] but not for HMBC experiments.", "Sensitivity-enhanced homonuclear decoupled HMBC spectra can be obtained by using band-selective decoupling during acquisition [43–46] for a selective region of the proton spectrum [47].", "The resolution for the determination of long-range proton–carbon couplings is of course still limited by the signal line-width.", "We have recently shown that unresolved homonuclear proton scalar couplings can be visualized by real-time J-upscaling [48].", "This approach enables the determination of scalar coupling constants with high resolution from the direct proton dimension, even in cases of limited digital resolution.", "Here we show that real-time heteronuclear J-upscaled HMBC type spectra can be used to directly visualize small and even unresolved long-range JHC values.", "To prevent interference from homonuclear coupling it is combined with band-selective decoupling in the proton dimension.", "With this approach, up to 6-bond splittings (of less than 0.3 Hz) can be observed almost base-line separated.", "The enhanced resolution of scalar coupling constants by real-time J-upscaling results from the refocusing of magnetic field inhomogeneity effects during the interruption delay.", "While for large scalar coupling constants line-broadening by residual field inhomogeneity is negligible, it can significantly affect the determination of small coupling constants, even in well-shimmed systems [48].", "An additional advantage of real-time J-upscaling is the lower digital resolution needed in the detected dimension since the effective splittings are larger.", "Chunking artifacts arise in the spectrum around the signals because of residual evolution of homonuclear scalar coupling during the data chunks and the resulting steps between the individual acquisition blocks.", "The position of these artifacts is at the reciprocal of the data chunking length.", "By varying the length of the data chunks from scan to scan, the artifacts are reduced significantly [56].", "The maximum scaling factor is determined by the relaxation during the interruption.", "For small molecules up to ∼20–30-fold is possible.", "When using larger scaling factors it is necessary to reduce the length of the interruption delay (and in parallel the chunking size), in order to prevent scalar coupling evolution beyond 1/2J during 2τ.", "To demonstrate the limits of conventional, magnitude mode HMBC experiments [13] and the enhanced scalar coupling resolution of the heteronuclear J-upscaled HSQMBC technique, we applied the different experiments on a degassed phenylacetylene sample (50 mg in 600 μl DMSP-d6).", "The conventional HMBC is shown in Fig. 2.", "Correlations for the methine proton at 4.14 ppm are seen to all carbons in phenylacetylene.", "The one, two and three-bond couplings from H1 to C1 (J = 252 Hz), C2 (J = 50.4 Hz) and C3 (J = 4.0 Hz) can be extracted from a conventional HMBC since H1 does not show any homonuclear couplings in this spectrum.", "The splitting to C4 (J = 1.9 Hz) can only be seen in a band-selectively decoupled HOBS-HSQMBC after removal of unresolved homonuclear couplings.", "But also, this very powerful technique fails when dealing with very small heteronuclear coupling constants such as the ones to C5 and C6, due to linewidth limitations.", "Simple upscaling (with λ = 20) of a HSQMBC yields very broad peaks due to the combination of small homo- and heteronuclear couplings.", "However, the combination of band-selective decoupling with J upscaling provides the resolution necessary to determine the remaining five- and six-bond couplings to C5 (J = 0.37 Hz) and C6 (J = 0.25 Hz).", "Twenty-fold real-time upscaling allowed these couplings to be visualized in the direct dimension baseline-separated, while they are hidden in the linewidth of a conventional HMBC and even a homonuclear decoupled HSQMBC.", "Upscaling does increase the linewidth as can be seen in Fig. 2, but not by as much as the coupling is upscaled.", "Due to this broadening the intensities are also reduced compared to a conventional HMBC.", "However, band-selective decoupling increases the intensity by collapsing homonuclear splittings, like in the HOBS-HSQMBC.", "To demonstrate the validity of the coupling constants obtained from the upscaled HMBC spectra, a signal whose coupling constant could be extracted from a conventional HMBC has been upscaled.", "The C3–H1 cross peak of phenylacetylene shows a splitting of 4.0 Hz in a conventional NMR experiment.", "In Fig. 3 upscaled spectra of this cross peak are shown with λ ranging from 2 to 12.", "By dividing the measured coupling constant by the corresponding scaling factor the results are only varying by 0.07 Hz, with an average of 4.15.", "The slightly larger value compared to the 4.0 Hz obtained from a magnitude-mode conventional HMBC probably results from the broad components of the doublet in the latter spectrum, which leads to extracted coupling constants which are smaller than their actual values.", "One limiting factor of real-time J-upscaling experiments is the data chunk duration.", "We used a chunking length of approximately 10 ms, which allows upscaled coupling constants of up to 100 Hz (=1/10 ms) to be measured.", "A larger splitting would show the same evolution as a correspondingly smaller splitting according to the Nyquist theorem.", "Therefore, the maximum scaling factor that can be used depends on the size of the actual coupling.", "Signals with smaller J-values can be upscaled with larger λ values.", "Additionally, artifacts are becoming more severe with increasing scaling factor.", "To get an idea of the sensitivity penalty and artifact evolution during J(up)-scaling the C4–H1 correlation is compared in Supporting Fig. S1 without and with 20-fold upscaling.", "Although the signal intensity has dropped by a factor of 4 the extracted coupling constant is the same.", "In general, the amount of signal reduction depends not only on the scaling factor, but also the transverse relaxation time of the investigated signal.", "For slowly relaxing signals the reduction is lower [48].", "Small heteronuclear coupling constants can also be determined by in-phase–anti-phase (IPAP) type spectra, as implemented in the HOBS-HSQMBC sequence [47].", "To compare the potential of the J(up)-HSQMBC versus an IPAP HOBS-HSQMBC we applied the latter technique to the H1–C5 and H1–C6 couplings of phenylacetylene.", "The F2-traces of the in-phase spectrum (black) and an anti-phase spectrum (red) are shown in Fig. 4a.", "The heteronuclear scaling coupling constant is obtained by measuring the shift difference between the addition (black) and subtraction (red) of the two individual traces (Fig. 4b).", "The signal splittings obtained for very small coupling constants from an IPAP-HOBS-HSQMBC depends heavily on the data processing, in particular the phasing.", "Small differences in the phasing yield huge differences in the extracted signal splitting.", "Additionally, the weighting of both traces is quite tricky, as differences in the relative weighting of the in-phase and anti-phase signal also have a huge impact on the relative size of such small J values.", "The IPAP HOBS-HSQMBC yielded coupling constants of 6J = 0.21 Hz and 5J = 0.18 Hz, which are not only quite different from the ones obtained using J(up)-HSQMBC, but also (unrealistically) larger for the 6-bond compared to the 5-bond couplings.", "It should also be mentioned that the quantitative HMBC approach proved completely unreliable in an attempt to measure these small 5- and 6-bond couplings because after the very long coupling evolution delay needed there was basically no signal left due to relaxation losses.", "For well isolated proton signals, relatively short selective pulses can be used for decoupling during acquisition to keep relaxation losses during the interruptions to a minimum.", "The use of longer, more selective, pulses leads to broader signals due to faster relaxation during acquisition.", "However, Gaussian pulses of up to 20 ms (corresponding to an excitation width of 45 Hz) allow the extraction of J values below 0.3 Hz in a 20-fold upscaled spectrum (Fig. 5).", "The signal intensities decrease upon increasing pulse selectivity.", "For singlet proton signals without any other proton coupling partners, homonuclear decoupling is of course not necessary.", "An example is the application of the J(up)-HSQMBC for the measurement of two- and three-bond proton-carbon couplings of the central proton of the dye D131 (2-Cyano-3-[4-[4-(2,2-diphenylethenyl)phenyl]-1,2,3,3a,4,8b-hexahydrocyclopent[b]indol-7-yl]-2-propenoic acid) [15 mg in 600 μl pyridine-d5] in Fig. 6.", "For the assignment of the (2,2-diphenylethenyl)phenyl moiety, especially the stereospecific assignment of the phenyl rings b and c, the correlations of the locally isolated central hydrogen at 7.21 ppm to the phenyl and ethenyl carbons was key.", "In a conventional HMBC the correlations to C1b and C1c both show a splitting (9.2 and 7.2 Hz).", "However, the direct measurement of coupling constants would not be very accurate due to the rather broad doublet signals.", "The coupling to C2 is not resolved in an HMBC, and in the HOBS-HSQMBC the value is too small (1.9 Hz) because the splitting value is close to the linewidth.", "By upscaling the HSQMBC spectrum to λ = 4 not only the doublet to C1c is properly defined (3J = 7.0 Hz), it is now also possible to measure the coupling constant to C2 (2J = 2.6 Hz).", "All two- and three-bond coupling constants, which were essential for the full assignment, especially of the individual aromatic rings are indicated in Fig. 6.", "It might be worth mentioning, that in this case homonuclear band-selective decoupling does not improve the quality of the spectrum, because the long-range coupled protons are too close in frequency and the necessary selective pulse would be too long.", "In conclusion we have presented a method to record “real-time” heteronuclear J-upscaled HSQMBC spectra.", "This technique minimizes the effect of magnetic field inhomogeneities on scalar coupling and allows their direct visualization in cases where the splitting is hidden in the signal linewidth or is unresolved due to a limited digital resolution of a spectrum.", "All experiments were carried out on a Bruker Avance III 500 MHz spectrometer using a 5 mm TXI probe with z-axis gradients at 298 K. All chemicals were purchased from Sigma Aldrich at 98% purity or above.", "For the band selective excitation/refocusing of the protons a 1 ms 180° Gauss pulse was used.", "Sine shapes were used for all pulsed field gradients.", "Further experimental details are given for the respective spectra." ]	[ "Easy determination of real-time upscaled heteronuclear coupling constants in the acquisition dimension.", "Lower digital resolution necessary for a certain coupling constant.", "Band-selective decoupling increases sensitivity and removes homonuclear couplings." ]
Phase morphology, variants and crystallography of alloy microstructures in cold dwell fatigue	This paper examines microscale crystal slip accumulation, cold creep, and stress redistribution (load shedding) related to dwell fatigue in a range of α–β Ti alloy microstructures. The role of basal slip and prism slip is evaluated in load shedding in a rogue grain combination. The results enrich the Stroh dislocation pile up interpretation of dwell by accounting for the anisotropic rate dependence of differing slip systems together with morphology. Microstructural morphology has been found to play an essential role in cold creep and load shedding in dwell fatigue. Basketweave structures with multiple α variants have been shown to give the lowest load shedding for which the mechanistic explanation is that the β lath structures provide multiple, small-scale α variants which inhibit creep and hence stress relaxation, thus producing more uniform, diffuse stress distributions across the microstructure through microscale kinematic confinement, imposed by multi (α)-to-single (β) BOR relations (i.e. multiple α variants sharing the same parent β grain). The critical consequence of this is that alloys typically having multi-variant basketweave structure (e.g. Ti-6246), remain free of dwell fatigue debit whereas those alloys associated with globular colony structures (e.g. Ti-6242) suffer significant dwell debit. This understanding is important in microstructural design of titanium alloys for resisting cold dwell fatigue.	[ "ENGI", "MATE", "MATH" ]	[ "Basketweave", "Burgers orientation relationship", "Cold dwell fatigue", "Creep", "Microscale kinematic confinement", "Titanium alloy", "Variants" ]	[ "2209", "2210", "2211", "2500", "2611" ]	[ "Component failure due to cold dwell fatigue was first recognized from in-service behaviour in RR RB211 engine Ti-alloy discs on Lockheed Tristar aircraft in the 1970s [42].", "In this problem, the defining characteristics is that the inclusion of a dwell period in the stress loading history is found to reduce the fatigue life substantially (sometimes orders of magnitude), and has been argued to be through cyclic accumulation of plastic strain during cold creep [5] and Lefranc et al. [28,29] both in air as well as in high vacuum.", "The mechanisms of fatigue facet nucleation in titanium alloys have been studied by several authors including Dunne and Rugg [14].", "In the latter, the load shedding within a rogue grain (soft-hard) combination was thought to play a significant role in facet fracture near basal planes [47,56], and which potentially leads to extreme values in fatigue indicator parameters [40,41].", "The Stroh model [50] for the stresses developing at the termination of an active slip band at a grain boundary has been used to explain fatigue facet formation in titanium alloys by many authors [5,17].", "However, the Stroh model was based on 2D isotropic elastic plane theory [48], and does not consider either crystal slip nor its strain rate sensitivity (SRS) giving rise to local creep even at low temperature.", "Multiple slip systems, but predominantly basal and prism systems, may be activated by applied loading in α-β titanium alloys [8], and the intrinsic anisotropy of multiple slip systems operating may be associated with the pile up of dislocations.", "Pilchak et al. [37,38] addressed non-dwell facet growth in Ti–6Al–4V colonies and found facets growing along basal planes, although deviations as high as 35° were also measured.", "In the context of rogue (hard-soft) grain combinations, researchers have often assumed prismatic slip in the soft grain adjacent to the hard grain [34], since it has been believed to be more readily activated in titanium alloys.", "In contrast, Evans and Bache [17] proposed a scenario where the dislocation pile-up occurs on a basal slip plane in a soft grain which potentially leads to facet nucleation, and this has also been discussed by Sinha et al. [48].", "In addition, faceting on the adjacent basal plane of a hard grain has been identified as the most critical damage mode in leading to fracture in a commercial α/β-forged Ti–6Al–4V alloy [9].", "An explanation was proposed recognising the resolved shear stress, reflecting the local Schmid factor and the normal stress in relation to the elastic anisotropy of the α-phase.", "It has also been found that the elastic and plastic anisotropies are important in material rate dependence and in localization of plastic slip and microcrack nucleation [12,36].", "An anisotropic rate dependence in differing slip systems and phases has recently been reported for titanium alloys [59,60] where basal slip has been found to be intrinsically more rate sensitive at 20 °C than for prism slip.", "Pilchak [39] pointed out that in order to characterize the dwell fatigue crack initiation, the strain rate sensitivity and strain accumulation during the dwell period have to be recognised.", "The strain rate sensitivity is itself strongly dependent on microstructure which in turn primarily results from the bulk deformation forming and heat treatment processes utilised for the material manufacture.", "Microtextured regions, or macrozones are also a consequence for forming history.", "Woodfield et al. [56] have pointed out that material microstructures in the Ti alloys are critically related to the component dwell fatigue life.", "Processing route in the Ti alloys potentially leads to a substantial range of microstructures, including primary alpha, colony, bi-modal and Widmanstätten structures, resulted in very different uniaxial yield and ultimate strengths, tensile ductility [49] and crack development [32].", "Microstructures consisting of large regions of aligned, crystallographically oriented alpha plates, often referred to as macrozones [20] have been argued to be most susceptible to dwell fatigue [19,54,56].", "Britton et al. [10] noted that geometrically necessary dislocation density was found to be twice as high in the macrozone region than outside of it.", "However, macrozones have been suggested to contribute more significantly to crack coalescence and growth rather than to crack nucleation [27,30].", "With differing cooling rate, dual phase (α-β) titanium alloys may have remarkably different morphologies.", "For example, a cooling rate of 1 °C/min may lead to colony structures but a much faster rate of 8000 °C/min produces a Widmanstätten basketweave structure [31].", "Air cooling of near-α Ti-6242 produced by α/β processing may generate equiaxed structures [26].", "Interestingly, basketweave microstructures, containing multiple colonies of α and β laths within prior β grains, were more creep resistant than the bimodal microstructure in Ti-6242Si [7].", "The samples with basketweave structure have been found not to be dwell sensitive in β processed Timetal 685 [16], or in Ti-6242 alloys [51].", "Miller et al. [33] found creep strains to be smaller in basketweave structures compared to colony structures in Ti-6Al-2Nb-1Ta-0.8Mo alloy.", "They argued that larger colony sizes led to higher creep strains and that smaller colony size provided shorter distances to accommodate slip, and that the basketweave structure exhibited limited slip compatibility.", "In addition, the thickness of α platelets has been reported to affect creep behaviour [46].", "Recent pillar tests also show that thicker β laths produce more impedance to slip transfer through α-β-α phase boundaries than thinner laths [59] and the basketweave structure is less rate sensitive in α-β titanium [57] than that for colonies.", "In addition, alloy Ti-6246 often used for compressor discs in engines has been found to have less dwell sensitivity as opposed to Ti-6242 which has a very strong dwell debit [43], although it is noted that this may relate more to the resulting microstructures in each alloy as opposed to their chemistry differences.", "Hence the recent studies showing that Ti-6246 exhibits a significant time-dependent plastic strain accumulation, which is comparable to Ti-6242 during the stress holds [22], are interesting.", "Very recent work by Zhang and Dunne [57] finds the strain rate sensitivity to be lower in the basketweave structures, specifically in Ti-6246 with a greater number of α variants, than the usual colony structures in Ti-6242.", "Note that basketweave microstructures could have up to twelve α variants sharing a single β grain (multi (α)-to-one (β) BOR relation).", "In contrast, the colony structure has sandwiched α-β phase laths with each α-β interface having the same one (α)-to-one (β) BOR relation.", "These findings suggest an explanation for the long standing problem in dwell debit difference between alloys Ti-6242 and Ti-6246.", "The former is typically near-α (β volume fraction less than 15%), and most often has colony and lamella structures.", "Ti-6242 may also take a basketweave form which has been reported to have greater creep resistance than colony structures [51].", "Ti-6246, however, an α-β alloy having up to 45% β [3,24], is known to be dwell insensitive.", "A potentially important factor is that the greater β phase volume fraction is accompanied by more α variants thus likely to provide stronger microscale kinematic confinement effects.", "Hence it is suggested that the dwell sensitivity in commercial Ti alloys depends on multiple factors but that particularly, α-β microstructure (including morphology as well as crystallography) plays a critical role [57].", "It is argued herein that the alloy chemistry plays a lesser part, so that previous hypotheses that Ti-6242 is dwell sensitive whereas alloy Ti-6246 is not are over-general and do not recognise the importance of processing giving rise to microstructural differences.", "However, when the microstructural differences from processing are reflected in the material behaviour, then strong differences in dwell behaviour are indeed observed [4,6,43,57,59,61].", "Hence, the effectiveness of differing microstructures needs to be assessed systematically in cold creep behaviour both at the grain or structural unit-level and at the transgranular or polycrystalline-level so as to provide useful insights into cold dwell fatigue and the optimisation of dwell-resistant alloys.", "This paper presents a systematic study of the slip accumulation, constraint and stress redistribution (load shedding) in key Ti microstructures from pure α systems through to α–β colonies, Widmanstätten and basketweave structures.", "Basketweave microstructures are here differentiated from Widmanstatten as those containing multiple α variants compared to the single variant in the latter structures, whilst all variants satisfy the separate Burger Orientation Relationship (BOR).", "In addition, in the α–β microstructures, the β thickness with respect to the α laths is also investigated in respect of cold creep and stress redistribution.", "The methodology is to employ the dislocation based crystal plasticity modelling method in which basic phase property data has been established from direct micro-pillar testing to give intrinsic slip strengths and phase strain rate sensitivities.", "In this study, we adopt a rate dependent crystal plasticity with updated lattice rotation [15,59].", "The strain hardening is included through the evolution of geometrically necessary (GND) and statistically stored (SSD) dislocation density.", "This allows for the proper representation of the strain gradient effect introduced by grain/phase boundaries of multiphase polycrystal materials.", "Rate dependent cold creep leads to stress redistribution from ‘soft’ grains orientated favourably for slip on to hard grains not well orientated.", "Microscale stress redistribution takes place as a result of slip with differing rate sensitivity responding to the local stress state.", "It is therefore appropriate to examine in more detail the activation of slip in the soft grains and to establish the local stress redistribution, or load shedding, which takes place, and the role of more rate-sensitive local basal slip with respect to prismatic slip.", "This has been a long-standing question of relevance to understanding cold dwell fatigue.", "Soft-hard α grain interactions are governed by dislocation pile-up at the grain boundary which provides local stress build up and hardening.", "The Stroh [50] pile-up model has often been invoked to explain the stress concentration established at a soft-hard grain boundary by the slip taking place in the soft grain developing a pile-up at the soft-hard grain boundary [17].", "However, this model neglected the material rate dependence leading to creep in Ti alloys and hence local grain-level stress redistribution, or load shedding, and in addition did not address the local intrinsic anisotropy in slip system rate sensitivity.", "Researchers have often assumed the dislocation pile-ups occurred on prismatic slip systems [34].", "Recent experiments and numerical studies have shown distinctly different strain rate sensitivities in basal and prismatic slip systems [60] as discussed above.", "Most dwell fatigue facets have been reported to develop close to hard grain basal planes with the facet normal within about 15°from the loading direction [47].", "It is thus interesting to study the strain localisation and load shedding due to the soft grain slip resulting from both basal and prismatic slip systems, respectively.", "Firstly, the study is focused on the slip accumulation adjacent to the hard grains.", "As given in Fig. 1(b) and (c), the grains of interest are illustrated with unit cell orientations in white.", "The hard grains are labelled g1, g2, and g3 (less hard), where g1 and g2 are strictly hard as the normal to the basal plane (c-axis) is parallel to the loading direction.", "In this orientation, typically only pyramidal slip may be activated, but this is difficult since the associated critical resolved shear stress is about three times that of the basal and prism systems [21].", "Grain g3 is less hard with its c-axis about 10° to the loading direction potentially to allow basal slip when the resolved shear stress is sufficiently high.", "As shown in Fig. 1, slip is initiated in grain g0, and the crystal orientation for g0 is chosen to enable basal slip activation in Fig. 1(b) and (d) and prismatic activation in Fig. 1(c) and (e), respectively.", "In addition, they produce the same global Schmid factor of 0.5, and the active plane normal remains identically 45° to the Y axis and slip directions are exactly the same, highlighted by solid white arrows in Fig. 1(b)–(e), so as to introduce a sensible comparison of basal and prism slip accumulation.", "It is noted that the slip activation and accumulation adjacent to a boundary depends on the crystal orientation combinations and the local stress states developed (through elastic constraint) and the corresponding ‘yield’ strength and rate dependence are also orientation dependent [57], as demonstrated by Savage et al. [45] who reported orientation dependent creep behaviour in Ti-6242 colonies.", "Fig. 1(b) shows basal slip activation in grain g0 which initialized at point 1, and develops towards the soft-hard grain boundary.", "Here, the basal slip continues to develop from point 2 local to the grain boundary towards point 3.", "The stress state developed triggers new basal slip within grain g3 shown in the insert next to Fig. 1(b).", "Finally, the plastic flow develops back into the soft grain g0.", "In Fig. 1(c), prismatic slip is favoured because of the chosen crystallographic orientation.", "The development of accumulated slip follows a similar path of 1-2-3-4 as in Fig. 1(b).", "Comparing Fig. 1(b) and (c), it is evident that there is slightly higher slip accumulation established due to basal slip than for prismatic slip.", "This reflects recent micropillar experiments and numerical studies, where the intrinsic strain rate sensitivity is higher in the basal slip than for the prismatic slip [60].", "This is consistent with the work by Amouzou et al. [2], where the basal slip systems were found to be more rate sensitive than the prismatic in their numerical model in order that the macroscopic response matched the constant strain rate experiments and work hardening tests in their TD and RD textured samples.", "Changing the crystallographic orientation of the previously hard grain g1 to a soft grain as shown in Fig. 1(d) and (e) generates quite different slip fields.", "Given that slip may now be activated without being inhibited by the presence of a hard grain, a band of slip is seen to develop in a 45° direction, and the plastic strain magnitudes are much greater than that in Fig. 1(b) and (c).", "The slip development across grains due to the basal slip at grain g0, shown in Fig. 1(d), is more significant than that as a result of prism slip in Fig. 1(e).", "The independent slip system progressive contributions to slip accumulation and band development, represented by the instantaneous effective plastic strain, are shown in Fig. 2.", "Fig. 3 shows the loading direction stress, before and after the stress hold in the dwell test, along path A-A′ given in Fig. 1(a), and the (soft-hard) grain orientations are shown in Fig. 1(b) and (c), respectively for basal and prismatic slip.", "Basal slip activation in the soft grain during the stress hold leads to the redistribution of stress from soft g0 to hard g1 grains increasing the stress magnitude within the hard grain considerably (by 275 MPa) as shown in in Fig. 1(a), which is greater than that which occurs when prism slip is activated in the soft grain for which the redistribution of stress, or load shedding, is shown in Fig. 1(b).", "In this case, the increase in stress during the dwell is 240 MPa in the hard grain.", "Hence it is the case that basal slip activation in a soft grain at a soft-hard pair leads to more damaging load shedding than that for prism slip activation, because of the higher intrinsic SRS of basal slip compared to prism slip.", "A consequence is that dwell fatigue facets may preferentially develop at soft (basal) – hard grain combinations, although soft(prism.)-", "hard grain pairs may also be possible.", "The α-β morphology in Ti polycrystals has been demonstrated to affect global rate dependence [57], since the β laths act potentially to inhibit the motion of dislocations and dislocation path.", "TEM studies have been carried out by Suri et al. [52] providing great insight into local deformation mechanisms.", "The same microstructures were examined by Zhang and Dunne [57] who investigated microscale kinematic confinement, owing to polycrystalline heterogeneity, grain boundaries and the BOR relations, and showed that these features can reduce the resulting global rate dependence compared to what would follow from, for example, an unconstrained micro-pillar single crystal response.", "Hence here we study the grain, or structural unit level cold creep and dwell behaviour in pure α phase, α-β colony, and basketweave (multiple α variant) structures with differing β thickness.", "For the grain-level morphological study of creep behaviour, the polycrystal model in Fig. 1(a) is again utilised but with differing morphological manifestations in grain g3 as shown schematically in Fig. 4(a).", "Grain g3 is firstly a pure α rain in Fig. 4(c), but is also then chosen to represent a simple α–β colony structure, a simple four-variant basketweave structure with thin β laths, and finally a simple four-variant basketweave structure with thick β laths.", "The β laths modelled are thicker than would sometimes be observed (to facilitate CP modelling) in eg a Widmanstätten structure, but including two thicknesses allows comparisons to be drawn.", "The four-variant (α1 to α4) basketweave orientation combinations are shown schematically in Fig. 4(b), where for simplicity the different habit planes of the α-β interfaces have not been explicitly illustrated.", "When subjected to stress-controlled dwell loading, the resultant accumulated slip development is shown for each structure in Fig. 4(c) for pure α, Fig. 4(d) for a simple α–β colony structure, Fig. 4(e) for four-variant basketweave with thin β laths and Fig. 4(f) for four-variant basketweave with thick β laths.", "The thin β lath thickness is selected as 1.6 μm, within the 0.5–2 μm range in Ti-6242 colonies [44].", "It is doubled to 3.2 μm for the thick β laths.", "What is starkly apparent in Fig. 4 is the dramatic effect of the four-variant basketweave structure ((Fig. 4(e) and (f)) on inhibiting slip development during stress dwell as compared to the pure α grain and the simple α–β colony (Fig. 4(c) and (d)).", "When multiple α variants are present, growing out of the same original β matrix, each α variant tends to rotate differently from the β matrix under deformation though each of them initially remains a semi-coherent phase boundary satisfying the BOR relation to the parent β phase.", "Multiple variants generate a shorter slip length, interlocking, and inhibition of slip and cold creep accumulation resulting in microscale kinematic confinement.", "It is also very clear that the β lath thickness appears not to play a significant role in inhibiting slip accumulation (Fig. 4(e) and (f)) during dwell such that the over-riding conclusion is that it is the presence of the α variants in the basketweave structures, as opposed to the presence of the β laths, which is inhibiting the dwell effect and slip accumulation (cold creep) in these microstructures.", "This is consistent with recent findings by Kasemer et al. [25] that the lamellar width has little to no effect on yield strength, macroscopic ductility or hardening in dual phase Ti-6Al-4V alloy.", "Naturally, the β laths are indirectly important since their presence gives rise to the potential for many α variants, but the creep-inhibiting mechanism is mainly provided by the α variants themselves.", "Fig. 5(a) and (b) show the considerable load shedding which takes place during the stress dwell from soft (g3) structure to adjacent hard (g4) grain when the soft g3 structure is a pure α grain and a simple α–β colony structure.", "Peak stresses in the adjacent hard grain for these cases exceed 1200 MPa.", "In clear contrast, however, the stress redistribution or load shedding occurring during the stress dwell for the four-variant basketweave structure (g3) is significantly reduced leading to a much more uniform spatial stress distribution across the hard-soft grain pairing.", "Hence the role of the basketweave, and most particularly the α variants, is to inhibit slip accumulation (cold creep) within the α phase during dwell, such that stress redistribution is inhibited and load shedding minimised.", "This provides the most compelling mechanistic explanation, it is argued, for the observation that the basketweave structures are vastly superior to the colony structures in inhibiting cold creep and hence load shedding, and ultimately in demarcating why the dwell fatigue debit can be large (factor of ∼10 on life) for some Ti-6242 alloys but negligible for some Ti-6246 alloys in aero-engine service, depending crucially on their microstructures.", "The observed behaviour resulted not directly from the chemistry but from the two materials’ differing microstructures.", "This mechanistically explained the recent discrete dislocation work by Zheng et al. [61] in which the homogenised activation energies and volumes led to the inhibition of load shedding in a particular Ti-6246 alloy versus marked load shedding in hard-soft grain pairs in a given Ti-6242 alloy.", "The improved performance of basketweave structures in inhibiting slip lengths and pile-up and the transfer of slip across multiple grains is apparent in Fig. 6.", "This is addressed in more detail for primary α, colony and basketweave structures in Fig. 6 which shows the crystallographic variation along path C-C′ marked in Fig. 4(a) for the three structures considered, namely in Fig. 6(a) pure α, Fig. 6(b) simple α–β colony, and Fig. 6(c) simple four-variant α–β basketweave structure showing the BORs.", "For primary α in Fig. 6(a), the slip takes place on the same (single) prismatic plane, while in the α-β colony in Fig. 6(b), due to the presence of one (α)-to-one (β) BOR relation, limited inhibition of slip occurs across the α and β phases, as shown in Fig. 6(e).", "Recent experiments addressing slip transfer across an α-β phase boundary showed that the slip system with the smallest mismatch in plane normal is preferably activated for slip transfer across the phase boundaries [59].", "Since the first four laths along C-C′ in the basketweave structure in Fig. 4(e) have the same α1-β orientations as in the colony case, the mismatch of plane normal remains the same at 14°.", "As shown in Fig. 6(d), the first α2 variant gives a mismatch angle of 25° and other variants give mismatch angle between 25° and 35°.", "Therefore, the basketweave structure facilitates much more diffuse slip direction development, which is reflected by the more uniform strain distribution for the basketweave structure shown in Fig. 6(e).", "The accumulated plastic strain during creep remains limited to about 0.02 in the basketweave structures, 0.03 for the colony structures and 0.04 for the primary α in the g3 grain representations.", "Importantly, with thicker β lath in the basketweave structure, the creep strain can be decreased further compared to a thinner lath.", "Note that the initial α variants and β matrix crystal orientations shown in Fig. 6(c) in the basketweave structure are the same for the case of the thin and thick β lath study, and the very small difference shown in Fig. 6(d) arises due to the lattice rotation update which occurs with deformation.", "The thicker β lath modifies the local stress redistribution during creep since the high strain rate hardening in the β phase strengthens the β lath and reduces overall strain accumulation.", "Here, the heterogeneity of the basketweave α variant orientations is made clear with respect to that for colony and pure α structures.", "Fig. 6(e) shows the corresponding spatial variation of accumulated slip along path C-C′ after dwell fatigue for each of the microstructures demonstrating the dramatic reduction in cold creep in the soft grain (g3) for the α variant basketweave structures.", "A consequence of the dramatic inhibition of cold creep occurring during the stress dwell in the basketweave (g3) grain, which is well-orientated for α slip, is likely that the stress distributions borne by this grain are different than would be the case if it were pure α or α–β colony.", "This in turn has consequences for the stress redistribution, or load shedding, on to the adjacent hard grain (i.e. badly orientated for slip).", "A further consequence of the α–β morphology is the potential disruption of the local stress states important in nucleating slip.", "This is investigated through the distributions of local maximum principal stress shown within the soft grain (g3), and are examined firstly along path C-C′ (see Fig. 4(a)) in Fig. 7 for the pre-dwell stage (i.e. during the 20 s load-up) and post-dwell (after the 2 min stress hold) for each of the four microstructures discussed above.", "The loading history is shown in Fig. 1(f).", "The arrows indicate the directions of the maximum principal stresses.", "The distribution density of these arrows indicates the local stress concentration.", "For instance, localized stresses become more uniform in basketweave structures in Fig. 7(g) and (h), but rather concentrated in the grains in Fig. 7(e) and (f).", "There is some indication that the stresses tend to be preferentially carried by the β laths during the load-up shown in Fig. 7(a) to (d), particularly for the colony structure in (b).", "It is also noticeable that for the basketweave structures in (c) and (d), the stresses are much more uniformly distributed than for either pure α or a colony structured grain, indicating the role of the α variants together with the β laths is to cause a more diffuse and uniform stress distribution in the soft grain.", "The uniform stress distribution remains the case after the stress hold (dwell) for the basketweave structures in (g) and (h) particularly but in contrast, much bigger intragranular variations develop in both the pure α grain and the colony structure, suggesting much more significant stress redistribution into the adjacent grains during the dwell period because of the developing (cold) creep in the soft grain.", "The stress redistribution is investigated by considering the stresses which develop along path B-B′ (shown in Fig. 4(a)) which therefore provides information both for the soft grain (g3) and an adjacent hard grain (g4).", "The results are shown at the beginning and end of the stress hold in Fig. 5 for the range of grain g3 structures (pure α, α–β colony, four-variant basketweave with thin β laths and four-variant basketweave with thick β laths).", "The final part of this study examines truly representative morphologies associated with alloys Ti-6242 and Ti-6246 obtained from EBSD characterisation.", "In order to avoid reproducing figures, readers are referred to the earlier work of Zhang and Dunne [57], where the full geometric details of Ti basketweave structures with and without α variants are described in detail.", "The α-β colony structures in Ti-6242 and basketweave structures in Ti-6246 are illustrated in Fig. 8(a) and (b), respectively.", "The cold dwell loading utilised above is applied to these structures, with load-up to 90% of their 0.2% proof stress followed by stress dwell for two minutes to drive cold creep.", "The material behaviour is again taken to be that given by the α and β properties in Table 1.", "Here the macroscopic creep strain, obtained by averaging over the entire polycrystal finite element model, is examined to explore the effect of morphology in cold dwell fatigue resistant material design.", "Fig. 9 shows the results obtained for the four morphological structures assessed.", "Entirely consistent with the results obtained above for the model morphological structures, the basketweave structure with most α variants produces the lowest creep strain accumulation.", "Single phase α polycrystal structures, colony structures, basketweave structures without variants all give higher macroscale creep rates during the stress dwell, and the results confirm that the basketweave structure with multiple α variants leads to considerably reduced creep strain evolution, and that the creep rates obtained for α-β colony structures are in fact close to those for basketweave structures without variants.", "A more statistical study is carried out on the impact of the number of variants in the basketweave structures on the creep behaviour.", "Fig. 10 shows the distribution of effective plastic strains obtained from all points over the full polycrystal plasticity model in order to investigate the full range of strains obtained for the basketweave morphologies in which multiple α variants, labelled as “variant”, and single α variant, labelled as “no variant”, are included in the geometric model.", "The histogram frequency given in the vertical axis is the number of finite elements normalized by the total number elements in the crystal plasticity model giving rise to the strain levels indicated on the horizontal axis.", "It is found that the majority of elements in the basketweave structure without variants have developed plastic strains from 0.005 to 0.025.", "On increasing the α variant number to five in the basketweave structure, referred to as basketweave with variants in Fig. 10, the plastic strains are found to reduce significantly to lie between 0 and 0.01 in most elements.", "The average macroscale creep strain accumulation is seen to be diminished considerably by the multiple α variants, in effect reducing it by half.", "The effect of multiple α variants is also to increase the bimodality of the distribution leading to a substantial peak in microstructural locations where the creep strain is confined effectively to near-zero during dwell, in complete contrast to that for the single variant basketweave structure.", "This observation is consistent with the grain-level study in Figs. 7(g) and 7(h) in that more α variants generate a much homogenous stress state.", "This study provides a micromechanistic basis for why (colony) Ti-6242 suffers from substantial dwell debit while (basketweave) Ti-6246 is largely dwell insensitive.", "In addition, Ti-6246 may have up to 45% β volume fraction within which the α variants grow within larger parent β grains [24].", "The total number of α variants may be as high as twelve within a single prior β grain, and multiple α variants in the parent β grain are often observed.", "The Ti-6242 considered, however, is a typical near α titanium alloy with less than 15% β phase locating in α grains of about 10–20 μm, such that basketweave structures may have been inhibited, giving its non-optimal dwell performance.", "The findings from this study are potentially important in guiding choice of material microstructures to achieve better cold creep resistance and dwell fatigue life.", "They are summarised as follows.", "At a hard-soft grain pair under cold dwell loading, basal slip activation in the soft grain gives rise to more damaging stress redistribution, or load shedding on to the hard grain, than that for prism slip because of the basal slip system’s higher intrinsic rate sensitivity.", "The α–β colony structures, and basketweave structures with and without Burger Orientation Relationship variants all give rise to differing polycrystal strain rate sensitivities.", "However, it has been shown that basketweave structures with multiple α variants give the lowest SRSs and that it is the α variants which in the main drive the reduction.", "β laths within these structures provide the opportunities for multiple, small-scale α variants, hence indirectly contribute to the reduction of SRS.", "But the primary mechanistic basis is that the α variants inhibit slip accumulation and localisation, and hence stress redistribution and load shedding leading to a much more uniform and diffuse stress distribution across the microstructure.", "This, it is argued, is the mechanistic basis for why basketweave structures typical of alloy Ti-6246 do not show significant dwell fatigue debit whereas colony structures, often associated with alloy Ti-6242, show a very considerable dwell debit.", "Hence by careful microstructural design, new dwell-resistant Ti alloys are within reach." ]	[ "The Stroh model was revisited for the anisotropic rate dependence in hexagonal metals.", "In rogue grain combination, basal slip activation gives higher cold creep.", "Of the Ti alloy microstructures, multi-variant basketweave inhibits cold creep most.", "Multi-variant basketweave inhibits dwell fatigue by microscale kinematic confinement." ]
Social housing of non-rodents during cardiovascular recordings in safety pharmacology and toxicology studies	Introduction The Safety Pharmacology Society (SPS) and National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) conducted a survey and workshop in 2015 to define current industry practices relating to housing of non-rodents during telemetry recordings in safety pharmacology and toxicology studies. The aim was to share experiences, canvas opinion on the study procedures/designs that could be used and explore the barriers to social housing. Methods Thirty-nine sites, either running studies (Sponsors or Contract Research Organisations, CROs) and/or outsourcing work responded to the survey (51% from Europe; 41% from USA). Results During safety pharmacology studies, 84, 67 and 100% of respondents socially house dogs, minipigs and non-human primates (NHPs) respectively on non-recording days. However, on recording days 20, 20 and 33% of respondents socially house the animals, respectively. The main barriers for social housing were limitations in the recording equipment used, study design and animal temperament/activity. During toxicology studies, 94, 100 and 100% of respondents socially house dogs, minipigs and NHPs respectively on non-recording days. However, on recording days 31, 25 and 50% of respondents socially house the animals, respectively. The main barriers for social housing were risk of damage to and limitations in the recording equipment used, food consumption recording and temperament/activity of the animals. Conclusions Although the majority of the industry does not yet socially house animals during telemetry recordings in safety pharmacology and toxicology studies, there is support to implement this refinement. Continued discussions, sharing of best practice and data from companies already socially housing, combined with technology improvements and investments in infrastructure are required to maintain the forward momentum of this refinement across the industry.	[ "PHAR" ]	[ "3Rs", "Dog", "Methods", "Minipig", "Non-human primate", "Non-rodents", "Safety pharmacology", "Social housing", "Telemetry", "Toxicology" ]	[ "3004", "3005" ]	[ "The assessment of cardiovascular function (electrocardiogram (ECG), heart rate and blood pressure) within a non-rodent species is a regulatory requirement for all new chemical entities (NCEs) prior to first administration in man (ICH, 2001 S7A; ICH, 2005 S7B).", "This is usually performed as a safety pharmacology telemetry study (Leishman, Beck, Dybdal, Gallacher, Guth, Holbrook, Roche, & Wallis, 2012) in species such as the dog (Chaves, Zingaro, Yordy, Bustard, O'Sullivan, Galijatovic-Idrizbegovic, Schuck, Christian, Hoe, & Briscoe, 2007; Gauvin, Tilley, Smith, & Baird, 2006), Göttingen minipig (Authier, Gervais, Fournier, Gauvin, Maghezzi, & Troncy, 2011; Markert, Stubhan, Mayer, Trautmann, Klumpp, Schuler-Metz, Schumacher, & Guth, 2009) or cynomolgus macaque (Authier, Tanguay, Gauvin, Di Fruscia, Fournier, Chaurand, & Troncy, 2007; Chaves, Keller, O'Sullivan, Williams, Fitzgerald, McPherson, Goykhman, Ward, Hoe, Mixson, & Briscoe, 2006).", "With the development of non-invasive jacketed telemetry for dogs (Chui, Fosdick, Conner, Jiang, Bruenner, & Vargas, 2009; Prior, McMahon, Schofield, & Valentin, 2009; Ward, Milliken, Patel, & McMahon, 2012; Kremer, Bills, Hanke, Chen, Meier, Osinski, & Foley, 2015), minipigs (Ascah, Abtout, Pouliet, Bassett, & Authier, 2015; Diehl, Nungester, & Regalia, 2015) and cynomolgus monkeys (McMahon, Mitchell, Klein, Jenkins, & Sarazan, 2010; Derakhchan, Chui, Stevens, Gu, & Vargas, 2014), cardiovascular endpoints may be integrated into toxicology studies (Authier, Vargas, Curtis, Holbrook, & Pugsley, 2013; Redfern, Ewart, Lainée, Pinches, Robinson, & Valentin, 2013; ICH, 2009a M3(R2)), providing valuable data following repeat-dosing.", "For biopharmaceuticals or anti-cancer agents the toxicology studies may be the only source of cardiovascular data, as a stand-alone safety pharmacology study is not always required (ICH, 2011 S6(R1); ICH, 2009b S9; Vargas, Bass, Breidenbach, Feldman, Gintant, Harmer, Heath, Hoffmann, Lagrutta, Leishman, McMahon, Mittelstadt, Polonchuk, Pugsley, Salata, & Valentin, 2008).", "Housing practices that support good welfare are a top priority in designing a scientifically sound study to obtain high quality cardiovascular data.", "To generate more representative baseline physiology, avoid abnormal behaviour and for psychological wellbeing, social species, including many commonly used laboratory mammals, should be housed in stable, compatible pairs or groups (social housing).", "Dogs, minipigs and NHPs exist in groups in nature and social interaction is known to be required for psychological well-being (Prescott, Morton, Anderson, Buckwell, Heath, Hubrecht, Jennings, Robb, Ruane, Swallow, & Thompson, 2004; Ellegaard, Cunningham, Edwards, Grand, Nevalainen, Prescott, & Schuurman, 2010; Jennings & Prescott, 2009).", "When housed in social groups within the laboratory environment, these animals are able to cope more effectively with potential stressors, exhibit less stereotypical behaviour and have a more balanced temperament (see DiVincenti & Wyatt, 2001 for a review).", "Additionally, pair-housed dogs have been shown to have lower heart rates and fewer vocalisations (Klumpp, Trautmann, Markert, & Guth, 2006), or fewer stereotypic behaviours (Hetts, Clark, Calpin, Arnold, & Mateo, 1992) than dogs housed alone, whilst isolated pigs show a chronic stress response when compared to group housed pigs (Kanitz, Tuchscherer, Puppe, Tuchscherer, & Stabenow, 2004; Ruis, te Brake, Engel, Buist, Blokhuis, & Koolhaas, 2001a).", "Accordingly, the regulations and policies surrounding housing of non-rodents specially mention the requirement for social housing (e.g., Directive 2010/63/EU, 2010; NC3Rs, 2006; Institute for Laboratory Animal Research, 2011).", "These are often supplemented by non-governmental systems of oversight, such as voluntary participation by institutions in accreditation programmes (e.g., AAALAC international), company global animal welfare processes (e.g., AWERB/IACUC), and the peer review processes and grant terms and conditions of research funding bodies (e.g., ARRIVE guidelines; Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010; McGrath, Drummond, McLachlan, Kilkenny, & Wainwright, 2010).", "The most current updates to international animal welfare regulations specifically state ‘except those which are naturally solitary, shall be socially housed in stable groups of compatible individuals' (Directive 2010/63/EU, 2010) and ‘single housing of social species should be the exception’ (Institute for Laboratory Animal Research, 2011).", "Although it is a general practice to socially house non-rodents on safety pharmacology and toxicology studies on non-telemetry recording days, the majority of the pharmaceutical industry will separate animals for data collection on the specific telemetry recording days within a study (Ewart, Milne, Adkins, Benjamin, Bialecki, Chen, Ericsson, Gardner, Grant, Lengel, Lindgren, Lowing, Marks, Moors, Oldman, Pietras, Prior, Punton, Redfern, Salmond, Skinner, Some, Stanton, Swedberg, Finch, & Valentin, 2013; McMahon, Mitchell, Klein, Jenkins, & Sarazan, 2010; Ward, Milliken, Patel, & McMahon, 2012).", "This separation of animals is considered by some to be justified to allow conduct of the specific scientific procedure (telemetry recording), such that the overall objectives of the experiment can be achieved.", "Although animals are usually acclimatised to these instances of periodic isolation, this can contribute to increases in heart rate (Xing, Lu, Hu, Wang, Zhao, Zheng, Schofield, Oldman, Adkins, Yu, Platz, Ren, & Skinner, 2015) which may impact study data.", "However, with the goal of social housing as default, the industry has come together to investigate ways to move towards these preferred conditions.", "Integration of cardiovascular recordings into repeat-dose toxicology studies, and/or the requirement for longer duration or more frequent recording periods within a study (e.g., for compounds with long pharmacokinetic half-lives, or for biologicals with unique mechanisms of action), may lead to increasing amounts of isolation time compared to ‘standard’ safety pharmacology studies.", "This further emphasises the importance of refinement of housing practices during telemetry recordings.", "Historically, single housing of animals has partly been a consequence of limitations in the equipment used (e.g. spatial separation of animals was required to avoid cross-talk between signals transmitting on the same frequency).", "However, some systems have been available that allowed and supported recording from multiple animals within the same area due to transmissions on multiple frequencies (Klumpp, Trautmann, Markert, & Guth, 2006; Markert, Trautmann, Groß, Ege, Mayer, & Guth, 2012; Ward, 2009) but it has not been a standard feature across telemetry manufacturers.", "Recent technological advances now offer multiple frequencies to be transmitted and recorded as standard, thus increasing the feasibility of wider adoption of recordings from socially housed animals (Bétat, Coquelin, Infanti, Delpy, & Drieu la Rochelle, 2014; Kearney, Appleby, Kieper, & Atterson, 2016 (this issue); Milano, Bory, Dupuis, Chalencon, Lege, Fant, Webb, & Singer, 2013; Niehoff, Sternberg, Niggemann, Sarazan, & Holbrook, 2014; Tichenor, Regalia, Holzgrefe, Lilly, Wilcox, & Kaiser, 2016 (this issue)).", "Although this technological advance would address one of the major barriers to implementation of social housing during telemetry recordings, many companies still require additional information before investing in new equipment and infrastructure.", "One key barrier to change is that the validation of telemetry data acquisitions systems and generation of subsequent historical data sets have been mainly from single housed animals.", "There is also a perception that recordings from socially housed animals may be more variable than (or otherwise distinct from) single-housed animals due to increased interactions with pen/cagemates, leading to a decreased sensitivity (statistical power) to detect changes in cardiovascular parameters.", "Nevertheless, some companies have started to validate processes and approaches for running safety pharmacology and toxicology studies using animals grouped together in pairs, threes or fours for the duration of the study (Kaiser, Tichenor, Regalia, York, & Holzgrefe, 2015; Prior, Billing, Wallace, South, Oldman, Moors, Edmunds, & Milne, 2015; Xing, Lu, Hu, Wang, Zhao, Zheng, Schofield, Oldman, Adkins, Yu, Platz, Ren, & Skinner, 2015), in addition to those companies for whom this has been the standard for many years (Klumpp, Trautmann, Markert, & Guth, 2006).", "In 2015 the SPS (http://www.safetypharmacology.org) and the NC3Rs (http://www.nc3rs.org.uk) established a working group to collect information on current housing practices of non-rodents during the collection of cardiovascular telemetry data on both safety pharmacology and toxicology studies.", "Further objectives were to share experiences and canvas opinion on the study procedures/designs that could be used for socially housing animals during telemetry recording periods and what barriers exist preventing this.", "The working group is facilitated by the NC3Rs and currently represents seven global pharmaceutical companies and six CROs (see author affiliations).", "The working group contains some companies with experience of successfully implementing social housing to others with no experience of this.", "The data within this manuscript represent the outcome of an international industry survey conducted in the summer of 2015 and a workshop at the SPS Annual Meeting in October 2015.", "The questionnaires were sent to targeted individuals within the safety pharmacology and toxicology communities worldwide, with responsibility for either running studies in-house or for outsourcing this work.", "Different questionnaires were sent to CROs and Sponsors, to allow for the capture of information from groups who run studies themselves (categorised as CRO or Sponsor in-house) and those that outsource studies (Sponsor outsource).", "Questions were asked around the themes below, for safety pharmacology and toxicology studies separately, and with separate responses for dog, minipig and NHP (specified as the cynomolgus macaque) as appropriate:", "How do you house non-rodents on telemetry recording and non-recording days?", "What are the barriers preventing the social housing of non-rodents on recording days?", "(respondents were requested to choose multiple reasons from a list and then indicate the major reason)", "What are the barriers preventing the utilisation of a companion animal on recording days in safety pharmacology studies?", "Do you have any positive or negative experiences with social housing during recording sessions?", "Is there an impact on the data quality if the animals are socially housed during telemetry recording?", "How many animals, and which sex, do you use for safety pharmacology telemetry studies?", "Comment boxes were provided throughout the survey to allow submission of additional information and expression of any concerns.", "Definitions were clarified within the survey as follows:", "Social housing: an animal is housed with at least one pen/cage mate for 24 h a day (apart from veterinary reasons for single housing);", "Partial social housing: an animal is housed with at least one pen/cage mate for the majority of the day, apart from during study procedures (e.g., separation for recording of food consumption or clinical sign observations immediately post-dose);", "Individual housing: an animal is housed alone in the pen/cage (note, the animal may or may not have contact with other animals through the bars or adjoining pens/cages).", "Companion animal: an additional animal is housed in the same pen/cage as the recorded animal.", "This animal may be part of the study (e.g., dosed and recorded on a different day) or may be an undosed animal (e.g., part of the stock colony) provided solely for companionship.", "Respondents were requested to provide answers focussed on the most representative/most frequently run cardiovascular telemetry study designs at their facility.", "Therefore, the responses may relate to early (non-GLP) investigative studies and/or ‘regulatory’ GLP studies intended for regulatory submission.", "Respondents were requested to provide answers for routine regulatory toxicology studies.", "Multiple replies were received from some companies, reflecting the presence of these companies at different sites worldwide.", "As these sites may have different standard practices, they were included in the analysis as separate replies.", "Where a Sponsor only outsourced studies, their opinions regarding the barriers to the adoption of social housing were included in the results.", "Eighteen CRO and 21 Sponsor sites responded.", "72% ran safety pharmacology telemetry studies and 44% ran toxicology studies with cardiovascular telemetry recording in their facility in at least one of the species.", "The number of replies across the different species and types of studies are presented in Fig. 1a.", "A larger number of Sponsors conduct safety pharmacology studies in-house when compared to toxicology studies, which the majority of Sponsors appear to outsource.", "Replies were received mainly from companies within USA (41%) or Europe (51%), (Fig. 1b).", "The majority of respondents used four animals of a single sex for safety pharmacology studies.", "However, there was some variation in the number of animals used and evaluation of both sexes (Fig. 2), increasing the number up to eight animals per study.", "There was little difference in the number of animals used according to species, with the majority of respondents using the same number of animals regardless of species (i.e., 4, 6 or 8).", "Acclimatisation procedures vary widely across the respondents, according to where in the facility animals are housed in relation to stock colonies.", "54% of responses indicated that animals are housed in the same room as the recording pen/cages, but might move to another pen within the room for the recording period.", "Some animals are housed in a different room outside of studies, but move to recording pens/cages in a dedicated room for studies.", "For these animals, acclimatisation times to the recording pen/cage range from 0 to > 12 days, however most respondents (75%) acclimatise for 1–6 days.", "The majority of respondents (79%) indicated that the animals then remain in the recording room throughout the study rather than being moved back to a stock area between recording sessions.", "The majority of respondents social house or partially-social house their animals on non-recording days (84, 67 and 100% of respondents for dogs, minipigs and NHPs respectively).", "However, this reduces on recording days to 20, 20 and 33% of respondents, respectively (Fig. 3).", "The majority (7/9) of respondents that house socially during the recording period are from European countries and this practice is slightly more prevalent in studies using NHPs than dogs (5 vs 8).", "Reasons for not housing animals socially during recordings were similar across species (Table 1, Safety Pharmacology Telemetry column).", "The top three major reasons were i) limitations of the recording equipment, ii) study design requirements and iii) temperament/activity of the animals.", "The majority of respondents listed four to six reasons preventing social housing, indicating that although the limitations in recording equipment is the major barrier, there are still many other barriers.", "If the equipment does not allow recording of telemetric data from multiple animals in the same area, one option to maintain social housing is to use a companion animal.", "Some respondents had experience implementing this approach (7/25 for dog, 3/15 for minipig and 6/23 for NHP respondents).", "Reasons for not housing animals with a companion were similar across the species and included the risk of cross-contamination, sponsor expectations, fate of the un-dosed animal, impact on costs/size of colony and size of cages/number of available cages (Table 1, Companion Animal Telemetry column).", "The majority of respondents listed one to three reasons preventing social housing with a companion, indicating that although the risk of cross contamination to the un-dosed animal is the major barrier, there are still other reasons and barriers to overcome.", "The majority of respondents with experience of social housing for safety pharmacology studies indicated that the data quality was the same or better than data obtained from individually housed animals (9/10 for dog, 4/4 for minipig and 10/11 for NHP respondents, respectively).", "Further expansion of these qualitative answers was not requested within the survey.", "Overall, the industry is receptive to social housing during recordings on safety pharmacology telemetry studies and some companies have already implemented social housing (CROs: 3 dog, 2 minipig and 5 NHP respondents; Sponsors: 2 dog, 1 minipig and 3 NHP respondents).", "Other companies are considering social housing in the future (Fig. 4); however, a different pattern is evident between CROs and Sponsors running studies in-house.", "For CROs, 10/12 dog respondents, 6/7 minipig respondents and 9/11 NHP respondents who had not yet implemented social housing were actively planning to do so.", "Conversely for Sponsors, only 1/5 dog respondents, 0/1 minipig respondents and 1/2 NHP respondents who had not yet implemented social housing were actively planning to implement this.", "There are a number of possible methods utilised for the recording of cardiovascular data within regulatory toxicology studies:", "snapshot approach (use of external ECG electrodes and blood pressure cuff attached for short periods of time at specific timepoints and days within the study),", "use of a jacketed telemetry system (external ECG electrodes attached, sometimes with blood pressure cuff or minimally-invasive blood pressure implants in addition, covered with a jacket holding the hardware for continuous telemetric transmission on specific days within a study), and", "implanted telemetry device (surgical implantation of telemetry hardware for continuous telemetric transmission of ECG and blood pressure waveforms on specific days within a study).", "The majority of respondents have the capability to utilise all three of these methods within their facilities (Table 2).", "However, the percentage of studies performed with jacketed telemetry technology was low, with most respondents (65%) indicating that only 1–40% of their studies used jacketed telemetry (Table 3).", "For the remainder of the questions the focus was only on studies using jacketed telemetry, as this method of recording may impact the housing of the animals. [", "Snapshot ECG collections (the second most commonly used toxicology method) has no impact to housing practices, as animals are removed to procedure rooms for this.", "Implanted telemetry technology will encounter the same barriers as the safety pharmacology recordings described previously.]", "Unlike safety pharmacology studies, the majority of respondents indicated that the animals remain in the same pen/cage for the recording sessions as for the rest of the toxicology study (e.g. animals are either individually housed throughout the study, or the pens for socially housed animals are divided and animals are kept separate for recordings).", "The majority (75%) of respondents acclimatise the animals to the jackets prior to the first recording occasion only.", "The number of acclimatisation sessions ranged from 0 to 9, with the majority of respondents indicating that they acclimatised for 1–3 occasions prior to the first recording session.", "There were no apparent differences in the number of acclimatisation sessions used for the different species.", "The majority of respondents social or partially-social house their animals on non-recording days (94, 100 and 100% of respondents for dogs, minipigs and NHPs respectively).", "However, this reduces on recording days to 31, 25 and 50% of respondents, respectively (Fig. 5).", "For respondents that house socially during the recording period there was a similar number of European (5) and non-European (4) countries, however, similar to the safety pharmacology results this practice was more prevalent in studies using NHPs than dogs.", "Reasons for not housing animals socially during recordings were similar across the species (Table 1, Toxicology Telemetry column).", "The top four major reasons were i) potential damage to the recording equipment, ii) technical limitations of the recording equipment, iii) food consumption recording and iv) temperament/activity of the animals.", "The majority of respondents had several reasons for not housing socially, with some citing up to eight reasons, indicating that although the potential damage to equipment is the major barrier, there are still many other barriers.", "The majority of respondents with experience of social housing during toxicology studies indicated that the data quality was the same or better than from individually housed animals (8/9 for dog, 3/3 for minipig and 8/9 for NHP respondents, respectively).", "Further expansion of these qualitative answers was not requested within the survey.", "Although a low number of responses were received for this question, some companies are considering social housing during cardiovascular recordings within toxicology studies in the future.", "For CROs, 2/4 dog respondents, 1/1 minipig respondents and 2/3 NHP respondents who had not yet implemented social housing were actively planning to do so.", "For Sponsors, 1/3 dog respondents and 0/1 NHP respondents who had not yet implemented social housing were actively planning to implement this.", "A similar number of CROs and Sponsors have already implemented social housing (CRO: 4 dog, 2 minipig and 5 NHP respondents; Sponsor: 1 dog, 0 minipig and 3 NHP respondents).", "The purpose of this survey and the subsequent workshop was to describe current practices for housing non-rodents during cardiovascular telemetry recordings in safety pharmacology and toxicology studies, with the further goal of identifying opportunities for the expansion of social housing across entire study durations.", "Although the majority of respondents already house animals socially on non-recording days, most revert to individual housing on days when cardiovascular data is acquired.", "However, many respondents were in favour of implementing social housing during recordings, with some companies already evaluating conducting studies this way, and others considering this change.", "A number of concerns were raised within the survey, which will require the drug development industry as a whole to address in order to increase acceptance and adoption of this refinement.", "Whilst most of the respondents were based within Europe and the USA, we received an appropriate number of responses from the facilities actively running these studies (within both CROs and pharmaceutical companies), and from the companies outsourcing this work to CROs to have a representative view of the industry at-large.", "With the recent decline in R&D productivity (increases in R&D costs with output of new drugs remaining static, Cook, Brown, Alexander, March, Morgan, Satterthwaite, & Pangalos, 2014) better and more efficient strategies are urgently required.", "However, changes to established ways of working can require careful consideration and validation.", "Ideally, a change in drug development processes would result in a reduction in costs and timelines, whilst improving data quality.", "In reality, although it is not always possible to improve all three, it is still advantageous to improve one whilst maintaining the others.", "The challenge for the industry is to identify and apply the benefits of social housing in terms of animal welfare, quality of science, cost and drug development timelines.", "As seen in Table 1, there is a wide range of reasons cited for not implementing social housing during telemetry recordings.", "These reasons can be generally divided into ‘physical aspects’ (e.g. the size of pens/cages, or the telemetry equipment available within the facility) or ‘risk perceptions’ (e.g. the expectation of reduced data quality or the risk of cross-contamination).", "Sharing of experiences and demonstration of the successful use of socially housed animals in these studies may help overcome some of the assumed risks.", "Information within this manuscript is intended to provide reassurance and potential solutions to those facilities that are planning to use socially housed animals in the future.", "For facilities that have not yet considered social housing, information within this manuscript may lead to some discussions for interim measures and/or future refinements.", "This survey has highlighted an increase in outsourcing of studies encompassing cardiovascular measurements compared with previous surveys (Ewart, Gallacher, Gintant, Guillon, Leishman, Levesque, McMahon, Mylecraine, Sanders, Suter, Wallis, & Valentin, 2012; Lindgren, Bass, Briscoe, Bruse, Friedrichs, Kallman, Markgraf, Patmore, & Pugsley, 2008), as organisational changes within the pharmaceutical industry continue the trend for consolidating work at CROs (Ewart, Milne, Adkins, Benjamin, Bialecki, Chen, Ericsson, Gardner, Grant, Lengel, Lindgren, Lowing, Marks, Moors, Oldman, Pietras, Prior, Punton, Redfern, Salmond, Skinner, Some, Stanton, Swedberg, Finch, & Valentin, 2013).", "The survey also shows a preference of pharmaceutical companies to use socially housed animals in studies involving telemetry recording.", "However, the lack of capital investment to support these changes in-house may be partly responsible for the expansion in outsourcing.", "The survey indicated that very few pharmaceutical companies run all their safety pharmacology studies in-house, with even fewer running toxicology studies in-house.", "Most rely either entirely on CROs to run these studies, or in combination with in-house work.", "Therefore, the increased implementation of social housing may lie primarily within the CRO sector.", "However, the views and acceptance of Sponsors (mainly within the pharmaceutical and biologicals industry) will also be an important factor, as their requirements will influence the CRO market.", "The survey indicated a need to improve communication on this topic as Sponsors commented that they would appreciate the availability of social housing options at CROs, whereas comments from CROs indicate there are few requests for these conditions from Sponsors.", "The main barriers to adoption of social housing are discussed below and are summarised in Tables 4, 5 and 6.", "Social housing is facilitated when using a telemetry system having independent frequencies for each animal.", "Although the recent introduction of new technologies should enable the concept and methods for social housing during telemetry recordings to be available to a wider number of companies, the principles of this have been available to users of other technologies for some time.", "Whilst some groups using these alternative technologies have socially housed during safety pharmacology (Klumpp, Trautmann, Markert, & Guth, 2006; Markert, Trautmann, Groß, Ege, Mayer, & Guth, 2012) or toxicology studies (Ward, 2009), others did not (Prior, McMahon, Schofield, & Valentin, 2009; Chaves, Zingaro, Yordy, Bustard, O'Sullivan, Galijatovic-Idrizbegovic, Schuck, Christian, Hoe, & Briscoe, 2007; Sivarajah, Collins, Sutton, Regan, West, Holbrook, & Edmunds, 2010).", "This difference is likely to be due to many of the reasons highlighted within this survey.", "Many of the companies surveyed will upgrade to new equipment in the future that allows for social housing, as the sale of and/or support for older systems are phased out by the manufacturers.", "Therefore, the most common technical barrier to the adoption of social housing during recordings will disappear over time.", "Individual facilities are unique in the way their telemetry recording rooms and stock colony holding rooms are arranged, and it can be difficult to make major changes to this infrastructure (requiring time and capital investment) unless there is a specific reason or desire to do so.", "European facilities comply with, or exceed, the updated animal welfare laws (Directive 2010/63/EU, 2010) so these pens tend to have larger available areas per animal, which may facilitate social housing during recordings.", "Many of these companies have other facilities worldwide (or exchange studies within different countries, so may harmonise practices), and this may increase the uptake of social housing through staff exchanges, technical talks, in-house 3Rs awards, etc.", "The business/financial decisions to make these infrastructure changes would be strengthened by 1) a change in regional directives (e.g. as observed in Europe), 2) scientific justification/acceptance within the industry and 3) Sponsor requests to CROs.", "Some facilities hold animals between studies in large groups in separate rooms, then move animals to individual pens/cages for recording.", "If these pens/cages have doors/hatches to open up to allow at least two animals to socialise during periods of non-recording, with suitable equipment it should also be possible to allow this to continue during recordings.", "If facilities have pens/cages of sufficient size to house two animals (complying with local laws) but their telemetry equipment is not able to record from both simultaneously, the inclusion of an unrecorded companion animal should be considered.", "One option is for the companion animal to also be part of the study, but dosed and recorded on a different day.", "However, this could prolong the study duration due to the staggered dosing.", "Alternatively, the companion animal may be an undosed animal and not included in the study (e.g. part of the stock colony), but instead provided solely for companionship.", "However, there is a potential risk of cross-contamination of the companion animal, which could restrict possible future use of this animal on another study.", "There are also concerns that inclusion of a companion animal could increase the overall number of animals used and potentially the size of stock colonies, however, as the companion animal is not undergoing study procedures (dosing, etc), this is still considered a refinement for the study animal.", "Study designs and concerns around abnormal/increased activity due to the second animal are addressed below.", "There is a growing pool of data available comparing cardiovascular data from socially housed animals with data from individually housed animals, in both safety pharmacology and toxicology study settings.", "Studies showing no significant difference in cardiovascular data in socially housed compared with individually housed animals have been published in dogs (Kearney, Appleby, Kieper, & Atterson, 2016 (this issue); Prior, Billing, Wallace, South, Oldman, Moors, Edmunds, & Milne, 2015; Sadekova, Boudreau, & Norton, 2015; Sadekova, Boudreau, & Norton, 2016 (this issue)) and cynomolgus monkeys (Kaiser, Tichenor, Regalia, York, & Holzgrefe, 2015; Tichenor, Regalia, Holzgrefe, Lilly, Wilcox, & Kaiser, 2016 (this issue)).", "In contrast, other publications indicate that lower heart rates were obtained in socially housed cynomolgus monkeys (Bétat, Coquelin, Infanti, Delpy, & Drieu la Rochelle, 2014; Kreckler, Grosh, Schneider, Niehoff, Foley, & Osinski, 2015; Xing, Lu, Hu, Wang, Zhao, Zheng, Schofield, Oldman, Adkins, Yu, Platz, Ren, & Skinner, 2015) and dogs (Klumpp, Trautmann, Markert, & Guth, 2006; Ward, 2009) compared to individually housed animals.", "A change in heart rate for socially housed animals could be interpreted a number of ways.", "For instance: an increase could indicate higher stress levels, or it could be a consequence of more interactions between the animals and/or more movement in what might be a slightly larger pen/cage than used for individual animals.", "A decrease in heart rate might indicate lower stress in a social setting, or it might indicate animals have ‘settled’ quicker after disturbances as they have a companion to buffer stress (Sanchez, McCormack, & Howell, 2015; Ruis, de Groot, te Brake, Dinand Ekkel, van de Burgwal, Erkens, Engel, Buist, Blokhuis, & Koolhaas, 2001b).", "It will be important for those implementing social housing to understand and interpret the effects of social housing on cardiovascular data in their own facilities.", "The standard study design for safety pharmacology studies is the Latin-square cross-over design (Guth, Chiang, Doyle, Engwall, Guillon, Hoffmann, Koerner, Mittelstadt, Ottinger, Pierson, Pugsley, Rossman, Walisser, & Sarazan, 2015), which is widely accepted as conferring the best statistical power (sensitivity to detect changes in cardiovascular parameters of interest; Aylott, Bate, Collins, Jarvis, & Saul, 2011; Leishman, Beck, Dybdal, Gallacher, Guth, Holbrook, Roche, & Wallis, 2012).", "Some facilities performing studies with socially housed animals have retained the Latin-square design, whereby each animal within the pen receives a different dose level, with no reports of cross-contamination in over 15 years of using this approach, based on drug exposure confirmation with each study (Guth, 2015, personal communication).", "Other facilities have adopted a slightly more conservative approach, whereby a ‘partial’ Latin-square is used, such that the two animals within the pen receive the same dose level (Prior, Billing, Wallace, South, Oldman, Moors, Edmunds, & Milne, 2015) and report similar sensitivity to previous Latin-square designs.", "Alternatively, an ascending dose design, whereby all the animals within the pen/cage receive the same dose level may be an even more conservative option, and this design can exhibit similar statistical power (Ewart, Milne, Adkins, Benjamin, Bialecki, Chen, Ericsson, Gardner, Grant, Lengel, Lindgren, Lowing, Marks, Moors, Oldman, Pietras, Prior, Punton, Redfern, Salmond, Skinner, Some, Stanton, Swedberg, Finch, & Valentin, 2013; Prior, Billing, Wallace, South, Oldman, Moors, Edmunds, & Milne, 2015; Guth, Bass, Briscoe, Chivers, Markert, Siegl, & Valentin, 2009) to Latin-square designs, although may be confounded by day effects.", "The adoption of a Latin-square study design may be dependent on the known properties of the compound, the dose levels chosen, and the species.", "For example, it is known that dogs in the laboratory may ingest vomit and/or faeces from pen/cagemates and cynomolgus monkeys spend substantial time grooming/licking pen/cagemates.", "If the chosen dose level is not expected to cause emesis (e.g. a well-characterised compound, or a test compound where the maximum tolerated dose (MTD) is known) then the risk of cross-contamination may be considered low.", "Inclusion of minimal blood sampling for confirmation of drug-exposure may be useful to identify any deviations due to cross-contamination within socially housed animals and publications exploring the incidence of cross-contamination using these study designs would be valuable.", "As study sensitivity and test article-characteristics are unique to each facility, the study design for socially housed safety pharmacology studies needs to be assessed within the validation studies and on a study-by-study basis.", "The measurement of food consumption from individual animals is not a requirement within safety pharmacology studies, however it is often recorded as a measure of general clinical condition of the animal.", "Depending on the time of day food is offered, and the timing in relation to any critical periods of cardiovascular monitoring, animals could be separated for a short period of time (e.g. 1 h) to allow these data to be obtained, and some facilities are using this approach (this was the definition for partial social housing within the survey).", "Other facilities simply provide each animal with a separate food allowance and make careful observations on the consumption.", "Collection of food consumption data is often a requirement for toxicology studies; however, as animals are generally socially housed on other days within the study, the cardiovascular recording should not impact these data if the same procedures are followed.", "Efforts should be made, however, to reduce the disturbance to the animals these procedures may cause on the cardiovascular recording days.", "Whole group or cage assessments for food consumption are acceptable within toxicology studies, particularly for NHPs.", "Automated systems may be implemented on these types of studies to measure food consumption from socially housed non-rodents, similar to those already available for rodents (Krohn, Farlov, & Hansen, 2011) or to those used within academia (Wilson, Fisher, Fischer, Lee, Harris, & Bartness, 2008) or more sophisticated systems used in pig production facilities e.g. Young & Lawrence (1994).", "Clinical signs can be monitored in safety pharmacology and toxicology studies via closed circuit television cameras (CCTV), but a method of individual identification may be required in socially housed animals.", "Animals could be marked non-invasively on the flank or the head, or different colour jackets could be worn (e.g., see Rose, de Heer, Korte, van der Harst, Weinbauer, & Spruijt, 2012).", "Clinical signs are typically monitored on non-recording days from socially housed animals on toxicology studies, thus if the same processes are adopted, then it should also be possible on the telemetry recording days.", "Jacketed telemetry has been used within the industry for over a decade (Hunter, Schofield, Gracie, Moors, Philp, Carter, Prior, Valentin, & Hammond, 2004) and has allowed the integration of quality continuous cardiovascular assessments into toxicology studies.", "However, implementation of jacketed telemetry into socially housed conditions could present some issues.", "Even single housed animals are able to tamper with their jackets and damage/destroy the ECG leads, leading to loss of data (Chui, Fosdick, Conner, Jiang, Bruenner, & Vargas, 2009) and socially housed animals may increase the incidence of this, due to interactions with equipment of pen/cagemates.", "However, comments within the survey from companies implementing (or validating) social housing suggest that the animals are less prone to jacket destruction, perhaps as they are sufficiently ‘enriched’ by their companion(s) (Kaiser, Tichenor, Regalia, York, & Holzgrefe, 2015; Sadekova, Boudreau, & Norton, 2015; Xing, Lu, Hu, Wang, Zhao, Zheng, Schofield, Oldman, Adkins, Yu, Platz, Ren, & Skinner, 2015).", "The keys to successful jacketed telemetry are (whether animals are singly or socially housed) appropriate jacket design, size and placement, along with sufficient acclimatisation to the jackets, as these will reduce the risk of damage of the jackets during the study.", "There are a number of articles with thorough descriptions of acclimatisation regimens (Derakhchan, Chui, Stevens, Gu, & Vargas, 2014; Prior, McMahon, Schofield, & Valentin, 2009; APV Scientific Advisory Committee Guidelines).", "Some ‘tips’ and solutions for successful recordings include introducing animals to wearing jackets from a young age (e.g., at the breeders facility), wearing an undershirt or bandaging or another jacket below the outer jacket (Ascah, Abtout, Pouliet, Bassett, & Authier, 2015; Chui, Fosdick, Conner, Jiang, Bruenner, & Vargas, 2009; Ward, Milliken, Patel, & McMahon, 2012; Xing, Lu, Hu, Wang, Zhao, Zheng, Schofield, Oldman, Adkins, Yu, Platz, Ren, & Skinner, 2015) to protect the electrodes, modifying the jackets to hide zips (Xing, Lu, Hu, Wang, Zhao, Zheng, Schofield, Oldman, Adkins, Yu, Platz, Ren, & Skinner, 2015).", "Additional acclimatisation sessions to jackets before each recording session (particularly important for longer studies where there may be a gap of a number of weeks between recording sessions) could also be used.", "Additionally, some practices may be transferrable from ambulatory infusion toxicology studies which are frequently run in social housed conditions.", "Although the survey was primarily interested in practices and views on social housing during telemetry recordings, a number of answers highlighted some interesting opportunities for additional animal welfare improvements.", "These are discussed below.", "Most companies use four animals in their safety pharmacology telemetry studies, usually of the same sex.", "However, some companies use six or eight animals and a mix of both sexes.", "The number of animals used should depend on the study sensitivity required for a given study type at each specific facility, however, a geographical trend indicated European companies often use fewer animals.", "Whilst the use of four animals can deliver acceptable test sensitivity for the majority of the measured parameters, there can be small improvements in test sensitivity when increasing to eight animals (Chiang, Bass, Cooper, Engwall, Menton, & Thomas, 2007; Sivarajah, Collins, Sutton, Regan, West, Holbrook, & Edmunds, 2010; Guth, Bass, Briscoe, Chivers, Markert, Siegl, & Valentin, 2009).", "If social housing leads to less variability within the data, there could be opportunities for facilities to reduce the number of animals used, dependent on study requirements.", "Reducing the number of animals used will have associated reductions in study costs and timelines (technician time, data processing time, etc.).", "The survey highlighted that in 100% of respondent facilities, NHPs are socially housed as stock or on non-recording days.", "However, dogs are still housed individually in a small proportion of facilities, even as stock or on non-recording days in safety pharmacology studies or for the entirety of toxicology studies.", "This may be due to infrastructure reasons or concerns around compatibility/conflict within groups.", "For guidance on successful social housing, please refer to NC3Rs, 2006 and Prescott, Morton, Anderson, Buckwell, Heath, Hubrecht, Jennings, Robb, Ruane, Swallow, & Thompson, 2004.", "Some respondents expressed concerns about changing the housing conditions within a study, preferring to maintain the animals in a stable condition at all times rather than switching the animals between social and single housing on recording and non-recording days, respectively.", "These comments provide support for social housing throughout the study.", "As most facilities are now social housing NHPs on non-recording days, this should increase the adoption of social housing on recording days where technically feasible.", "There is also the potential for this practice to extend to the other non-rodent species.", "An unexpectedly high number of respondents run safety pharmacology and toxicology telemetry studies in the minipig (20 and 13 companies, respectively).", "When the dog is considered unsuitable as the non-rodent for use, the minipig is often the second species of choice and can potentially be a suitable model for many different toxicology study types (Bode, Clausing, Gervais, Loegsted, Luft, Nogues, & Sims, 2010).", "The acceptable cardiovascular data from these animals (Authier, Gervais, Fournier, Gauvin, Maghezzi, & Troncy, 2011; Markert, Stubhan, Mayer, Trautmann, Klumpp, Schuler-Metz, Schumacher, & Guth, 2009) and the stability of pairings means that minipigs can be considered as a suitable non-rodent species for telemetry studies in socially-housed conditions.", "The implementation of social housing during telemetry recordings in safety pharmacology and toxicology studies is an achievable goal.", "However, individual companies will have varying challenges according to their current practice and infrastructure, which will be unique to each specific facility.", "For example, changing to social housing may require review/modification of physical aspects such as pen/cage sizes, room availability and telemetry hardware, and/or process aspects such as acclimatisation of animals to each other and to recording areas, stock colony/pair compositions and study designs.", "Each facility will need to investigate the optimal conditions to ensure the cardiovascular data obtained from socially housed animals is of similar, or better, quality and sensitivity than from individually housed animals.", "If Sponsors do not wish to invest in their in-house facilities, then the trends identified in the survey shows increased opportunities to use CROs for socially housed studies.", "CROs need to meet market expectations but concerns over possible decreases in capacity/number of studies that can be offered when using social housing will need to be addressed.", "CROs may therefore retain capabilities to run studies according to all possible requests as an interim measure.", "Further demonstration of cost or time-efficiencies, in addition to an increased data quality, will accelerate the implementation of socially housed studies.", "The implementation of social housing during telemetry recording may be most straightforward for standard studies initially, e.g. oral dosing studies when using cautious dose levels (to minimise emesis and cross-contamination) if the MTD is already known.", "There may be specific studies where, in order to ensure the likelihood of successful completion, animals will need to be singly housed (e.g., if using a technical dosing route or a specific compound which may cause aggressiveness or other clinical signs which may affect the welfare of pen/cage mates).", "However, the overall intention should be to consider social housing as normal/default from the start, and ensure robust scientific rationale is used to deviate from this.", "Fig. 6 shows a flow diagram with decision points and suggestions for housing options for individual studies.", "Additionally, pair housing of animals may be more achievable in the short term for some companies than housing and recording from larger groups of animals.", "In conclusion, although the majority of the industry does not yet have the experience or capabilities for recording from socially housed animals in safety pharmacology and toxicology studies, there is support to implement this refinement.", "Continued discussions, sharing of best practice and data from companies already socially housing, combined with technology improvements and investments in infrastructure are required to maintain the forward momentum of this refinement across the industry.", "There are no known conflict of interests for any authors." ]	[]
Sustainable Management and Action in China under the Increasing Risks of Global Climate Change		[ "CENG", "COMP", "ENER", "ENGI", "ENVI", "MATE" ]	[]	[ "1500", "1700", "2102", "2200", "2305", "2501" ]	[ "In the past 100 years, earth’s changing climate has resulted in widespread and significant impacts, with the strongest and most comprehensive evidence of climate change being seen in natural systems.", "Some impacts on human systems have also been observed, including impacts on water resources and hydrological systems, species shifting and migration, and negative impacts on crop yields.", "The most noticeable impacts are believed to result from extreme climate-related events such as those that have recently occurred, including heat waves, droughts, floods, tropical cyclones, and wildfires.", "The impacts of such climate-related extreme events also include the alteration of ecosystems, disruption of food production and water supplies, damage to infrastructure and settlements, morbidity and mortality, and consequences for mental health and human wellbeing [1].", "The impacts of extreme events are of primary concern, as they can often induce severe and abrupt hazards and, in particular, compound disasters [2].", "Climate-related hazards exacerbate other stressors, often with negative outcomes for livelihoods, especially for those of people living in poverty; direct negative outcomes include reduction in crop yields and the destruction of homes, while examples of indirect negative outcomes include increased food prices and food insecurity.", "In the future development of humanity and human society, many kinds of risks will be met.", "The six highest environmental risks confronting humanity are as follows:", "Extreme weather and climate events, and especially the emergence of compound disaster events;", "The failure of human mitigation of and adaptation to climate change;", "Large-scale loss of biodiversity and collapse of ecosystems;", "Increasing supply shortage of water resources and food, particularly for developing countries;", "Large-scale natural disasters, including rapid sea-level rise and Arctic sea ice melting; and", "Anthropogenic environmental damage and disasters, including persistent air and water pollution.", "Each of these risks is closely or directly related to climate change.", "Therefore, global climate change is a core risk that is ranked as the second most serious global risk.", "Fig. 1 shows the global mean annual temperature anomaly for 1850–2016.", "Global warming over the past 100 years is clearly characterized by an increasing trend of the time series of temperature, although two accelerating warming periods (1915–1945 and 1980–1998) are visible, along with two warming hiatus periods (1950–1979 and 2000–2015).", "The trend of the annual surface temperature anomalies averaged in China for 1901–2016 (Fig. 2) is very similar to the trend shown Fig. 1, implying that the climate in China shows a good regional response to global climate warming.", "Fig. 3 presents recent atmospheric carbon dioxide (CO2) concentrations at representative stations in the United States and China (Mauna Loa in Hawaii, USA, and Waliguan Mountain in Qinghai Province, China).", "The two CO2 curves are very close to each other, and show a linear upward trend.", "In 2016, the CO2 concentration exceeded 400 ppm by volume (ppmv), the highest value (by about 280 ppmv) in the past 800 000 years.", "Detection and attribution studies have shown that anthropogenic CO2 emissions are a dominating contributor to external forcing to global climate warming, as well as to climate warming in China [3] (Figs. 4 and 5 [4]).", "As the Intergovernmental Panel on Climate Change (IPCC)’s Fifth Assessment Report (AR5) (2014) states, the warming of the climate system is unequivocal and, since the 1950s, many of the observed changes are unprecedented over decades to millennia.", "In climate change studies, key risks and potentially severe impacts are referred to as the results of “dangerous anthropogenic interference with the climate system.”", "Risks are considered to be key risks if they present a high hazard or expose a high vulnerability in society or systems, or both.", "Some climate-change-related risks are considerable at 1 °C or 2 °C above preindustrial levels.", "However, global climate-change-related risks are rated as “high to very high” with a global mean temperature increase of 4 °C or more [5].", "The ultimate objective of the United Nations’ Framework Convention on Climate Change (FCCC) in 1992 was, first, to achieve “stabilization of greenhouse gas concentrations in the atmosphere at a level that would prevent dangerous anthropogenic interference with the climate system.", "Such a level should be achieved within a time frame sufficient to allow ecosystems to adapt naturally to climate change, to ensure that food production is not threatened, and to enable economic development to proceed in a sustainable manner.”", "However, at the time of the FCCC’s publication, dangerous anthropogenic greenhouse gas (GHG) levels were not well defined.", "After 17 years, a consensus on limiting global climate change to a 2 °C threshold was established at the Copenhagen conference on climate change in 2009 in Denmark.", "A few years later, in 2015, the 2 °C threshold for global temperature rise was formally set up as the common global target; that is, it was decided that the temperature change caused by anthropogenic GHG emissions should be kept to less than 2 °C or, preferably, to below 1.5 °C relative to preindustrial levels.", "Fig. 6 [6,7] presents a time series of the annual mean surface air temperature (SAT) anomalies in China in 1861–2005, along with a time series of the annual mean SAT anomalies in China under the IPCC’s Representative Concentration Pathway (RCP) 2.6 (relative to the 1871–1900 period) [6].", "In fact, in the last 100 years, the mean temperature rise in China has been less than 1 °C (Fig. 6(a)).", "However, in the 21st century, the temperature rise will exceed 1.5 °C under RCP 2.6, which is the lowest emission scenario.", "Thus, the future emission pathway for China will, at least, follow this emission scenario in order to limit the temperature rise to below 2 °C (Fig. 6(b)).", "Fig. 7 [7,8] presents the emission pathway under different scenarios of global climate change from 1990 to 2100.", "China will reach its emission peak in 2030, or even earlier.", "Other emission pathways will cause the global temperature rise to exceed 2 °C.", "As its total target for the implementation of the Paris Agreement in China, China will: ① reach its carbon emission peak before 2030; ② transform into and enter an era of low-carbon and green energy development; and ③ pursue the sustainable development and management of society and the economy by greatly improving the state of the environment, ecosystems, human health, water supplies and security, and so forth.", "Under this target, China must take an irreversible road of low-carbon, green, and clean energy development (Fig. 8 [8]).", "To achieve this goal, China will expend great efforts to rapidly develop renewable energy and geo-engineering.", "Many countries have recently taken steps to develop new renewable energy sources.", "Low-carbon energy sources in China accounted for 17.9% of the total energy consumption in 2015.", "Renewable energy production accounts for 25% of the total energy production in China, making China the world leader in renewable energy production in terms of scope [9–13].", "The future of the earth depends on the world’s efforts to reduce GHG emissions, and on the adaptation policies we adopt.", "There are two possible futures for the generations to come: a peaceful, secure, and sustainable future; or an irreversible, degraded, and disastrous one.", "A sustainable future implies clean air and a comfortable climate for future generations (Figs. 9 and 10 [1]).", "As the IPCC has stated (Fig. 10) [1]: “① Our world is threatened by multiple stressors that impinge on earth’s resilience from many directions, represented here simply as biophysical and social stressors.", "Stressors include climate change, climate variability, land-use change, degradation of ecosystems, poverty and inequality, and cultural factors.", "② Opportunity space refers to decision points and pathways that lead to a range of ③ possible futures with differing levels of resilience and risk.", "④ Decision points result in actions or failures-to-act throughout the opportunity space, and together they consistent the process of managing or failing to manage risks related to climate change.", "⑤ Climate-resilient pathways (in green) within the opportunity space lead to a more resilient world through adaptive learning, increasing scientific knowledge, effective adaptation and mitigation measures… ⑥ Pathways that lower resilience (in red) can involve insufficient mitigation, maladaptation, failure to learn and use knowledge…; and they can be irreversible in terms of possible futures” [1]." ]	[]
Electrophysiological and Contractile Effects of Disopyramide in Patients With Obstructive Hypertrophic Cardiomyopathy: A Translational Study	Disopyramide is effective and safe in patients with obstructive hypertrophic cardiomyopathy. However, its cellular and molecular mechanisms of action are unknown. We tested disopyramide in cardiomyocytes from the septum of surgical myectomy patients: disopyramide inhibits multiple ion channels, leading to lower Ca transients and force, and shortens action potentials, thus reducing cellular arrhythmias. The electrophysiological profile of disopyramide explains the efficient reduction of outflow gradients but also the limited prolongation of the QT interval and the absence of arrhythmic side effects observed in 39 disopyramide-treated patients. In conclusion, our results support the idea that disopyramide is safe for outpatient use in obstructive patients.	[ "MEDI" ]	[ "QT interval", "action potentials", "arrhythmias", "diastolic dysfunction", "hypertrophic cardiomyopathy", "safety" ]	[ "2705" ]	[ "Detailed methods are available in the Online Appendix data supplement.", "In vitro studies were performed at the University of Florence.", "Protocols were approved by the ethical committee of Careggi University Hospital (2006/0024713; renewed May 2009).", "We enrolled 20 patients with HCM regularly followed by our Cardiomyopathy Unit and consecutively referred to surgical myectomy for relief of drug-refractory symptoms related to LVOT obstruction.", "Among the 20 patients, 12 agreed to undergo mutational screening in sarcomeric genes.", "Clinical and genetic data are found in Supplemental Table 1.", "Cellular mechanisms of disopyramide action on dispersion of repolarization were investigated in a population of human ventricular cardiomyocyte models (n = 250) in control versus HCM remodeling, calibrated against human AP and Ca2+-transient data, as previously described (23).", "Unpaired or paired t tests, as well as linear fitting with r-squared calculation, were employed to analyze data from populations of models.", "Mechanisms of dispersion at the whole ventricular level were investigated in a cardiac magnetic resonance–based anatomical model of a patient with obstructive HCM, under realistic human activation sequence and heterogeneity in repolarizing currents (24).", "From October 1, 2015, to January 31, 2018, 39 patients with limiting heart failure symptoms and elevated LVOT gradients were initiated on sustained release disopyramide at New York University Langone Medical Center as previously described (4–6,9).", "Initial disopyramide dosage was 250 mg twice a day, followed by drug titration up to a maximum dosage of 300 mg every 12 h. Electrocardiography (ECG) was performed at each visit by recording 3 12-lead 15-s strips (collected at least 5 min apart), and QTc interval was calculated from each strip by averaging the QT intervals measured by a blinded operator from the 12 leads and correcting them for heart rate using the Bazett and the Fredericia methods.", "QTc values from the 3 repeated ECG strips were then averaged to obtain the final value.", "Echocardiography was performed to measure transmitral flow velocities and mitral annular diastolic velocities and to calculate resting LVOT gradients and LV ejection fraction before and after disopyramide.", "Clinical data from patients are expressed as mean ± SD.", "Comparisons of clinical variables before and after disopyramide were performed using Student’s paired t tests.", "To assess the relationship between the initial QTc and the increase in QTc with disopyramide, we calculated the Pearson correlation coefficient (r) and its significance level using MedCalc (version 19.0; MedCalc Software, Ostend, Belgium).", "Septal specimens from 20 consecutive surgical patients were collected from the surgeon and immediately washed with cardioplegic solution and processed within 30 min from excision.", "Each sample was given a unique anonymous identification number (ID 1 to ID 20).", "Endocardial trabeculae suitable for mechanical measurements were dissected and the remaining tissue was minced and subjected to enzymatic dissociation to obtain viable single myocytes, as previously described (17).", "A perforated patch whole-cell current clamp was used to measure membrane potential, as previously described (15). [", "Ca2+] variations were simultaneously monitored using the Ca2+-sensitive fluorescent dye FluoForte (Enzo Life Sciences, Farmingdale, New York).", "A whole-cell ruptured patch voltage clamp was used to record peak and late Na+ current, L-type Ca2+ current (ICa-L), and delayed rectifier K+ current (IK), using appropriate protocols and solutions (15).", "Ventricular trabeculae were mounted between a force transducer and a motor for muscle length control (15), and isometric force was recorded under different stimulation protocols.", "In brief, we evaluated the inotropic responses to increased pacing frequencies and the kinetics of isometric twitches.", "Resting sarcomere length was 1.9 ± 0.1 μm.", "Ventricular trabeculae were skinned by exposure to 0.5% Triton X100 solution.", "Demembranated trabeculae were employed to obtain calcium concentration (pCa)-tension curves as previously described (15,18,19).", "Sarcomere energetics was assessed in demembranated trabeculae by simultaneous measurement of isometric force and adenosine triphosphatase activity with an enzyme-coupled assay (15,18,19); measurements were repeated in the presence of disopyramide.", "For experiments on isolated cardiomyocytes and trabeculae, disopyramide was used at the concentration of 5 μmol/l, unless otherwise specified.", "Test recordings in presence of the drug were performed after >3 min from the beginning of drug exposure.", "Afterward, the drug was washed out for >5 min and measurements were repeated.", "Of note, all the effects of disopyramide in isolated cardiomyocytes and trabeculae disappeared after 5 min of drug washout.", "Ventricular myocytes were isolated from the hearts of 4 transgenic HCM mice carrying the R92Q troponin-T mutation, as previously described (18,19).", "Myocytes were permeabilized with saponin (20) and resuspended in an intracellular buffer containing 150 nmol/l free [Ca2+] and 5 μmol/l of the Ca2+-sensitive dye Asante Na+-green K+-salt (Teflabs, Austin, Texas).", "The frequency of spontaneous Ca2+ sparks was evaluated with a confocal microscope through line scan along the longitudinal cell axis (20).", "Myocytes incubated with vehicle were compared with cells exposed to 5 μmol/l of disopyramide.", "The rate of sparks was calculated from confocal line-scan recordings using the SparkMaster ImageJ plugin (National Institutes of Health, Bethesda, Maryland) for automated analysis (21).", "None of the 20 consecutively collected patient samples was excluded from the final analysis.", "However, we were unable to perform all the different experimental procedures described herein in all 20 samples.", "Therefore, each dataset comprises results from cells or trabeculae isolated from 3 to 11 patient samples.", "For each dataset, we indicated in the respective figure legends, the total number of cells/trabeculae included, as well as the number (and ID) of patients’ samples from which they were isolated.", "Results from each dataset are expressed as mean ± SEM.", "Statistical analysis, taking into account non-Gaussian distribution, inequality of variances and within-subject correlation, was performed as previously described (15,18).", "In brief, to reduce the risk of type I errors resulting from the stronger interrelationship among cells/trabeculae isolated from the same patient sample, we used hierarchical statistics including 2 nested levels (patients and cells/trabeculae) (22), plus a third hierarchical level (presence or absence of disopyramide in the same cell/trabecula) to assess the effects of drug treatment in a pairwise fashion.", "This approach was implemented using linear mixed models in Stata 12.0 (StataCorp LLC, College Station, Texas).", "The p values were calculated using linear-mixed models.", "For categorical data (e.g., occurrence of cellular arrhythmias), we used the Fisher exact test.", "We considered p < 0.05 statistically significant.", "Thirty-nine patients were begun on disopyramide (age 66 ± 10 years; 59% were female).", "The mean daily dosage of disopyramide after 3 months was 497 ± 87 mg/day.", "No patient had clinically significant arrhythmia or organ toxicity during the study period.", "In 9 patients (23%), disopyramide was ineffective or there were limiting vagolytic side effects that led to termination of drug before 3 months.", "ECG parameters before and at a median of 96 days after disopyramide initiation are shown in Table 1.", "There were prolongations in the QTc, QRS, JT, and PR intervals, but no significant change in heart rate at 3 months.", "The mean QTc interval increased from 458 ± 22 to 486 ± 27 ms (p < 0.001).", "Mean QTc prolongation was 27 ms, which corresponds to a 5.8% increase from baseline.", "Patients with an initially longer QTc interval (greater than the median 457 ms) had a smaller increase in QTc interval than did patients with shorter initial QTc interval (ΔQTc: 18 ± 4 vs. 34 ± 8 ms, p = 0.007).", "There was an inverse correlation between the initial QTc interval and the increase in QTc interval with disopyramide (Pearson r = −0.44, p = 0.008).", "Initial resting LVOT gradients were 58 ± 49 mm Hg.", "After 96 days of disopyramide, gradients were reduced to 25 ± 26 mm Hg (p < 0.001).", "There were no differences in tissue Doppler variables, transmitral flow velocities, and in the calculations from tissue Doppler and transmitral flow velocities used to estimate left atrial pressure (Table 2).", "Ejection fraction was minimally reduced by disopyramide from 72.2% to 68%, an average decrease by 5.8 ± 3.1% (Table 2).", "Samples from 20 patients with HCM undergoing surgical myectomy for drug-refractory symptoms were assessed.", "Clinical data at pre-operative assessment are shown in Supplemental Table 1.", "Disopyramide was added to standard perfusion solution while recording isometric tension from intact contracting trabeculae dissected from the endocardial surface.", "As expected, the drug displayed a consistent negative inotropic effect (Figure 1A).", "To assess the concentration dependency of this effect, we exposed the muscles to different concentrations of disopyramide (Figure 1B).", "Calculated disopyramide concentration at 50% of maximal effect on isometric twitch amplitude was 5.29 ± 1.55 μmol/l.", "We therefore decided to employ the drug at 5 μmol/l for all the following experiments; notably, 5 μmol/l corresponds to the average plasma concentration of disopyramide measured in patients under a standard treatment regimen (25).", "Importantly, 5 μmol/l disopyramide hastened isometric twitch kinetics in HCM trabeculae: both time to peak and relaxation time were reversibly shortened by the application of the drug (Figures 1A and 1C).", "We tested the effects of disopyramide at different stimulation frequencies (Figure 1D): the reduction of steady-state isometric twitch force was more pronounced at higher pacing rates as compared with lower rates.", "Isometric twitch force was reduced by 33 ± 5% at 0.5 Hz (30 beats/min) and by 62 ± 10% at 1.5 Hz (90 beats/min; p = 0.015 vs. 0.5 Hz; data from 13 trabeculae in 10 patients, calculated using linear-mixed models).", "We tested the effects of disopyramide on demembranated trabeculae from 3 patients with HCM (Figure 1E).", "Disopyramide (5 μmol/l) did not affect maximal force obtained when exposing trabeculae to an activating solution with pCa 4.5 (Figure 1F).", "Force generation at lower [Ca2+], determining submaximal tension development, was also unaffected by disopyramide; we concluded that disopyramide does not modify myofilament Ca sensitivity (Figure 1F).", "Moreover, disopyramide did not alter the energy cost of tension generation (Supplemental Figure 1).", "The same results were obtained in demembranated trabeculae from healthy donors.", "Disopyramide was tested at 5 μmol/l in patch-clamped ventricular cardiomyocytes while simultaneously recording Ca2+ transients and AP during stimulation at different frequencies (Figures 2A and 2C).", "The drug reduced the amplitude of Ca2+ transients at all frequencies (Figure 2B), hastened the kinetics of Ca2+ transient rise and decay (Figures 2C and 2D, Table 3) and reduced diastolic Ca2+ concentration (Figures 2A and 2B, Table 3).", "Disopyramide (5 μmol/l) shortened the duration of AP in HCM cardiomyocytes (Figures 2C and 3A, Table 3) at all frequencies (Figure 3B) and the effect was more pronounced at lower rates.", "For instance, average AP shortening at 0.1 Hz was 27 ± 5% and is comparable to that observed in human HCM cardiomyocytes with 10 μmol/l of ranolazine (17).", "In addition, disopyramide reduced AP amplitude and upstroke speed (Table 3).", "Finally, disopyramide reduced the incidence of spontaneous afterdepolarizations in HCM cardiomyocytes, both early afterdepolarizations ([EAD], occurring during the AP plateau) and delayed afterdepolarizations ([DAD], occurring during the diastolic phase), occurring during 3 min of regular stimulation (Figures 3C and 3D).", "We previously have shown that in HCM cardiomyocytes there is markedly enhanced INaL, slightly increased ICa-L, and lower IK, leading to prolonged AP duration (15).", "We assessed Na+ currents in HCM cardiomyocytes during voltage clamp on depolarization to −10 mV.", "Peak current was measured in the first 10 ms of depolarization (Figure 4A), whereas INaL was estimated by integrating the residual inward current (50 to 800 ms after onset) (Figure 4B).", "In HCM myocytes, disopyramide (5 μmol/l) reduced peak Na+ current by 22 ± 4% and greatly decreased INaL integral by 45 ± 6% (21 myocytes, 5 patients).", "We then assessed the effects of disopyramide on IK, measured at steady state during depolarization at different potentials (Figure 4C).", "Disopyramide (5 μmol/l) exerted a small but significant inhibitory effect on IK currents in HCM cardiomyocytes (Figure 4D).", "Notably, the density of steady-state IK at baseline is reduced in our HCM myocytes as compared with control myocardium (Supplemental Figure 2).", "Moreover, disopyramide reduced the amplitude of ICa-L in HCM cardiomyocytes (Figures 4E and 4F); the average ICa-L reduction was 16 ± 4% (mean of 16 myocytes from 4 patients).", "Taken together, the observed effects of disopyramide on Na+, K+, and Ca2+ currents explain the net reduction of AP duration by disopyramide (Figure 3).", "In HCM septal cells, in the presence of lower IK (15), slightly increased ICa-L, and markedly enhanced INaL, the effect of disopyramide is a net decrease of depolarizing currents, ultimately leading to AP shortening.", "On the contrary, in control human ventricular myocytes, where the expression of IK is preserved and INaL is small (15), inhibition of human ether-à-go-go-related gene (hERG) K+ current by disopyramide (26) prevails over the reduction of ICa-L and INaL, ultimately leading to a slight AP prolongation (Supplemental Figure 3), in agreement with previous reports (27).", "Similar to other class I antiarrhythmics (28), disopyramide exerts an inhibitory effects on the ryanodine receptor (RyR).", "We tested the effects on RyR channels by evaluating the rate of Ca sparks in permeabilized ventricular myocytes isolated from a transgenic HCM mouse model carrying the R92Q-TnT mutation (Figure 5A).", "The frequency of Ca2+ sparks in myocytes incubated with 5 μmol/l of disopyramide was lower than that observed in vehicle-treated cells (Figure 5B).", "The effects of 5 μmol/l of disopyramide were modeled in silico based on the above-mentioned characterization of drug action.", "In agreement with the experimental findings, 5 μmol/l of disopyramide yielded AP shortening in HCM endocardial cardiomyocytes and left AP duration unaltered in control endocardium (Figures 6A and 6B).", "Due to transmural differences in ion channel expression, this trend was reversed in epicardial cardiomyocytes, where disopyramide slightly prolonged AP duration in control cells (Figures 6A and 6B).", "The magnitude of AP duration at 90% of repolarization shortening in HCM endocardial cells was inversely correlated with basal AP duration at 90% of repolarization (Figure 6C).", "Further analysis of responders to drug action indicates that INaL density is the primary determinant of the extent of AP shortening due to disopyramide (Figure 6D).", "In the reconstructed ventricles of a patient with obstructive HCM, 5 μmol/l disopyramide reduced conduction velocity, increasing total activation time by 11 ms (Supplemental Figure 4), comparable to clinical data (QRS prolongation at ECG, see Table 1).", "In the region of septal hypertrophy, 5 μmol/l of disopyramide markedly decreased dispersion of repolarization times (baseline: 529 ± 62 ms, 625 ms maximum; 5 μmol/l of disopyramide: 504 ± 50 ms, 585 ms maximum) and slightly prolonged repolarization in the nonhypertrophic epicardium (Figure 6E).", "This heterogeneous ventricular action resulted in QT shortening in septal precordial ECG leads along with modest QT prolongation in the lateral ones (Figure 6E), hence explaining the apparent discrepancy between AP shortening and the clinical QTc prolongation observed in patients with HCM following disopyramide intake.", "In the present work, we have investigated the effects of disopyramide in patients with obstructive HCM and on isolated HCM cardiomyocytes harvested from patients undergoing surgical septal myectomy.", "The results shed light on the agent’s biophysical mechanisms of benefit, and its demonstrated clinical safety in patients with HCM.", "In addition, the suppressive effect of disopyramide on afterdepolarizations in vitro raises the possibility that disopyramide may reduce ventricular arrhythmia propensity in vulnerable patients with HCM.", "In patients with obstructive HCM, the beneficial effect of disopyramide is that it decreases LV contractility, specifically by decreasing early LV ejection flow acceleration (29).", "In patients with obstructive HCM, the mitral valve apposes with the septum very early in systole, and the duration of mitral-septal contact correlates with gradient magnitude, because of the amplifying feedback loop in which the presence of a gradient pushes the valve further into the septum.", "Thus, to reduce obstruction-related gradients, the impact of any negative inotropic medication must be very early in systole, before the onset mitral-septal contact.", "By decreasing early systolic ejection acceleration, disopyramide decreases early drag forces on the mitral valve, delaying or abolishing mitral-septal contact.", "Systolic anterior motion results from an equilibrium between the displacing anteriorly directed force of ejection flow striking the mitral valve, versus restraining forces, from the papillary muscles and chordae (30).", "Disopyramide displaces this equilibrium toward restraint by dint of its effects on early flow acceleration.", "Interestingly, despite the decreased velocity of pressure generation (dP/dt) during early systole and the lower peak LV pressure, disopyramide results in a modest reduction of global systolic function (5% to 6% decrease of ejection fraction in our study, in line with previous observations).", "In normal subjects, disopyramide causes a prolongation of total ejection time (1); in subjects with HCM, however, where ejection time is prolonged due to obstruction, the relief of obstruction by disopyramide leads to a 12% shorter ejection time (31).", "Moreover, disopyramide has a more potent gradient-lowering effect than other negative inotropic agents given for obstructive HCM (beta blockers and verapamil).", "In a head-to-head comparison in individual patients with HCM by intravenous administration of the 3 agents on sequential days, investigators found a 59% reduction of gradient with disopyramide, a 19% reduction with propranolol, and only 8% reduction with verapamil (11,32).", "On average, 60% to 70% of patients started on disopyramide will experience a significant drop in LVOT gradient and improvement in symptoms (5,6).", "Whereas the gradient-lowering effect of disopyramide shows a clear dependency on dose and plasma levels (1,3,33), the negative inotropic effect is always very modest and does not vary within the clinically relevant range of doses (up to 300 mg twice daily).", "These observations suggest that symptomatic gradients can be effectively lowered by negative inotropic agents without largely reducing ejection fraction.", "This may be related to the fact that most patients with obstructive HCM are pathologically hyperkinetic.", "In HCM hearts, negative inotropic drugs may counteract LV hypercontractility and reduce early ejection acceleration while at the same time allowing a more efficient LV emptying due to loss of obstruction.", "Thus, the impact of negative inotropic drugs on end-systolic volume will be very limited, explaining the modest reduction of ejection fraction.", "In the current study, disopyramide lowered resting LVOT gradients from mean 58 to 25 mm Hg after 3 months.", "This was similar to a prior report in 221 patients using disopyramide with an average dose of 500 mg/day, where resting LVOT gradients decreased from 63 to 25 mm Hg after 4.5 years (6).", "Symptoms and gradient in 64% of patients of this cohort could be managed pharmacologically, while 36% ultimately underwent septal reduction.", "Our in vitro force assessments using human HCM samples confirm the negative inotropic action of disopyramide observed in patients.", "The results of our studies show that this effect is the result of the inhibitory action of disopyramide on multiple ion channels within the cardiomyocyte.", "Disopyramide does not modify the maximal force generated by the contractile apparatus in HCM and control trabeculae, and it does not change myofilament sensitivity to Ca2+, suggesting that the drug does not directly target contractile proteins (Figure 6).", "Therefore, disopyramide must reduce force through modifications of the cardiomyocyte excitation-contraction coupling, that is, intracellular Ca2+ handling.", "By contrast, mavacamten (MYK-461) produces a negative inotropic effect by virtue of direct inhibitor of cardiac myosin (34) with no apparent off-target side effects.", "Mavacamten was recently tested in a small number of patients with obstructive HCM in a phase II study, showing that a clinically meaningful reduction of post-exercise LVOT gradients can be achieved with relatively small doses of the drug, while leading to a modest reduction of LV ejection fraction, on the order of 6% to 10% on average.", "This is in line with the idea that a large reduction of LVOT gradients in patients with obstructive HCM can be achieved using negative inotropic drugs with a minimal loss of global LV function.", "The negative inotropic effect of disopyramide is a consequence of the reduction of systolic intracellular Ca2+ levels in the cardiomyocyte, thus determining lower Ca-mediated activation of myofilaments and, ultimately, lower force.", "We found a number of concurrent mechanisms contributing to the reduction of Ca2+-transient amplitude.", "Early studies demonstrated that Na+ channel block (e.g., by tetrodotoxin) has a slight negative inotropic action per se in the myocardium.", "Interestingly, these studies compared the negative inotropic potency of different drugs with their efficacy in slowing down AP upstroke (a direct consequence of peak Na+-current inhibition) (35,36).", "The ratio of negative inotropic efficacy versus Na+-channel blocking potency was the lowest for tetrodotoxin (0.23) and the highest for disopyramide (2.2).", "Disopyramide has the most negative inotropic effect of all Class I antiarrhythmics, higher than mexiletine, procainamide, and quinidine (35).", "As tetrodotoxin is a pure Na+-channel blocker, these results suggest that Na+-channel inhibition only slightly contributes to the negative inotropic action of disopyramide, and that disopyramide has additional Na+-channel–independent mechanisms that make it the most potent negative inotropic agent among class I antiarrhythmics (35).", "Nonetheless, Na+-channel inhibition may independently contribute to the reduction of Ca2+-transient amplitude by disopyramide through modification of the activity of the NCX (37).", "When membrane potential is positive (peak and plateau of the AP) and subsarcolemmal [Na+] is high (due to large Na+ influx by peak INa), NCX works in reverse mode, in other words, letting Ca2+ enter the cell in exchange for Na+ (38).", "The contribution of reverse-mode NCX to Ca2+ transients is particularly high in HCM cardiomyocytes (16).", "In the presence of peak and late INa inhibition by disopyramide, maximal AP voltage is reduced (Table 2) and intracellular [Na+] is diminished (16): these changes lead to a decrease of reverse-mode NCX activity, thus reducing Ca2+ entry (39).", "Moreover, reduction of diastolic [Na+] may enhance the diastolic extrusion of Ca2+ via the NCX (forward mode) (15,18).", "The 2 mechanisms (reduced reverse- and enhanced forward-mode NCX) may contribute to reduce total Ca2+ load of the SR (15) and thus Ca2+-transient amplitude.", "Selective inhibition of INaL without block of peak INa (e.g., with ranolazine or eleclazine) is not sufficient to induce a negative inotropic effect under basal conditions (15,16).", "Therefore, we can conclude that simultaneous inhibition of peak and late INa is required for the negative inotropic action of disopyramide.", "Disopyramide exerts a slight but relevant inhibitory effect on ICa-L, which is substantially lower than that of verapamil.", "Nonetheless, the observed 15% inhibition of peak ICa-L by disopyramide contributed to reduce Ca2+-transient amplitude in ventricular myocytes, the effect being larger at higher stimulation rates.", "A similar rate-dependency of the negative inotropic effect is typical of pure Ca2+-channel blockers such as verapamil (40).", "At variance with verapamil (41), however, disopyramide has minimal effects on heart rate and atrioventricular conduction (Table 3), and thus can be safely combined with -beta blockers.", "Na+ channel blockers flecainide and propafenone stabilize the closed state of cardiac RyR channels (42).", "We observed that disopyramide reduced the rate of spontaneous Ca2+ sparks in permeabilized myocytes, in the presence of fixed intracellular [Ca2+]: the reduction in the spontaneous RyR opening rate is likely due to a direct effect of disopyramide on RyR channels.", "Notably, selective inhibition of RyR channels using JTV-519 in human ventricular trabeculae led a slight negative inotropic effect (43).", "Similarly, inhibition of RyR-mediated systolic Ca2+ release by disopyramide may contribute to the reduction of Ca2+-transient amplitude.", "Early studies in healthy myocardium showed that disopyramide prolongs ventricular AP (13) due to decreased IKr current by direct inhibition of hERG channels (44).", "Similarly, we observed a slight AP prolongation in cardiomyocytes from patients without HCM (Online Figure 3), also confirmed by modeling studies (Figure 6A).", "Interestingly, the AP prolonging effect in control cardiomyocytes was shown to be prevalent at concentrations below 8 μmol/l, whereas at higher concentrations, disopyramide tended to shorten AP (13,45).", "At low concentrations, hERG inhibition prevails over INaL and ICa-L block, leading to a net decrease of repolarizing currents and AP prolongation; at higher concentrations, however, after maximal hERG inhibition is reached, the inhibition of depolarizing Ca2+ and Na+ currents becomes prevalent, leading to AP shortening in healthy myocardium.", "Based on our observations, we can speculate that the latter behavior occurs in HCM cardiomyocytes even at low, clinically relevant concentrations (such as 5 μmol/l, which we used in this work), due to the decreased expression of hERG channels in HCM myocardium (15).", "Moreover, INaL and ICa-L are both increased in HCM versus control myocardia (15); therefore, their inhibition by disopyramide leads to greater AP shortening, especially at low pacing rates.", "Simulation studies suggested that AP shortening by disopyramide is more pronounced in cells that have larger INaL and smaller IKr (Figures 6C and 6D).", "Taken together, the altered balance of depolarizing and repolarizing currents in cardiomyocytes from the hypertrophic septum of patients with HCM causes disopyramide to decrease net depolarizing currents (Na+ and Ca2+) without substantial reduction in repolarizing K+ currents, ultimately shortening AP plateau duration (Figure 2, Table 1).", "In the 39 patients begun on disopyramide, QTc interval increased from 458 ± 22 to 486 ± 27, an average 5.8% rise after 3 months.", "The shortening of AP observed in HCM cardiomyocytes in vitro is in apparent contrast with the slight QTc prolongation in patients.", "However, prolongation of QTc interval with disopyramide depends, at least in part, on the prolongation of QRS interval, reflecting delayed intraventricular electrical conduction velocity due to peak Na+-current inhibition (Table 1) (46).", "However, even if we consider only the repolarization phase (JT interval), disopyramide still has a significant prolonging effect.", "All the cardiomyocytes analyzed in this work were isolated from the subendocardial region of the basal septum, the most hypertrophied region in patients with obstructive HCM.", "If the degree of cardiomyocyte electrical changes is heterogeneous across the LV and mirrors the asymmetrical distribution of hypertrophy, cardiomyocytes from other regions of the LV are likely to be similar to control cardiomyocytes.", "Therefore, we constructed a 3-dimensional model of the whole LV from an obstructive patient (Figure 6E) where we assumed that the HCM-specific ion current abnormalities we observed in isolated cardiomyocytes from septal myectomy samples (15,23) were limited to the hypertrophied septal region.", "In agreement with this assumption, the spatial dispersion of QTc interval is greatly increased in patients with HCM with asymmetrical hypertrophy (47).", "In this model, we found that disopyramide shortens the duration of septal AP while slightly prolonging the AP of the remaining LV myocardium, ultimately resulting in a slight prolongation of the global QT interval in the reconstructed ECG trace.", "In line with that prediction, the average prolongation of QTc interval in patients with HCM with disopyramide (5% to 6%) is lower than the average QT prolongation in healthy patients taking a similar dose of disopyramide (10% to 15%) (1,48).", "The model predicts that disopyramide determines marked reduction of QT dispersion across the different LV regions, as well as transmurally.", "Because the magnitude of the AP-shortening effect of disopyramide is linearly related with the AP duration at baseline (Figure 6C), the effect would be greater in cells with longer AP, globally reducing the heterogeneity of AP duration among different LV regions.", "In line with that, we observed in patients that the QTc prolonging effect of disopyramide is inversely related to the baseline QTc interval: patients with an initially longer QTc interval (>457 ms) had a smaller increase in QTc interval after disopyramide than did patients with shorter initial QTc interval.", "Interestingly, spatial QT dispersion is a substrate for sustained re-entrant arrhythmias and is related with the occurrence of nonsustained ventricular tachycardia in patients with HCM (47); therefore, its reduction by disopyramide could be antiarrhythmic.", "We observed a reduction of the cellular triggers of arrhythmias in HCM cardiomyocytes treated with disopyramide, that is, EAD and DAD (Figure 3).", "The risk of EAD is directly associated with AP prolongation (49).", "The shortening of AP by disopyramide is therefore the main mechanism behind the reduction of EAD (15).", "DAD result from the activation of the electrogenic (depolarizing) NCX secondary to spontaneous diastolic Ca2+ release from the SR (50).", "The likelihood of DAD is therefore increased in all conditions leading to increased open probability of RyR during diastole, that is increased SR Ca2+ load, increased diastolic cytosolic [Ca2+] or altered intrinsic properties of the RyR (e.g., phosphorylation by Ca2+/calmodulin-dependent protein kinase II).", "All these features are present in HCM cardiomyocytes, explaining the observed increase of DAD with respect to control cells (15).", "Interestingly, we observed that disopyramide reduced diastolic cytosolic [Ca2+] (Figure 2) and SR Ca2+ content and stabilized the closed state of RyR (Figure 5), thus lowering the likelihood of diastolic Ca2+ waves.", "The multichannel inhibitory effects and the membrane stabilizing actions of disopyramide in vitro provide an explanation to the clinical observation that, even though the drug prolongs QT intervals, it does not increase arrhythmic propensity in HCM.", "In a multicenter study of disopyramide, with a relatively low dosage, there was a clear trend toward lower sudden cardiac death mortality in disopyramide-treated patients with HCM (5).", "Subsequent studies have shown that the incidence of sudden cardiac death, appropriate ICD discharges or resuscitated cardiac arrest in a cohort of over 200 patients treated with disopyramide is only 0.3% per year, as evaluated after an average follow-up of 5 years (4,6).", "This rate of lethal or potentially lethal events compares favorably with the natural history of HCM and even with the long-term outcome of patients with obstructive HCM treated with surgical myectomy (51).", "Disopyramide appears to have an intrinsic safety mechanism in HCM cells, whereby the QT prolonging effects are most evident in cells with short baseline AP duration and are overcome by QT shortening effects in those with longer AP.", "Combined with its potential protective action from ventricular arrhythmias, this provides disopyramide with a very favorable profile for clinical use in HCM.", "Following the results of this work, an investigation with prolonged arrhythmia event monitoring before and after disopyramide would be of great interest.", "In addition to its beneficial electrophysiological effects described herein, the hemodynamic effect of gradient reduction is likely to be antiarrhythmic by decreasing myocardial work and supply-demand ischemia, and by improving myocardial energy efficiency.", "Despite the demonstrated electrophysiologic and clinical safety, we continue to believe it is prudent to avoid concomitant administration of other QT-prolonging medications along with disopyramide (Figure 7).", "First, in this work, we only studied cardiomyocytes isolated from the hypertrophied upper septum; therefore, we could not verify whether the effects of disopyramide are different in cells from other less affected regions.", "Second, disopyramide hastens the decay of Ca2+ transients and accelerates myocardial relaxation in HCM myocardium (Figures 1 and 2), as a consequence of the increased Ca2+-extrusion activity of NCX (15,18) (Figure 5).", "Such effect is expected to improve diastolic function in vivo.", "However, echocardiographic data from our patients did not show any amelioration of active mechanical diastolic parameters (Table 4).", "This is in line with previous results obtained in patients with HCM using ranolazine, which failed to improve diastolic parameters, despite reducing circulating pro–B-type natriuretic peptide (52).", "Third, we did not directly evaluate the effects of disopyramide on atrial or ventricular arrhythmia burden in the patients included in the clinical study, as no ambulatory ECG monitoring was performed.", "Fourth, our results do not provide clear mechanistic insights into why up to 30% of patients with obstructive HCM started on disopyramide are nonresponders.", "Given the relatively small number of patient samples, we could not observe a subset of them showing minimal or no response to the drug.", "However, as the response to disopyramide appears to be higher in cells with more pronounced electrophysiological changes (e.g., increase of INaL and decrease of IK [see Figures 6C and 6D]) and the severity of electrical remodeling varies substantially among different patients (as we previously reported in [15]), interpatient variability in the severity of cellular abnormalities may underlie the differences in the clinical response to disopyramide.", "Moreover, prior clinical investigations have shown that a subset of patients with a combination of both high resting gradients >85 mm Hg and long anterior mitral leaflets ≥33 mm Hg have a suboptimal response to oral disopyramide due to a combination of adverse anatomy and excessive leaflet slack (6).", "Fifth, due to the limited number of patient samples and incomplete genetic data (Online Table 1), we were unable to correlate specific parameters of drug effectiveness with the different disease-causing mutated genes.", "As disopyramide does not directly interacts with sarcomeres (Figure 1), we believe it is unlikely that mutations in different sarcomeric genes affect the efficacy of the drug.", "Moreover, we previously observed that the degree of electrical abnormalities at cardiomyocyte level is similar in samples from patients carrying mutations in MYH7 and in MYBPC3 and patients with no sarcomeric gene mutations.", "As disopyramide targets ion channels, these results support the idea that the effects of the drug do not vary depending on the different causing mutations.", "Sixth, one-half of the patients with HCM included in this study were under disopyramide therapy prior to surgery (Online Table 1); as it is unknown whether long-term disopyramide treatment alters the expression or function of ion channels and other functional cardiomyocyte proteins, we cannot exclude that this may have influenced the results of this study.", "Disopyramide emerges as a safe drug due to its multichannel blocking effects.", "Disopyramide is an effective negative inotrope that avoids interference with sarcomere protein function.", "This promises to enrich the pharmacological armamentarium to control obstruction as well as arrhythmias.", "Indeed, our data suggest a potential protective effect of disopyramide from ventricular arrhythmias mediated by suppression of afterdepolarizations and transmural re-entry; future studies aimed at evaluating the antiarrhythmic potential of disopyramide in patients with HCM are warranted.", "The results of this study provide a measure of reassurance to clinicians still concerned with the use of disopyramide in a complex structural disease such as HCM.", "COMPETENCY IN MEDICAL KNOWLEDGE 1: The effects of disopyramide in the hearts of patients with HCM are mediated by the combined inhibition of peak Na+ current, INaL, Ca2+ channels, K+ channels, and RyR.", "Such multichannel inhibition explains the marked negative inotropic effect of the drug, which is responsible for the reduction of LV outflow gradients in patients with obstructive HCM.", "Moreover, these combined actions also result in shortening of the HCM cardiomyocyte AP, improvement of intracellular Ca overload, reduction of afterdepolarizations, and smaller dispersion of repolarization, conferring additional antiarrhythmic potential.", "COMPETENCY IN MEDICAL KNOWLEDGE 2: Our translational study provides the mechanistic explanation for the safety and efficacy of disopyramide in patients with obstructive HCM, which has been confirmed by over 40 years of clinical use.", "These results support the idea that disopyramide treatment can be safely initiated in the outpatient setting and requires only an initial evaluation of the changes in QTc interval, which are usually rather small and are not associated with an increased risk of arrhythmias.", "TRANSLATIONAL OUTLOOK 1: The negative inotropic effect of disopyramide does not depend on direct inhibition of myofilament contraction.", "As such, it does not appear incompatible with the new class of allosteric myosin modulators currently under investigation (mavacamten).", "As disopyramide and mavacamten act on different targets, they could potentially be combined with synergic effects, if the efficacy and safety of mavacamten on long-term administration is confirmed in the ongoing phase III studies.", "TRANSLATIONAL OUTLOOK 2: The use of drugs that inhibit hERG channels and cause drug-related QT prolongation are commonly considered to be more dangerous in patients with pre-existing structural heart disease, such as HCM.", "This assumption does not take into consideration the disease-related changes in cardiomyocyte ion channel expression.", "In HCM, the baseline expression of hERG channels is reduced, so the QT prolonging effect of disopyramide (and probably that of other hERG blocking drugs) is reduced.", "Paradoxically, the proarrhythmic consequences of hERG-blocking QT-prolonging drugs might be less severe in hearts with structural disease and profound electrical remodeling, as compared with healthy hearts.", "TRANSLATIONAL OUTLOOK 3: The promising antiarrhythmic properties of disopyramide in vitro suggest a role for clinical use also in patients without obstructive HCM, such as, as a less toxic alternative to amiodarone for control of atrial fibrillation." ]	[ "In patients with HCM and symptomatic LVOT-obstruction, first treatment with disopyramide leads to a marked reduction of LVOT gradients, with a slight decrease of resting ejection fraction and a modest increase of corrected QT interval, highlighting high efficacy and safety.", "In single cardiomyocytes and intact trabeculae from surgical samples of patients with obstructive HCM, in vitro treatment with 5 μmol/l disopyramide lowered force and Ca2+ transients while reducing action potential duration and the rate of arrhythmic afterdepolarizations.", "These effects are mediated by the combined inhibition of peak and late Na+ currents, L-type Ca2+ current, delayed-rectifier K+ current, and ryanodine receptors.", "In addition to the negative inotropic effect of disopyramide, in vitro results suggest additional antiarrhythmic actions." ]
Future changes, or lack thereof, in the temporal variability of the combined wind-plus-solar power production in Europe	Here we present the first assessment of climate change impacts on the temporal variability of the joint production of wind and solar photovoltaic (PV) power across Europe. For that we adopted regional and continental perspectives (assuming a single European electricity grid), considered several temporal frequencies (from daily to annual), used state-of-the-art regional climate projections together with a climate-production model, and assumed a future massive deployment of wind and PV power installations. Results support that the spatio-temporal complementarity between the wind and solar resources helps to minimize the temporal variability of the combined production under both present (1971–2000) and future (2070–2099) climate conditions similarly. Thus the projected changes are overall negligible (well below ±5%). However, an additional assessment of theoretical upper/bottom bounds for these changes indicated significant potential increases in the stability of the joint production ranging from 5 to 25% across regions, 15% at the continental scale. This would be subordinated to the feasibility of reaching, with the future deployment strategies, individual wind and PV power production series with a perfect temporal anticorrelation. These results may encourage stakeholders to take holistically optimized decisions.	[ "ENER" ]	[ "Climate change", "Solar photovoltaic power", "Supply variability", "Wind power" ]	[ "2105" ]	[ "The transition to renewables plays a key role in the climate change mitigation strategies [1], as well as in the response to the challenges of fossil fuel depletion [2,3] and air quality upkeep and improvement [4].", "Besides, national energy independence targets require increased shares of electricity production from the free, local and clean renewable energy resources, such as wind and solar [5,6].", "However, a high penetration of wind and solar power in the grid, such as proposed for future energy mix scenarios in Europe [7], increases the dependence of the power supply on weather and climate conditions.", "This dependence induces variability and, therefore, the need for backup and storage energy systems to prevent blackouts [8,9].", "This calls for holistic deployment plans aimed at exploiting the spatio-temporal complementarity of the resources [10–17] and thereby minimizing the uncontrollable variability of the renewable supply.", "On the other hand, the long-term projected changes in near-surface wind speed and cloudiness directly impact the amount of energy produced by wind and solar photovoltaic (PV) power farms respectively [18–23].", "Therefore, the vulnerability of the renewable production to climate change could pose an additional issue, not only in terms of changes in the mean production, but also in terms of changes in the temporal stability of the production.", "In terms of mean production, in Europe, previous works have shown that the climate change impact on PV and wind power is limited but overall negative.", "Projected reductions are in the range 0–10%, being, roughly, more pronounced northward for PV and southward for wind power [20,22–24].", "Still these depletions would be smaller than those estimated for the two other main sub-sectors of the electricity market, namely hydro and thermo-electric [25].", "In particular, the projected change in thermo-electric power generation using river water for cooling involves reductions as large as three times higher than for PV and wind power due to future river water scarcity and warming.", "Hence, it seems doubly beneficial to increase the wind and PV power share in combination with reducing thermo-electric power (primarily fossil-fuel based power) in the European energy mix, which, in turn, could increase the energy mix vulnerability to climate change in terms of the variability of the supply.", "The impact of climate change on the temporal variability of PV and wind power production, individually, over Europe, has been assessed in Jerez et al. [20] and Tobin et al. [23] respectively.", "For both powers, projected changes are essentially small (less than 10%) on either high (daily), medium (monthly) or low (yearly) time frequencies.", "As for the mean production, worse signals (higher variability in the future) were found northward for PV and southward for wind power.", "Either way, the whole issue, i.e. the behavior of the PV-plus-wind power generation in terms of its temporal variability under changed climate conditions, is more than the sum of its parts (shaped by the temporal co-variability between PV and wind power production series) and still remains unveiled.", "The main goal of this study is to shed light on the climate change impacts on the temporal variability of the combined electricity production from wind and solar PV power over Europe.", "This is done both at regional and continental scales (i.e. for the ideal case of a single European electricity grid, as encouraged from the European Commission; https://cordis.europa.eu/programme/rcn/664349_en.html) using a consistent approach based on common six-member ensemble of state-of-the-art regional climate model projections and considering future massive deployments of wind and PV power installations aimed at reaching the 80% renewable energy supply goal set by the European Climate Foundation [7].", "Impacts are further assessed under the ideal assumption of perfectly anticorrelated PV and wind power production series with the aim of providing, along with the actual projected changes in the stability of the joint PV-plus-wind power production, upper bounds for potential achievements based on a smart optimization of the spatio-temporal complementarity between the PV and wind powers in their response to the projected climate change.", "Six Euro-Cordex (https://www.euro-cordex.net/) simulations, spanning historical (1971–2000) and scenario (2070–2099) periods, were used (Table 1).", "The latter were developed under the most challenging Representative Concentration Pathway RCP8.5 [26].", "These simulations were chosen based on their previous and coincident use in the works by Tobin et al. [23] and Jerez et al. [20].", "As for these previous works, 3-hourly time series of 10-m wind speed (W), surface downward solar radiation (R) and near surface air temperature (T) were retrieved from the climate simulations and used for the estimation of wind and PV power production series.", "Estimations of power production require the knowledge of the penetration level of the variable renewables in the energy system.", "We considered the mixes defined by the European Climate Foundation [7] in its ambitious 80% Renewable Energy Supply (RES) low-carbon pathway.", "As part of the climate change mitigation strategies, the 80% RES pathway establishes high PV and wind power shares in the European energy mix for the year 2050 (specified per regions; see Table 2) contributing to about 50% of the total power supply (1/3 from PV, 2/3 from wind power installations).", "Wind and PV power production series were estimated at the grid point level (using the Euro-Cordex compliant domain) and thereof aggregated per regions.", "At the grid point level, they were obtained by combining weather-production models (driven by the simulated 3-hourly W, R and T series) and the spatial disaggregation of wind and PV power installations provided by the CLIMIX model [27] from the regional totals of installed capacity listed in Table 2, exactly as in Tobin et al. [23] and Jerez et al. [20].", "The obtained 3-hourly regional production series were then aggregated at the daily time-scale.", "The resulting PV and wind power series were finally added to construct the PV-plus-wind power production series analyzed here.", "The statistical significance of the differences between present and future values was evaluated using the Student's t-test, after the convenient F-transformation applied to the standard deviations of the series and Fisher's z-transformation applied to the temporal correlations, imposing p < 0.05.", "The ensemble mean projected signals are then considered robust when at least 3 individual signals (out of 6, 50% of experiments) are significant and agree in the sign, and any significant signal has opposite sign.", "If there are at least two significant signals with different signs, the ensemble mean signal is considered uncertain.", "If there are not significant signals disagreeing in the sign, but there are less than 3 significant signals with the same sign, the ensemble mean signal is considered negligible.", "This criteria follows Jerez et al. [20].", "Eq. (11) thus permits to assess the effect of changes in the temporal correlation between the PV and the wind power production series on the projected changes in the temporal variability of the joint PV-plus-wind production series, as well its upper and bottom limits.", "Fig. 1 shows the climatologies under present and future conditions of the mean and the temporal variability of the whole daily production series considering only the PV power output (yellow), only wind (blue), or the sum of both (PV-plus-wind; green), along with the projected changes in these magnitudes to the end of the century.", "Climatologies and projected changes for the temporal variability of the series at the time frequencies described in Section 2.4 (Y2Y, MIA, M2M and D2D) are analogously displayed in Fig. 2.", "Changes in mean production (Fig. 1b) are very similar to those reported in previous works [20,23,25], despite the particular sub-ensemble of climate simulations considered here.", "Changes in mean PV-plus-wind power production are negative and robust for all regions, but limited to below 5% according to the ensemble mean signals.", "In the Nordic region, where climate change impact on mean PV power production is highest (∼10%), the high share of wind power (see Table 2) reduces the impact on the total PV-plus-wind mean production (∼2%), as wind power is less affected.", "On the contrary, in the Iberian region, where the impact on wind power is highest (>5%), the high share of PV power (see Table 2) reduces the impact on the total PV-plus-wind mean production (<2%), as PV power is less affected.", "At the continental scale, projections for either PV, wind or PV-plus-wind mean power production involve reductions of a few (2–3) percentage points.", "The temporal variability (normalized standard deviation) of the whole daily PV-plus-wind power production series is lower than for the PV or wind power series individually (Fig. 1c).", "This holds for all European regions under both present and future climate conditions, revealing a certain degree of temporal complementarity between both renewable energies whatever the climate scenario is.", "At the continental scale, for instance, the variability of the PV power production represents about 50% of the mean PV power generation, about 30% for the wind.", "However, the variability of the PV-plus-wind power production represents “only” a 20% of the total mean production.", "To the future, SPV+WP is projected to undergo robust changes, mostly increases, while limited to a few percents all over Europe, with the ensemble mean signals well below 5% (Fig. 1d).", "Interestingly, in the regions of Benelux-Germany, Central Europe and France, also at the continental scale, the climate change signals for SPV+WP are lower than for the individual PV and wind power series, may indicating a certain increase in the complementarity between the WP and PV series in their response to climate change (i.e. ρ'<ρ, Eq. (9)).", "By contrast, the projected increase in the variability of the PV-plus-wind production series exceeds the projections made for the individual series of wind or PV production in the Nordic and Iberian regions.", "In the rest of regions (UK-Ireland, Poland-Baltic, South East Europe and Italy-Malta regions), the climate change signals for SPV+WP are in between those for SWP and those for SPV.", "Among the set of temporal frequencies analyzed here, the highest values of SPV+WP arise for the MIA and D2D terms (left column panels in Fig. 2; see also first column panels of Fig. 4), while also complementarity (i.e. SPV+WP < SPV and SPV+WP < SWP) outstands the most for these two terms, similarly in both the historical and the scenario period.", "In the northernmost regions, and also at the continental scale, complementarity works mostly at the MIA time frequency, indicating that the annual cycles of PV and wind power production are somehow inverted (the anticorrelation values at the MIA time-scale are indeed high, Fig. 3a,c).", "The higher share of wind power than of PV power in northern regions (see Table 2) also helps to reduce the MIA variability of the PV-plus-wind power series as compared to the individual power production series, as the MIA variability is lower for the wind power (Fig. 2c).", "Over central and southern Europe, complementarity outstands the most at the D2D time frequency, partly because of the anticorrelation between the two powers (although the anticorrelation values at the D2D time-scale are not very high, Fig. 3a,c), but mostly due to the higher share of PV power than of wind power (see Table 2), as the D2D variability is lower for the PV power (Fig. 2g).", "In terms of changes (right column panels in Fig. 2) the largest signals arise precisely at these two time-scales (MIA and D2D), being generally positive while small (<5%).", "Consequently, most of the ensemble mean signals are either negligible or uncertain (see also second column panels of Fig. 4).", "Still, it can be appreciated that the hypothesis of complementarity between PV and wind power in their response to climate change (i.e. ΔSPV+WP<ΔSPV and ΔSPV+WP<ΔSWP) holds, at least partially (i.e. ΔSPV+WP at least in between ΔSPV and ΔSWP), in almost all regions and for all the terms (with the exceptions of the changes projected in the MIA variability for Iberia and in the M2M variability for the Nordic region).", "This is particularly appreciable at the continental scale.", "It is also clear from this analysis that the reduced ΔSPV+WP as compared to ΔSPV and ΔSWP identified above for the WDS in the regions of Benelux-Germany, Central Europe and France (Fig. 1d) responds predominantly to the reduced impact on the MIA variability when the PV-plus-wind combined approach is considered (Fig. 2d).", "Notably, in Central Europe, the projected changes in the D2D variability are also lower for the combined series as compared to the signals for PV and wind power (Fig. 2h).", "PV and wind power production series are anticorrelated in time, which holds both in the historical and the scenario periods similarly at any time frequency (the only exceptions appear in South East Europe for the Y2Y and M2M terms and in the Italy-Malta region for the Y2Y term; Fig. 3a,c).", "This helps to reduce the temporal variability of the joint PV-plus-wind power series (Eq. (6)).", "Such anticorrelation outstands the most at the MIA time frequency, with ρ close to −1 in most regions and scenarios.", "As a result, the standard deviation of the MIA PV-plus-wind power production series is notably lower than it would be in the case of non-correlated individual production series (the reference state taken to quantify benefits derived from the PV and wind power complementarity, as described in Section 2.6).", "The gain is above 15% in the regions of Benelux-Germany and Poland-Baltic; 12% at the continental scale (Fig. 3b,d).", "At the D2D time frequency, gains are a bit milder but still notable, e.g. ∼10% at the continental scale.", "However, the upper theoretical limits for the gain in stability are still far.", "For the whole daily series, it could be up to almost 40% regionally, ∼25% at the continental scale, if totally anticorrelated individual production series could be achieved (see range-bars in Fig. 3b,d).", "In terms of changes, it has been already anticipated that the correlation between the PV and the wind power production series (ρ) is expected to remain basically as it is (Fig. 3e).", "This, together with the small changes projected for both SPV and SWP, explains the small changes projected for ΔSPV+WP.", "(Fig. 1d and right panels in Fig. 2).", "As the effect of the projected changes in ρ on the projected changes in SPV+WP is thus quite limited (Fig. 3f), the interest here is to evaluate how it could be given the range of variation allowed for the correlation.", "Results show that negative change signals in ρ would allow to enlarge the stability of the total production daily series by, as much, 10–20% in several regions (e.g. Benelux-Germany, Iberia, Poland-Baltic, France) and at the European level (see range-bars in Fig. 3f).", "Benefits would come mostly from a larger stability at the D2D time frequency (up to 10% in several regions, e.g. Iberia, Poland-Baltic, Central Europe, Italy-Malta and France), since ρ is already close to the ideal −1 value at the MIA time frequency in both present and future scenarios, and the two other terms, Y2Y and M2M, have actually revealed as the least important ones (Section 3.1).", "A synthesis analysis is presented in Fig. 4.", "First column depicts SPV+WP in the historical period, second column the actual ensemble mean projected changes in SPV+WP, and third column the potential ensemble mean projected changes in SPV+WP for an ideal value of ρ = −1 in the future period (i.e. F(-1) according to the notation of Section 2.6, Eq. (9)).", "Gray and black colors mask the negligible and uncertain signals, respectively.", "The variability (standard deviation) of the PV-plus-wind power production series in the historical period ranges from 20 to 40% of the mean production across regions (Fig. 4a).", "At the European scale, it drops to a bit below 20%, which is likely due to the higher wind-and-solar spatio-temporal complementarity potential over wide regions.", "The highest contribution to the variability of the whole daily series come from the MIA and the D2D terms, the former being highest in southern regions (Fig. 4g), the latter in northern ones (Fig. 4m).", "Projected changes are well bellow 5% and negligible for most time frequencies and over most regions, which holds also at the continental scale.", "Robust positive change signals appear over the south-western and northernmost regions for the variability of the whole daily series (Fig. 4b), and over central-eastern and northern regions for the D2D variability of the series (Fig. 4n).", "Robust negative signals appear over Iberia for the M2M and D2D variability of the series (Fig. 4k,n).", "Finally we investigated how changes could be assuming that the PV and the wind power production series could reach, in the future, a perfect anticorrelation.", "Under such an ideal theoretical assumption, the variability of the whole daily series of PV-plus-wind power production could be reduced between 5 and 25% (15% at the European level, Fig. 4c), with the D2D variability term holding the best expectations: 5–15% of reduction all over central and southern Europe (Fig. 4o).", "Previous works have been devoted to assess the spatio-temporal complementarity between wind and solar resources [10–17,28].", "Others have paid attention to the climate change impact on either wind or PV power production, individually [20,22,23,29].", "However, how their combined response to climate change is, in terms of the temporal variability of the total production, still remained unveiled, at least for Europe.", "Here we further demonstrate the temporal complementarity (anticorrelation) of the wind and solar PV production series in their climatological behavior over Europe, which outstands the most because of their typically inverted annual cycles.", "This holds similarly under both present and future climate conditions.", "Thus, given that their temporal variability is not projected to undergo important changes individually, small changes in the temporal variability of the combined PV-plus-wind power production are projected: well bellow 5%, negligible for most time frequencies, from daily to annual, and over most regions.", "This implies that the energy background and storage strategies needed to guarantee the supply stability, in a scenario with a high penetration of the variable renewable in the grid, can be designed considering the current climate behavior and will still work under changed climate conditions.", "Additionally, we investigated how these changes would be under an ideal future scenario where the individual PV and wind power production series were perfectly anticorrelated in time, while maintained their characteristic mean and standard deviation values (as these magnitudes are, essentially, not projected to change).", "This scenario drew potential significant reductions in the variability of the daily PV-plus-wind power production series, upper-bounded at 5–25% across regions, 15% at the European level, with the highest benefits rebounding on the high frequency day-to-day variability of the combined production series.", "These are just upper theoretical bounds, but could motivate smart deployment plans that look for highly complementary locations (holistically) for the new PV and wind power installations, instead of highly rated ones (individually), as this could actually enhance the self-regulation of the stability of the wind-plus-solar power production system.", "Alternative approaches for minimizing the variability of the combined production could be based on minimizing the variability of the PV and wind power production series individually, or could investigate how to reach a better balance between them by modifying their regional shares.", "In any case, while a stable production is clearly a sign of energy security, in practice, the time-varying demand is the driving factor, and production should follow it.", "Hence future research is needed on the feasibility of the various supply-security approaches, including the energy demand factor, adopting holistic and practical perspectives tailored to the challenges of the energy sector, and taking into account geophysical constraints [30].", "Also, efforts should be allocated to improve the regional climate projections in order to enhance the reliability of the climate change impact assessments and narrow the uncertainties around the ensemble mean signals.", "Currently, regional climate models miss (or may miss) important processes and feedbacks that are likely to have an important fingerprint in the simulation of the renewable potential, such as the aerosol-radiation and aerosol-clouds interactions [31–33] and the radiative effect of the greenhouse gases increase at regional level [34].", "The Authors declare no conflict of interests." ]	[ "Spatio-temporal complementarity between wind and solar resources drops the variability of the mix production similarly under present and future climates.", "Climate change impacts little on the stability of the combined PV-plus-wind power production.", "Stability could grow up to 15% at the European continental scale (5 to 25% across regions) by exploiting the spatio-temporal complementarity of the resources." ]
Growing N-doped multiphase TiO2 nanocomposites on reduced graphene oxide: Characterization and activity under low energy visible radiation	Reduced graphene oxide (G) was used as a platform to grow a mixed catalyst made of brookite and rutile nanoparticles doped with nitrogen, resulting in excellent performance for the oxidation of 4-nitrophenol (4-NP) in water under low energy (>425 nm) radiation. The samples were fully characterized by X-Ray Diffractometry (XRD), Raman Spectroscopy, Electron Microscopy, X-Ray photoelectron spectroscopy (XPS), photoluminescence (PL), Z-potential analysis, UV-vis Diffuse Reflectance Spectrophotometry (UV-vis DRS), and porosimetry. The improved hole-electron separation, demonstrated by PL, is boosted by the exceptional properties of reduced graphene oxide, which attracts and conveys electrons to dissolved oxygen, in turn initiating the oxidation process. The optimal amount of reduced graphene oxide was found to be 1% w/w based on 4-nitrophenol (4-NP) conversion rates. No leaching of carbon into water was revealed, even under irradiation, pointing to the suitability of the composite catalyst in water.	[ "CENG", "ENVI" ]	[ "Multiphase TiO2", "N-doping", "Photocatalysis", "Reduced graphene oxide-TiO2", "Visible radiation" ]	[ "1501", "1508", "2310", "2311" ]	[ "Photocatalysis is an increasingly popular technology to mitigate pollution and environmental issues [1–3].", "As such, it enables the degradation of organic species in water without the use of aggressive oxidizing chemicals.", "TiO2 has been the most widely reported photocatalyst owing to its low cost, high efficiency and good stability.", "However, the ability of TiO2 to be activated for oxidation processes is restricted to the need of UV light.", "Consequently, its modification with nitrogen has been proved as one of the best way to boost reactivity under visible light radiation, which is the majority of solar spectrum, and the only light available indoors [4].", "The most used crystal phase of TiO2 is anatase (with a band gap of 3.2 eV), very active in the UV region; however, rutile and brookite-rutile nanocomposites have been recently reported to be active in the visible, especially upon nitrogen doping [5,6].", "Rutile has a narrower band gap (3.0 eV) than anatase and, coupled with brookite, it can enhance charge separation.", "Nitrogen atoms can be incorporated either interstitially or substitutionally, acting as attraction centers for holes [7], and then foster the formation of oxygen vacancies, thus promoting absorption of visible light and, eventually, reactivity.", "Beside nitrogen doping, addition of reduced graphene oxide (G) can significantly enhance reactivity under visible radiation [8] due to its remarkable properties, such as high electron mobility, high surface area to volume ratio, because of its 2-dimensional nature, and transparency [8].", "To the best of our knowledge, there was only one attempt to obtain a N-doped TiO2 synthesized on graphene through a sol-gel route, giving rise to anatase phase; moreover, the catalyst was not used for water remediation purposes [9].", "While the modification of anatase or anatase-rutile TiO2 with reduced graphene oxide has been reported in the last years by a number of scholars [10,11], the present study deals with a new composite material (N-TiO2-G) obtained by synthesizing N-doped brookite-rutile nanoparticles in the presence of reduced graphene oxide (G).", "The particles form and grow on the reduced graphene oxide sheets, resulting in a number of advantages in terms of 4-NP photodegradation in water, irradiated by visible LED, as detailed in the following.", "To the best of our knowledge, TiO2 modified with reduced graphene oxide has never been used before to oxidize 4-NP.", "A modified Hummer method was used to prepare graphite oxide by using graphite flakes as the precursors.", "3 g graphite flakes, 2.5 g potassium persulfate and 2.5 g phosphorous pentoxide were poured into 12 mL concentrated sulfuric acid (H2SO4) and stirred vigorously for 4.5 h at 80 °C.", "The mixture was cooled down to room temperature naturally.", "Afterwards, 0.5 L deionized (DI) water was added and aging was prolonged for 12 h under stirring.", "The solution was filtered, washed and dried to obtain a black solid.", "The pretreated graphite flakes underwent oxidation by 15 g potassium permanganate (KMnO4) in 120 mL concentrated sulfuric acid (H2SO4) in an ice bath for 1 h.", "The mixture was stirred at 35 °C for 24 h to obtain a very viscous dark-brown paste, diluted with a slow addition of 250 mL of DI water and further stirred for 4 h 30 mL (30 w%) of H2O2 were slowly added to quench the solution thus producing a golden-brown solution.", "The mixture was filtered and washed with DI water until a pH of 6 was obtained in the washing solution.", "The graphite oxide was dried at 40 °C.", "Following the oxidation process, graphite oxide (0.5 g L−1) was exfoliated in water using an ultrasound bath (100 W) for 10 h, ultimately producing a multilayered graphene oxide (GO).", "The reduction of GO was performed by using reducing agent, that is sodium borohydride (NaBH4), by stirring at room temperature for 5 h. Reduced graphene oxide (G) black powder was finally dried by a rotary evaporator at 50 °C.", "Washing with DI water 10 times with the aid of a centrifuge removed the residual ions producing a pure G powder.", "N-TiO2-G was prepared by using a sol-gel method.", "Titanium (IV) butoxide (TBOT) was used as TiO2 precursor.", "TBOT, 2-propanol and HCl (4 M) in a volumetric ratio of 1:5.461:1.283, respectively, were mixed until a clear solution (total volume: 116.24 mL) was obtained under stirring at 70 °C for 20 h.", "The samples were dried by means of rotary evaporator at 70 °C.", "Following that, the powder was calcined at 450 °C for 4 h under nitrogen flow to minimize oxidative/thermal degradation of G. G and ammonium nitrate, used as N-doping agent with 0.8% (w/w), were dispersed/dissolved in the water prior to mixing with TBOT, HCl and 2-propanol solution.", "When needed, ultrasound treatment and ultrafast (3000 rpm) magnetic stirring were applied to properly break down and disperse G. Bare N-doped TiO2 was prepared as well.", "The amount of G was fixed at 0.1%,0.5% 1%, 1.5%, 2% (w/w) with respect to TiO2 and the corresponding samples were denoted as N-TiO2/G_0.1%, N-TiO2-G_0.5%, N-TiO2-G_1%, N-TiO2-G_1.5% and TiO2-G_2%, respectively.", "A Nova NanoSEM was used to observe the morphology and dimensions of samples and to perform EDX mapping.", "Samples were prepared by dropping a water suspension on a stainless steel stub previously cleansed with water, acetone and ethanol under ultrasounds.", "Mapping of the constitutive elements of the composite materials was carried out by using a distribution mapping technique by energy dispersive X-ray spectroscopy (EDX) combined with SEM.", "EDX mapping was recorded using a spot size of 5 nm, X-ray energy of 20 eV with a 256 × 200 resolution and 200 μs dwells (64 frames).", "Carbon, titanium and oxygen mapping, using red, green and blue colors respectively, provides the visualization of elemental contents.", "Powder X-Ray diffraction (XRD) was performed in a 2θ range of 10–90° by using PANalytical Empyrean diffractometer with Cu Kα radiation of 1.54 Å at 45 kV and 40 mA to determine the crystal structure of the prepared samples.", "The powders were ground in a mortar before analysis, and a holder with fixed thickness of samples was used to get comparable results.", "Raman spectroscopy was performed by a Renishaw Raman spectroscope equipped with a 514.5 nm laser line and laser power below 0.5 mW.", "The Raman spectra were collected in back scattering configuration in the range 3500 cm−1–50 cm−1.", "The powder was pressed in a holder to get a homogeneous surface before the analysis.", "Fourier transform infrared (FT-IR) analysis was carried out by using a Bruker Vertex 80 v FT-IR (128 scans) in the 4000 cm−1–400 cm−1 region in diffuse reflectance mode (DRIFT).", "The powders were ground in a mortar before analysis and pressed in a holder to get a flat surface.", "X-ray Photoelectron Spectroscopy (XPS) was performed on an Escalab 250 × i from Thermo with a monochromatic Al Kα source (1486.6 eV).", "Photoelectrons are collected at an angle of 0° relative to the sample surface normal.", "Samples were fixed using double-sided carbon tape.", "Survey and high-resolution spectra were acquired using a spot size of 250 μm and pass energy of 150 eV and 20 eV, respectively.", "A flood gun with combined electron and low energy ions was also used during analysis to prevent surface charging.", "Surveys were measured in steps of 1.0 eV with 50 ms dwell time per data point.", "Nitrogen, carbon, and oxygen 1 s high-resolution spectra were measured within the spectral range of interest with 0.1 eV steps and 50 ms dwell time per data point.", "Analysis of the data was carried out with CasaXPS software.", "A Shirley background was used in curve-fitting along with a GL(30) line-shape (70% Gaussian, 30% Lorentzian using the Gaussian/Lorentzian product form) for the C 1s, N 1s and O 1s spectra.", "Samples were referenced to the C 1s emission by adventitious hydrocarbon contamination at 284.8 eV.", "TEM analysis was carried out by using a Tecnai G2 transmission electron microscope to observe the morphology and dimensions of samples.", "Samples for TEM were deposited on a Forvar/Carbon 300 or 400-mesh Cu grid purchased from Tedpella; the powder was previously suspended in water and 2 μL were dropped on the TEM grid twice (waiting for the complete drying before the second deposition).", "The adsorption/desorption isotherms of the catalysts were recorded by using a Quantachrome NOVA 2000e surface area and pore size analyzer by using N2 as adsorbent.", "The sample was degassed in static conditions under vacuum at 400 °C for 4 h prior to analysis.", "The specific surface area of catalysts were calculated using the multipoint Brunauer-Emmett-Teller (BET) method, as an average desorption and adsorption values in the P/Po range of 0–0.35.", "The pore size distribution was calculated from the desorption curve in the whole range of pressures by using two different models, BJH (Barrett-Joyner-Halenda) and DFT (Density Functional Theory).", "UV–vis diffuse reflectance spectra (DRS) were recorded by using a Shimadzu UV-2600 Spectrophotometer in a range 200–800 nm.", "The powder was pressed in a holder to get a flat surface before the analysis.", "The optical band gaps were calculated by considering indirect transitions, as typically applied to TiO2 based samples.", "Photoluminescence was recorded by using a Perkin Elmer LS-55, preparing the samples by pressing in a holder to get a flat surface before the analysis.", "The analyses were run by using dry powders with the following parameters: excitation wavelength: 300 nm, scanning speed of 500 nm min−1, excitation slit width 2.5 nm, emission slit: 7.5 nm.", "The point of zero charge was assessed by Brookhaven ZetaPALS (Zeta potential Brookhaven Instruments Corporation, USA).", "The Zeta potential of the samples was recorded at different pH’s in water suspensions of 1 mg/L at 25 °C and the equipment run 10 cycles per measurement.", "The pH of samples was adjusted by using 0.3 M HCl and 1 M NaOH and the same analysis was triplicated.", "The irradiation system employed for the reactivity study consisted of a flat visible LED source (electric power absorbed: 33.1 W) emitting at wavelengths greater than 425 nm (Fig. S1).", "A 400 mL beaker was placed containing 250 mL suspension mixed at a 1800 rpm by using a magnetic stirrer.", "The radiation intensity reaching the surface of the suspension was 203 W m−2 (measured in the 450–950 nm range by using a DeltaOhm 9721 radiometer and the matching probe).", "Before the runs, each sample was added progressively into 250 mL DI water (every time treating with ultrasounds for 2 min) until reaching a final transmitted radiation intensity from the bottom of the beaker of ca. 10% with respect to the transmitted light in the presence of only DI water: in this way it was assured that all particles were irradiated.", "The powder weight required to have this transmittance was the one used for the reactivity tests (Table S1), carried out in water suspensions of 5 mg L−1 of 4-nitrophenol (4-NP), at the natural pH of 5.5.", "At this pH, only molecular 4-NP exists in solution and the amount of 4-nitrophenate can be neglected.", "This is relevant since the latter one has a strong absorption in the visible radiation and would drastically affect the reactivity under radiation greater 425 nm, such that used in the present study.", "Oxygen was bubbled into the suspension for 2 h in the dark before turning the lamp on, after which the adsorbed 4-NP was checked.", "Then light was then switched on and oxygen was flowed during all the run.", "Samples were withdrawn periodically from the reactor by using a syringe and filtered using a 0.2 μm PTFE filter.", "The total irradiation time was 20.5 h.", "The extent of degradation of 4-NP was measured as a function of the visible absorbance of each sample at a wavelength of 315 nm by using a Thermo Scientific HPLC (Dionex UltiMate 3000 Photodiode Array Detector) with an Acclaim-120 C18 Reversed-phase LC column working at 25 °C (eluent: 33% water, 33% acetonitrile, 34% methanol; flow rate: 0.2 mL min−1).", "The reproducibility of the experimental runs was always over 95%.", "The stability of N-TiO2-G_1% was assessed by recycling the catalyst for 3 runs.", "After the each photocatalytic reaction, the suspension was centrifuged and filtered to recover the photocatalyst, which was washed with DI water, dried and used in the next cycle of 4-NP oxidation, in the same experimental conditions described above.", "GO was prepared by using a modified Hummer’s method [12] and reduced using sodium borohydride.", "After that, Titanium tetrabutoxide (TBOT) was hydrolyzed in a controlled way, in the presence of evenly dispersed G and a nitrogen source.", "The obtained powder was annealed at 450 °C under nitrogen flow to limit graphene oxidative thermal degradation.", "The thermal treatment resulted in the degradation of excess G layers built up over the nanoparticles (Figs. 1, 2 and S2 ) during the annealing of the composite catalyst, thus ensuring the photocatalytic activity of the materials.", "EDX mapping on titanium, oxygen and carbon on a representative composite shows the very different Ti signal before and after treatment (Figs. 1 and 2), due to the degradation of G over TiO2 particles during the annealing.", "Incidentally, it should be pointed out that EDX is not very sensitive for oxygen and, especially, for carbon at the low used loading.", "XRD allowed to verify the successful oxidation of graphite to graphite oxide first and to graphene oxide (GO) after exfoliation, and also the reduction of graphene oxide (GO) to reduced graphene oxide (Fig. S3).", "In fact, the characteristic diffraction peaks of graphite, corresponding to (002) and (004) plans, disappear after oxidation, and GO peak at 10.25° disappears after reduction.", "The diffraction peak at 8.2°, obtained for G, can be ascribed to the establishment of a 1.1 nm interplanar spacing in the stacking of the carbon layers, similarly to what previously observed [13].", "A broad (002) peak can be observed at ca. 29.0° and it can be attributed to the layer-to-layer distance (0.31 nm) [14].", "The broad diffraction peak at 43° is associated to a turbostratic signal characteristic of disordered carbon nanomaterials [15].", "Looking at the XRD diffractograms of N-TiO2 and N-TiO2-G (Fig. S4), we can observe all the characteristic peaks of brookite and rutile (25.4°, 30.8°, 48.1° for brookite; 27.4°, 41.2°, 54.3°, 56.6°, 69.0° for rutile), and exclude the presence of anatase (75.1° is an intense diffraction peak of the crystal plane (215) of anatase and, in this case, it is absent).", "Graphene peaks are not visible in the XRD of the composite materials, since its percentage is far below XRD detection limit.", "Using the Scherrer’s equation [6], the rutile crystallites was 22–29 nm in size, well larger than those of brookite crystallites (11–13 nm).", "The Raman spectra of graphite, graphite oxide, GO and G (Fig. S5) highlight that, upon graphite oxidization to graphite oxide, the characteristic sharp bands of the former disappear.", "The predominant Raman bands in GO and G are the D and G bands, and the ratio between the intensity of D and G bands looks greater for G, compared to GO, thus confirming the successful reduction of the latter one.", "Moreover, the 2D band is absent because both GO and G are highly defective [16].", "Raman spectra of the composite materials are shown in Fig. 3, where brookite signals at ca. 150 (A1g), 246 (A1g), 322 (B1g), 365 (B2g), 402 (A1g), 449 (B2g), and 640 (A1g) cm−1 can be clearly observed [17].", "Rutile signals are more difficult to be identified and they correspond to frequencies of 126 (A1g), 440 (Eg) and 611 (B1g) cm−1, as also suggested by literature [18].", "However, the exact determination of rutile frequencies is hampered by the high number of brookite bands, which partially overlap with rutile bands.", "The Raman signals of graphene cannot be seen, due to the small amount used, and to the TiO2 Raman background in the spectral region of the graphene Raman peaks.", "After reduction of GO, the FTIR highlighted the disappearance of the band centered at 1700 cm−1, assigned to CO stretching, pointing to the effective reduction of GO to G (Fig. S6).", "The difference between N-TiO2 and N-TiO2-G_1% is not obvious.", "The CC band centered at 1600 cm−1 partially overlaps with OH signals making it very difficult to appreciate any difference.", "XPS surveys of GO and G also support the effective reduction of the former to the latter.", "As shown in high resolution carbon spectra for GO and G (Fig. 4 a and b), the oxidized carbon peaks (CO at 286.8 eV and CO at 288.1 eV) decreased from 36 to 21.8% and from 5.4 to 4.5%, respectively, while sp2 carbon peak (CC at 284.8 eV) increased from 17.9 to 28.7% after the reduction.", "In Fig. 4c and d, high resolution (HR) carbon spectra for N-TiO2 with 0.1% and 1% G loadings are shown.", "Due to the very small G content in N-TiO2 and the possible contamination of adventitious carbon from the atmosphere, it is difficult to conclusively attribute the sp2 carbon signal to G. However, small increase of carbon content is observed for the higher G loading sample, possibly originated from increased G in N-TiO2.", "TEM characterization of graphite oxide, GO and G are shown in Supporting information (Fig. S7), where the 2-dimensional morphology typical of these materials with thinner and smaller particle sizes moving from graphite oxide down to G is evident.", "TEM of N-TiO2 samples modified with G (Fig. 5 and Fig. S8) highlighted the presence of bigger particles of rutile (20–100 nm) with respect to brookite (5–20 nm), as suggested by XRD as well.", "The significantly different size and shape of brookite and rutile was recently validated by the analysis of electron diffraction patterns (EDP) and d-spacing (through HRTEM) of analogous brookite-rutile samples prepared without G [6].", "Fig. 5 shows that brookite and rutile nanoparticles grow on G sheets with dimensions ranging from ca. 100 to 1000 nm, and the good contact among rutile, brookite and G is remarkable.", "This is obviously propitious for an efficient charge transfer and stable separation of electrons and holes, as further demonstrated by photoluminescence spectra shown in the following.", "In samples containing 1% G, it was possible to detect G in few points, although its high transparency to the electron beam, along with the masking effects of nanoparticles on it hampered a perfect visualization (Fig. 5c and d).", "The EDP of an area where brookite, rutile and G were present (inset of Fig. 5a) resulted in bright circles typical of G, along with distinct spots determined by crystalline particles.", "A hysteresis of type IV can be seen in the BET isotherms of all the samples (Figs. S9–14 in Supporting information), pointing to a capillary condensation in mesopores.", "The pore sizes are in the mesoporosity region, roughly ranging from 2.9 to 30 nm and pore size distributions are available as Supporting information.", "The specific surface area is smaller for the sample with 1% G, with wider pores compared to the other samples, as shown in Table 1.", "In UV–vis DRS (Fig. S15) we can notice a slightly better ability to absorb visible radiation with respect to N-TiO2, although the calculated band gap of N-TiO2 (2.95 eV) for indirect semiconductors is very close to that of N-TiO2-G_2% (2.93 eV), similarly to what previously reported for modified TiO2 [20,21].", "The photoluminescence (PL) emission upon excitation at 300 nm (Fig. 6) shades light on the strong improvement of charge separation on the composite materials compared to TiO2 and N-TiO2.", "G surface has a positive Mulliken charge, resulting in an opposite interface dipole at the border with TiO2 nanoparticles: consequently a strong tendency exists for electron transfer to G, resulting in an attenuation of charge recombination [22].", "Accordingly, a lower photoluminescence emission can be observed in the whole emission spectrum (380–585 nm) for N-TiO2-G_1% compared to N-TiO2 and TiO2.", "From the spectra in Fig. 6 one can notice that the shape of the emissions are similar and the main emission is centered at ca. 420 nm.", "The band-to-band transition occurs around this wavelength corresponding to the band gap energy.", "Emission at greater wavelengths is produced by electron trapping in TiO2 oxygen vacancies and transitions from intra-band gap states to valence band.", "However, the intensity of the peak in the presence of G is lower compared to N-TiO2, highlighting the ability of G to attract electrons on an energy level close to the conduction band of brookite and rutile and to convey them away from TiO2, thus limiting their recombination with holes.", "This is possible given the work function of reduced graphene oxide (−4.5 ± 0.2 eV) [23] compared to the conduction band of TiO2 which is −4.0 ± 0.1 eV (the N-doping does not affect this level much) [6,8].", "The point of zero charge of G in water is ranging from 3 to 5 due to the presence of carboxylic acid groups and phenolic hydroxyl groups in G [24].", "On the other hand, the point of zero charge of TiO2 can be drastically different depending on the surface characteristics of the semiconductor.", "In the present case, the Zeta-potential analysis performed on the composite materials indicated that Nitrogen doping and G loading had a minor effect on the point of zero charges, which ranged between 4.7 and 5.3 without any trend (Supporting information, Fig. S16 and Table S2).", "The minor change by doping with nitrogen can be ascribed to the electronegativities of nitrogen and oxygen in TiO2, which are not very different.", "On the other hand, G is not affecting the point of zero charge of photocatalysts most probably because it is located below the semiconductor nanoparticles, with a negligible interface with water molecules.", "Although the investigation of reaction mechanism was not the aim of the present study, extensive past research has shown that, in the course of 4-NP photocatalytic oxidation, NO2 group can be replaced by a hydroxyl group forming hydroquinone or alternatively an hydroxyl radical can enter the ortho-position activated by the phenolic group in 4-NP, producing 3,4-dihydroxynitrobenzene.", "Ring opening and conversion to CO2 are further oxidation steps, although the parallel conversion to CO2 of the starting substrate, without any formation of organic intermediates is also viable, as shown in a number of studies [25–27].", "Finally, the value of the presented catalysts in the oxidation of 4-NP under low energy visible radiation was underlined by the total lack of activity, which distinguished analogous experimental runs carried out in the presence of commercial catalysts, i.e. Evonik P25 and mixtures of Sigma-Aldrich rutile and brookite (50/50 w/w).", "The reported study showed that the contemporary i) N-doping and ii) growth of brookite-rutile samples on reduced graphene oxide yield a high efficiency in the oxidation of a model pollutant (4-NP) from water through photodegradation under UV-free visible radiation.", "The effect of reduced graphene oxide in the composite material is to: (i) improve the charge separation and (ii) extend the light absorption threshold.", "Likely, π-conjugation of reduced graphene oxide can play a major positive role." ]	[ "N-doped brookite-rutile were grown on reduced graphene oxide through a sol-gel method.", "4-Nitrophenol was used as a model pollutant in water.", "The optimal loading of reduced graphene oxide was found to be 1%.", "PL at 1% graphene is minimal due to limited electron-hole recombination." ]
Modelling water use efficiency in a dynamic environment: An example using Arabidopsis thaliana	Intrinsic water use efficiency (Wi), the ratio of net CO2 assimilation (A) over stomatal conductance to water vapour (gs), is a complex trait used to assess plant performance. Improving Wi could lead in theory to higher productivity or reduced water usage by the plant, but the physiological traits for improvement and their combined effects on Wi have not been clearly identified. Under fluctuating light intensity, the temporal response of gs is an order of magnitude slower than A, which results in rapid variations in Wi. Compared to traditional approaches, our new model scales stoma behaviour at the leaf level to predict gs and A during a diurnal period, reproducing natural fluctuations of light intensity, in order to dissect Wi into traits of interest. The results confirmed the importance of stomatal density and photosynthetic capacity on Wi but also revealed the importance of incomplete stomatal closure under dark conditions as well as stomatal sensitivity to light intensity. The observed continuous decrease of A and gs over the diurnal period was successfully described by negative feedback of the accumulation of photosynthetic products. Investigation into the impact of leaf anatomy on temporal responses of A, gs and Wi revealed that a high density of stomata produces the most rapid response of gs but may result in lower Wi.	[ "AGRI", "BIOC" ]	[ "Diurnal", "Dynamics", "Intrinsic water use efficiency", "Photosynthesis", "Stomatal conductance" ]	[ "1102", "1110", "1311" ]	[ "In order to meet the projected demand for cereal production by 2050, crop yields must improve by 1.16–1.31% each year; however, current estimates are well below this required rate [1].", "The primary determinant of crop yield is the cumulative rate of photosynthesis over the growing season and is determined by the ability of the plant to capture light and CO2, use this energy to convert the CO2 to biomass, and how much of this biomass ends in usable yield.", "Improving photosynthetic efficiency is recognised as an important but unexploited avenue to increase yield potential in crop plants [2].", "Increasing photosynthetic efficiency is accompanied by a higher CO2 demand, which can be limited by the resistance of CO2 diffusion into the leaf.", "Any attempt to decrease this resistance greatly increases the water loss by transpiration from the leaf.", "Photosynthetic productivity is linked to water consumed by the plant and often measured as water use efficiency (WUE).", "WUE can be defined at different scales of time and space and, at the leaf level, it is often assessed as the ratio of CO2 fixed per unit of H2O transpired (E).", "Intrinsic water use efficiency (Wi) is defined when stomatal conductance to water vapour (gs) is used instead of E.", "The use of gs to describe the stomatal control on the rate of E facilitates the comparisons between different leaves and environmental conditions.", "The photosynthetic capacity of the leaf determines the net CO2 assimilation (A) as a function of the variation in the micro-climate surrounding the leaf.", "Over the diurnal period, A is mainly determined by the irradiance absorbed by the leaf and the limitation of CO2 imposed by stomatal control.", "Under field conditions, environmental variables that affect both photosynthesis and stomatal behaviour are rarely constant.", "For example, light intensity (and spectral quality) alters in time scales of seconds to hours to which A and gs must respond.", "The temporal response of A and gs to a fluctuating environment are asynchronous, with gs response often an order of magnitude slower than A, which results in rapid variations of Wi.", "Thus, it is important when describing the kinetic response of Wi to use an approach that considers responses by A and gs simultaneously.", "Intrinsic water use efficiency (Wi) is dependent on the anatomy (e.g. stomatal size and density) and the physiology (e.g. behaviour) of stomata as well as the leaf biochemistry (e.g. activity of the Calvin cycle), all of which interact to determine the kinetics of CO2 and H2O gaseous exchange between the leaf and atmosphere.", "The dynamic nature of the interactions between the different components that determine Wi are not fully understood and need to be addressed if we are to successfully improve both A and Wi under dynamic field conditions.", "It is possible to conceptualise the inherent complexity of gas exchange over a fluctuating light regime through modelling, which will improve our understanding of the Wi response by simulating a number of gas exchange scenarios (e.g. changes in light intensity and humidity) that would normally be difficult to assess in a reasonable amount of time using experimental approaches.", "Current models focus on predicting gs in steady state [3] and cannot be used to infer the impact of stomatal behaviour on A or Wi under dynamic conditions.", "Although temporal responses of gs have previously been described using a dynamic model [4,5], the relationship between stomatal response and leaf level gas exchange was not clearly described.", "We propose to use a model that will take into consideration the anatomy and physiology of stomata to more accurately represent the stomatal control of Wi.", "To scale stomatal responses to leaf level gs, the two most important stomatal characteristics are aperture and density [6–8].", "A high stomatal density does not necessarily result in a higher gs as stomata ultimately control their aperture depending on the guard cell responses to the external (e.g. light intensity) and internal (e.g. mesophyll demand for CO2) stimuli [9].", "To link stomatal behaviour to leaf level gas exchange responses, we propose a ‘big stoma’ approach that consists of simulating the response of one stoma that is representative of the heterogeneous response of many stomata and scaling the response to the leaf level.", "This approach was incorporated in an enhanced version of the multi compartments model described by Noe and Giersch [10] to predict A and Wi.", "Scaling up the dynamic of the stomatal response to the leaf level, with the improved model for CO2 diffusion inside the leaf, will help to dissect Wi into traits of interest and predict potential gains in Wi.", "The objective of this study was to develop a new model combining our most recent knowledge of kinetics in stomatal behaviour and photosynthesis to describe the temporal response of Wi over the course of a day with natural dynamic variations in irradiance.", "All the parameters of the model described here incorporate a trait of interest for Wi and were adjusted using Bayesian inference.", "The model was validated using a dataset with a different irradiance pattern to assess the predictive power of the model.", "A sensitivity analysis was finally performed to show the interaction among the parameters and display the potential gain in Wi in the case of one or two parameters changing.", "We used the output of the model to understand how temporal responses in gs impacts A and Wi.", "The first differential equation (Eq. (1)) described the temporal variations of the stomatal pore area (a) with as the steady state target followed by a and two time constants, ki and kd, for an increase or a decrease of a respectively.", "Considering the spatial heterogeneity of the stomatal response, a top-down approach was used, signifying that the model simulated the response of one stoma representative of the sum of the individual stomatal responses and scaled it to leaf level instead of trying to integrate the response of each stoma.", "Eq. (3) described the variation of the CO2 concentration in the intercellular airspaces (Ci) with Ca the atmospheric CO2 concentration and Cc the CO2 concentration at the sites of carboxylation.", "The resistance of CO2 diffusion in the mesophyll cells (gm) was calculated following the equations described by Von Caemmerer et al. [11] and the values given for Arabidopsis thaliana.", "As the temperature of the leaf was regulated during the experiment, gm was considered stable.", "The last equation (Eq. (7)) described the negative feedback of sugar accumulation on as (Eq. (2)) and AG during a period of light, with Sa being a factor representing the influence of the rate of accumulation of sugars, and Se a factor representing the influence of the rate of export of sugars.", "The differential equations were solved using the function lsodes from the R package deSolve (v1.12).", "The initial absolute tolerance was set to 0.01 for all the outputs and decreased by 10 each time the solver failed to converge.", "The initial values of the differential equations were chosen based on the observed data.", "For the pore area, the initial aperture was calculated by solving Eq. (5) for the first recorded gs.", "Parameter values known to impact the diurnal variation of gas exchange were adjusted using the Bayesian inference (see below) to fit the observed data.", "During the diurnal period, gross CO2 assimilation (AG) was determined by maximum Rubisco activity (Vcmax), maximum electron transport activity (Jmax) and the proportion of light absorbed by PSII (α).", "The day respiration in presence of light (Rd) was used to calculate the net CO2 assimilation (A).", "The stomatal conductance to water vapour (gs) was determined as described above by amin, amax, αL and ki/kd.", "The diurnal variation of A and gs were linked by the negative feedback of sugar export described by Sa and Se.", "Parameter values known to remain unchanged during the diurnal period of measurement or that could be assigned a priori were treated as constants in the model, to facilitate the adjustment, and were chosen from the literature or measured on Arabidopsis thaliana (see below).", "The Michaelis constants and the temperature responses of the Farquhar model were parameterized using values from Walker et al. [13].", "The thickness of the leaf was set to 200 μm [14,15] and the ratio of airspace in the leaf was set to 0.255 [14,16].", "The value of θL in equation 2 was chosen at 0.7 by analogy with the similar equation describing the electron transport rate [17].", "The boundary layer conductance (gb) and atmospheric pressure (Patm) were set to the values provided by the LI-6400XT: 9.29 mol m−2 s−1 and 101.8 kPa respectively.", "Estimates of gb are computed as a function of leaf area and fan speed (LI-6400XT Instruction Manuals, page 3–93).", "The parameter values describing the membrane properties required to estimate gm followed that of Von Caemmerer et al. [18].", "Stomatal density (SD) was assessed on plants grown under the same environmental conditions, on both faces of the leaf (section 1.9).", "As the model simulated the average response of both faces, the densities of both faces were added and set to 400 mm−2.", "In equation 5, 5.89 μm was used for the depth of the stomatal pore (l) [7], the diffusivity of water in air at 25 °C (D) was 24.2e−6 m2 s−1, the molar volume of air at 25 °C (V) was 0.02446533 m3 mol−1 [19].", "The parameter values were estimated using Bayesian inference by comparing the model outputs to recorded gas exchange data [20].", "The inference was performed on two objective functions as A and gs were adjusted simultaneously and in this case, there was a risk of bimodal posterior distributions.", "Monte Carlo Markov Chain (MCMC) using inter-chain adaptation were previously able to sample from bimodal distributions and this technique was thus used to adjust our model (INCA, [21,22]).", "To remove any adaptive effect, the final algorithm used to sample the posterior distributions was the random walk metropolis (RWM) with the covariance matrix estimated previously with INCA as an input.", "The use of Bayesian Inference allowed us to use prior information available in the literature and shown in Table 1.", "The results of the inference were posterior estimates describing the probability density of the possible parameter values considering the error of prediction and the sensitivity of the model for these parameters and their inter-correlation.", "The posteriors were represented using quantiles of the posterior distribution at 2.5% and 97.5%, also called credible intervals (similar to a confidence interval).", "The medians were also shown as the most representative set of parameter values.", "Two chains with 30,000 iterations were thinned every 2 samples and tested for different criteria: the acceptance rate greater than 15% (to achieve a rapid mixing of the chain), the Effective Sample Size (ESS) > 100 (which is usually enough to describe 95% probability intervals) and the convergence using Monte Carlo Standard Error (MCSE) less than 6.27% (which allows the true mean to be within 5% of the area under a Gaussian distribution around the estimated mean; [23]).", "The algorithms used to perform the Bayesian inference were implemented using the R software (R Core Team, 2015, v3.2.2).", "The sensitivity analysis was performed using the same data set and parameter values estimated previously using Bayesian inference.", "Each parameter value was altered by ±50%, the impact on the model outputs was summarized by using the daily mean of A and gs divided by the corresponding values obtained using the original parameters values.", "This resulted in a factor centred on 1 representing the positive or negative impact of the parameter alterations on A and gs (e.g. a factor of 1.1 corresponds to an increase of 10% in the original parameter value).", "Arabidopsis thaliana ecotype Col-0 plants were grown in a controlled environment under fluctuating light conditions provided by a Heliospectra LED light source (Heliospectra AB, Göteborg, Sweden).", "The fluctuating light regime was reconstructed from natural variations in light intensity recorded during a day in July 2013 at the University of Essex, UK (Fig. 2a) assuming a constant spectral distribution.", "The growth environment was maintained at a relative humidity of 55–65%, a temperature of 21–22 °C and a CO2 concentration of 400 μmol mol−1.", "Plants were well watered, and positioning under the Heliospectra light was altered daily.", "Measurements were taken on the youngest fully expanded leaf.", "Using the set of parameter values previously estimated, the model was used as a simulator to assess the diversity of the temporal response of gs after a step change of light.", "The step change was performed by initially setting the light to an intensity of 100 μmol m−2 s−1 for 30 min followed by a step increase in intensity to 1000 μmol m−2 s−1 for 2 h. All other environmental conditions were kept constant, with the [CO2] maintained at 400 μmol mol−1 and a leaf temperature of 25 °C.", "Parameter values of SD, ki and αL were increased or decreased by a factor of 2 to illustrate their impact on temporal responses of gs, A and Wi.", "The rate of change of gs was estimated using a linear regression on the first 10 min of the variation.", "The impact of the temporal response of gs on A and Wi was characterised by calculating the percentage of variation of the mean A and Wi during the 2 h after the step change of light.", "Photosynthesis gas exchange parameters (A and gs) were measured using a Li-Cor 6400XT portable gas exchange system (Li-Cor, Lincoln, Nebraska, USA), with dew point and vapour pressure deficit maintained via a Li-Cor 610 portable dew point generator.", "Throughout the measurement cuvette, conditions were maintained at a CO2 concentration of 400 ppm (corresponding to ambient growth), leaf temperature of 25 °C, and a leaf to air water vapour pressure deficit of 1 (±0.2) kPa.", "The largest fully expanded mature leaf was used, with A and gs allowed to stabilize under the controlled cuvette conditions for a minimum of 30 min.", "At this point, the automatic 12 h light program (mirroring that of the growth conditions, Fig. 2a) was started, with A and gs recorded every 2 min.", "Wi was calculated as A/gs.", "For the model validation, the same measurement conditions were used in the cuvette with the exception of the light that reproduced a different pattern of light intensity (Fig. 2b).", "Following the dental impression methods of Weyers & Johansen (1985), negative impressions of the ab- and ad-axial leaf surface were made using Xantoprene Polysiloxane precision material (Heraesus Kulzer Ltd).", "Positive impressions were produced by coating nail varnish on the dry polymer and used to count the number of stomata in 9 field of view using an Olympus BX60 light microscope (Olympus Europa, Southend-On-Sea, UK).", "The area of view was measured using an eyepiece graticule to express the number of stomata by mm−2.", "Stomatal impressions of the leaf surface were taken on the same leaves as gas exchange measurements were conducted.", "The two chains (describing the posterior distributions) resulting from the Bayesian inference successfully converged and were used to calculate the credible intervals of each parameter shown in Table 1.", "The credible interval of amin was significantly higher and not overlapping the prior distribution, which was not the case for amax, suggesting that the observed data were informative for amin but not for amax.", "The model estimated αL precisely, despite the fact that the prior distribution covered a large range of possible values, suggesting that the model was particularly sensitive to this parameter.", "The time constants, ki and kd, displayed a strong asymmetry with values almost 3 times higher for ki compared to kd.", "Vcmax and Jmax values were sensibly higher than the prior means estimated from the literature, however the prior distribution and the credible interval overlapped, suggesting that these values were still in the range observed in the literature.", "The medians of the posterior estimates shown in Table 1 were used to represent the most representative prediction of the model (Fig. 3a–f).", "Under a diurnal fluctuating light regime, our dynamic model accurately described gs (Fig. 3b, RMSE: 0.014 mol m−2 s−1; Suppl. Fig. S1a in the online version at DOI: 10.1016/j.plantsci.2016.06.016, R2:0.984) and A (Fig. 3c, RMSE: 0.6 μmol m−2 s−1; Suppl. Fig. S1b in the online version at DOI: 10.1016/j.plantsci.2016.06.016, R2:0.993).", "Consequently, the rapid variations of Wi were also accurately reproduced (Fig. 3e, RMSE: 2.58 μmol mol−1; Suppl. Fig. S1c in the online version at DOI: 10.1016/j.plantsci.2016.06.016, R2:0.97).", "By adjusting the model to the observed gs, our ‘big stoma’ approach allowed us to predict the average behaviour of the stomata with a variation of the pore area from 4 to 14 μm2 (Fig. 3a).", "Under these environmental conditions A and gs displayed a decrease in efficiency of approximately 8% at the end of the diurnal period (Fig. 3d), which was described by Se and Sa.", "The model seemed to be less sensitive to Se as the credible interval was much larger than the one of Sa.", "The model successfully describes the variation of Ci but also predicts the variations of Cc that showed a larger variation compared to Ci due to low gm (Fig. 3f).", "The medians of the posterior estimates shown in Table 1 were used to represent the most representative prediction of the model (Fig. 3a–f).", "Under a diurnal fluctuating light regime, our dynamic model accurately described gs (Fig. 3b, RMSE: 0.014 mol m−2 s−1; Suppl. Fig. S1a, R2:0.984) and A (Fig. 3c, RMSE: 0.6 μmol m−2 s−1; Suppl. Fig. S1b, R2:0.993).", "Consequently, the rapid variations of Wi were also accurately reproduced (Fig. 3e, RMSE: 2.58 μmol mol−1; Suppl. Fig. S1c, R2:0.97).", "By adjusting the model to the observed gs, our ‘big stoma’ approach allowed us to predict the average behaviour of the stomata with a variation of the pore area from 4 to 14 μm2 (Fig. 3a).", "Under these environmental conditions A and gs displayed a decrease in efficiency of approximately 8% at the end of the diurnal period (Fig. 3d), which was described by Se and Sa.", "The model seemed to be less sensitive to Se as the credible interval was much larger than the one of Sa.", "The model successfully describes the variation of Ci but also predicts the variations of Cc that showed a larger variation compared to Ci due to low gm (Fig. 3f).", "Modelled (Mod.)", "vs observed (Obs.)", "stomatal conductance to water vapour (gs, mol m−2 s−1), net CO2 assimilation (A, μmol m−2 s−1) and intrinsic water use efficiency (Wi, μmol(CO2)/mol(H2O)) measured under: a–c light regime described in Fig. 2a for the model parameterization, d–f light regime describe in Fig. 2b for the model validation.", "Solid lines and dashed lines represented the 1:1 lines and linear regressions respectively.", "The coefficient of determination (R2) was derived from the linear regression.", "To validate the model, the same plant and set of parameter values (Table 1) were used to predict A, gs and Wi under a different light regime (Fig. 2b).", "Our dynamic model was able to accurately predict the observed gas exchanges (Fig. 3g-l): gs (Fig. 3h, RMSE: 0.023 mol m−2 s−1; Suppl. Fig. S1d in the online version at DOI: 10.1016/j.plantsci.2016.06.016, R2:0.983), A (Fig. 3i, RMSE: 0.69 μmol m−2 s−1; Suppl. Fig. S1e in the online version at DOI: 10.1016/j.plantsci.2016.06.016, R2:0.988) and Wi (Fig. 3k, RMSE: 2.95 μmol mol−1; Suppl. Fig. S1f in the online version at DOI: 10.1016/j.plantsci.2016.06.016, R2:0.961).", "Compared to the rapidity of the stomatal response (ki: 24 min and kd: 9 min), the light variations were too fast (on average a change every 8 min) to observe the complete stomatal response and, as a consequence, a followed the general tendency of the light intensity (Fig. 3g).", "The efficiency of A and gs during the diurnal period showed a decrease of around 10% similar to that of the estimate during the other diurnal period (Fig. 3j).", "To validate the model, the same plant and set of parameter values (Table 1) were used to predict A, gs and Wi under a different light regime (Fig. 2b).", "Our dynamic model was able to accurately predict the observed gas exchanges (Fig. 3g-l): gs (Fig. 3h, RMSE: 0.023 mol m−2 s−1; Suppl. Fig. S1d, R2:0.983), A (Fig. 3i, RMSE: 0.69 μmol m−2 s−1; Suppl. Fig. S1e, R2:0.988) and Wi (Fig. 3k, RMSE: 2.95 μmol mol−1; Suppl. Fig. S1f, R2:0.961).", "Compared to the rapidity of the stomatal response (ki: 24 min and kd: 9 min), the light variations were too fast (on average a change every 8 min) to observe the complete stomatal response and, as a consequence, a followed the general tendency of the light intensity (Fig. 3g).", "The efficiency of A and gs during the diurnal period showed a decrease of around 10% similar to that of the estimate during the other diurnal period (Fig. 3j).", "A sensitivity analysis of the model was undertaken to illustrate the impact of varying individual parameters on A and gs (Fig. 4).", "The parameters that displayed the most sensitivity for gs were (ordered by importance): SD, amin, αL, ki, and Sa.", "For A, the parameters were (ordered by importance): α, Jmax, Vcmax, gm, SD, amin, αL and Sa, showing the interdependence of A and gs.", "Even if the model was sensitive to the variation of a parameter, this variation would not necessarily improve Wi.", "For example, Vcmax only displayed variation in A that resulted in a decrease in Wi suggesting that under the conditions used here A is not limited by CO2 diffusion.", "The greatest impact on both A and gs was observed when SD was decreased, while increasing photosynthetic capacity (Vcmax and Jmax) by 50% only improved A by 10% under the environmental conditions used here.", "In order to determine the impact of stomatal characters on the temporal response of gs, A and Wi, the influence of the rapidity of the stomatal response (ki) and the steady states gs (SD, αL) to a step increase in light intensity were examined (Fig. 5).", "The initial slope of the gs response displayed large differences (e.g. 0.58–0.29 mol m−2 s−2, Fig. 5b) even if the rapidity of increasing stomatal aperture did not change (Fig. 5a).", "The different temporal responses of gs driven by SD, ki and αL limited A by 8.8% (Fig. 5c), 2% (Fig. 5g) and 6.1% (Fig. 5k) respectively.", "The impact on Wi was due to a larger decrease in gs rather than A following the step increase in light intensity, with an increase of Wi by 82% (Fig. 5d), 7.4% (Fig. 5h) and 19.2% (Fig. 5l) respectively.", "Over different diurnal light regimes, our new dynamic model was able to incorporate the rapid fluctuations in light and accurately predicted gs, A and Wi.", "To our knowledge, this is the first attempt to model stomatal behaviour and scale it up to leaf level to predict gas exchange under dynamic light regimes.", "This ‘big stoma’ approach to model gs provides a direct estimation of the rapidity of the stomatal response, whereas most current models consider only an instantaneous response [3].", "Moreover, the simulated average pore area (a) predicted by the model was in a similar range to those previously reported in the literature [24,25].", "Previous studies have attempted to directly model temporal responses of gs [4,5], but our approach models the speed of increasing pore area rather than the speed of gs increase, and is therefore more mechanistic and biologically relevant when describing the dynamics of gas exchange.", "Although we have validated the model using responses to fluctuations in light intensity, other environmental variables (e.g relative humidity) could be included in the future and would greatly improve the predictive power of the model.", "Our model also incorporates negative feedback on A and gs that revealed a 10% decrease in both by the end of the diurnal period.", "Noe and Giersch [10] suggested that the accumulation of sugars from photosynthesis could negatively regulate A.", "The decrease in gs and therefore subsequent decrease in guard cell turgor, could be related to the decrease of A [26–28].", "Such mechanisms for negative feedback are not well documented but are important for breeders, as any increase in photosynthetic capacity could lead to a great decrease in efficiency over the diurnal period.", "By accurately predicting diurnal gas exchange and dissecting Wi into the key parameters controlling dynamic responses, we believe our model will provide an important tool for future breeding strategies to identify targets for improved Wi under different environmental conditions.", "Previous studies have shown spatial heterogeneity in stomatal distribution and behaviours [29–32] that have not been included in the model.", "Instead, the model predicted the behaviour of a stoma that is representative of the sum of individual stomatal responses over the entire leaf surface.", "This simplification did not appear to impact on the quality of the predictions and it is difficult to assess the temporal and spatial heterogeneity in stomatal response; therefore, further investigations would be required to determine if and how such variations would impact model outputs.", "Stomatal density (SD) was shown to be the most sensitive stomatal parameter for improving Wi, with decreases in SD resulting in a substantial reduction in gs but minimal impact on A.", "This is in agreement with the work of Franks et al. [33], who showed significant variation of Wi in Arabidopsis mutants with different SD.", "The Bayesian inference and the sensitivity analysis revealed that the model was not sensitive to changes in amax, as this value was not reached at any point over the diurnal period.", "In contrast, amin and αL were important determinants of Wi but are rarely reported or examined in this context in the literature.", "The model predicts that, under dark conditions, amin is statistically greater than zero resulting in significant nocturnal transpiration, which has been reported previously [34] and has implications for overall plant water use.", "As SD cannot change over the diurnal period, αL could be an important determinant of plant water use by adjusting the magnitudes of change in gs as a function of the environment.", "Although the coefficient of light absorption by PSII (α) was an important determinant of A and Wi in the field, this parameter does not show a large diversity; therefore, Vcmax, Jmax and gm are the most promising targets for improving A.", "The model predicts that increasing gm could lead to similar improvements in A and Wi [35] as those observed when Vcmax and Jmax were increased through manipulating enzymes associated with the Calvin cycle (e.g. [36]).", "The sensitivity analysis performed here should be seen as a tool to determine the key parameter(s) for improving Wi under specific environmental conditions (e.g. fluctuating light intensity).", "Indeed, the parameter values estimated with the Bayesian inference are specific for this study and the variation around the determined parameter may change between individuals and/or species.", "For example, an individual with different photosynthetic capacity (Vcmax, Jmax) or SD will exhibit different thresholds of A limitations (Fig. 5c) and the results of the sensitivity analysis may also vary.", "To generalize the sensitivity analysis, ranges of biologically possible parameter values and the interactions between them (e.g. relationship between size of the pore and SD) should be used; however, to our knowledge, this information is not yet fully available in the literature.", "Our model simulations clearly showed that the temporal response of gs is the product of several parameters (SD, αL and ki/kd), and the rapidity of the stomatal response cannot be interpreted by comparing the initial slope of variation in gs, as is often reported in the literature [37,38].", "Changes in the initial slope of the gs response are correlated with SD, αL and ki/kd and therefore are not only indicative of the speed of changes in stomatal aperture.", "In terms of gas diffusion, stomatal size constrains the maximum pore area but the observed pore area also depends on the sensitivity of the guard cell (αL) responses to light intensity.", "As a result, it is theoretically possible to have large stomata that even at high light retain a small pore area.", "Considering the relationship between stomatal size and density [39], our results suggest that having a high density of small stomata will result in fast changes in gs but also in a high average gs value through the diurnal period, decreasing Wi.", "The model simulations also revealed large variations in Wi (>80%) when the parameter values controlling the temporal response of gs were altered.", "Indeed, the decrease in A due to greater limitation of CO2 diffusion was minimal compared to the improvement in Wi.", "The importance of the temporal response of gs was highlighted previously [40,41], but not the importance of the additive effects of parameter interactions on A and Wi as revealed by our model.", "Therefore, our model could be used to find the optimal parameter set that would decrease gs without impacting A and maximizing Wi to direct future breeding and research programmes to identify plants with reduced water usage, but the same or greater productivity.", "We assessed and validated a new dynamic model of leaf gas exchange that accurately predicts gs, A, and Wi under fluctuating light regimes such as those experienced by plants in the field.", "The model uses a unique framework that takes into account leaf anatomy, biochemistry and the physiological responses of stomata on Wi.", "The model revealed important negative feedback controls on A and gs towards the end of the diurnal period that resulted in decreases in A with implications for overall plant productivity.", "Importantly, the model enabled Wi to be dissected into key parameters such as stomatal sensitivity to light (αL) and minimal pore area under dark conditions (amin) that have previous been neglected and could provide new and realistic targets for future improvements in crop water use efficiency." ]	[ "A model scaling stoma behaviour at leaf level is proposed.", "The model is tested by reproducing natural variations of light intensity.", "Stomatal aperture and photosynthesis decrease as a function of sugar accumulation.", "Leaf anatomy influences the rapidity of stomatal conductance.", "The model dissects intrinsic water use efficiency into traits of interest." ]
Pulsed laser deposition of crystalline garnet waveguides at a growth rate of 20 μm per hour	We report pulsed laser deposition of high-quality crystalline yttrium aluminium oxide and yttrium gallium oxide with a growth rate approaching 20 μm per hour by using an excimer laser operating at a repetition rate of 100 Hz. This result demonstrates the capability of PLD at 100 Hz for upscaling deposition speeds to a rate that is industrially relevant. In addition, we show that use of this high repetition rate can cause additional heating of the substrate, which in turn affects the film composition. This effect is used as an additional control parameter on the composition, and thus refractive index, of the grown material.	[ "CHEM", "MATE", "PHYS" ]	[ "Integrated optics", "Laser materials", "Optical fabrication", "Planar waveguides", "Pulsed laser deposition", "Thin films" ]	[ "1600", "2505", "2508", "3104", "3110" ]	[ "Pulsed laser deposition (PLD) is a technique that uses laser pulses to ablate a target to form a plume of material which is then transferred to a substrate positioned nearby.", "PLD offers a variety of control parameters that can affect the thickness, quality and composition of the deposited material.", "As an example, the target composition, laser fluence and substrate temperature have been shown to affect the phase, stoichiometry and degree of crystallinity in a grown film.", "PLD has been used to deposit and grow a variety of materials, such as metal oxides [1], ferromagnetic materials [2], glasses [3] and, as discussed further in this work, crystalline optical materials [4,5].", "Crystalline optical materials grown via PLD, have been shown to have very low propagation losses down to values of ~ 0.1 dB/cm [6], though one limitation on growing optical films with desired properties is the ability to obtain the correct stoichiometry.", "However, as demonstrated previously, by pre-compensating a target to counteract specific elemental losses during transfer or evaporation during growth on the substrate, stoichiometric deposition can be achieved [7].", "Optical films made of garnet materials such as yttrium aluminium oxide (Y3Al5O12/YAG) are useful as solid-state laser hosts owing to their properties of high thermal conductivity and being optically isotropic [8].", "In particular, erbium-doped yttrium gallium oxide (Y3Ga5O12/YGG) is of interest owing to its spectroscopic properties, which are desirable in applications for both CO2 and methane gas detection in LIDAR (LIght Detection And Ranging) systems [9–11].", "One hurdle for PLD being used as an industrial and commercially viable process for films of thickness > 10 μm, is the rate of deposition.", "In this work, we present the hetero-epitaxial growth of single-crystal garnets on 〈100〉-oriented YAG substrates via pulsed laser deposition using laser repetition rates from 20 Hz up to 100 Hz.", "In this paper, we show that high-quality single-crystal garnets can be grown by PLD at a deposition rate of 20 μm per hour, and that, the increase in number of pulses when operating at 100 Hz contributes to heating of the substrate, which in turn affects the composition of the material in the grown film.", "Using a PLD setup discussed in previous work [12], laser pulses of ~ 20 ns duration from a Coherent COMPexPro 110 excimer, operating at 20–100 Hz, were focused into a stainless-steel vacuum chamber, which was back filled with oxygen, onto a target material at a pulse energy of 100 mJ.", "The focused laser pulses resulted in a fluence of 1.1 J cm− 2 on the target, which was rotated by an offset cam using a DC motor, in order to increase utilization of the surface area.", "The targets were 5-mm thick, 50-mm diameter, hot-pressed ceramic discs consisting of YAG, Er(1%):YGG or YGG.", "We also added an additional 2.5% Al2O3 to the YAG target, 9.4% Ga2O3 to the Er(1%):YGG target, and 12.7% Ga2O3 to the YGG targets, in order to compensate for loss of Al and Ga respectively that routinely occurs during deposition.", "The ablation plume produced was incident on surface-polished 〈100〉-orientated YAG substrates, of dimensions 10 mm × 10 mm × 1 mm, placed 6 cm away from the target.", "To enable crystalline growth of the deposited material, the substrates were heated using the output from a CO2 laser (10.6 μm wavelength (IR), with a maximum output of 38 W), which was incident on the rear surface of the substrate [13].", "The CO2 laser beam had a ‘top-hat’ spatial intensity profile, produced using a ZnSe tetraprism.", "This method has been routinely used to minimize both undesirable heat sinking and the total power required to heat the substrates, thereby avoiding undesirable vacuum chamber heating [14].", "The substrates used here were heated by a CO2 laser power of 17.5 W, corresponding to a substrate temperature of ~ 600 °C, before deposition.", "All the garnet films, grown under the same conditions, though at different repetition rates of 20 Hz and 100 Hz for YAG and Er(1%):YGG, and 20, 40, 60, 80 and 100 Hz for YGG, were compared.", "The number of pulses for each deposition was kept constant at 36000, which corresponded to ~ 2 μm-thick films, sufficient for reliable X-ray diffraction (XRD) analysis, which was carried out using a Rigaku Smartlab X-ray diffractometer and Bruker D2 Phaser.", "X-ray diffraction spectra of the YAG and Er(1%):YGG films grown at 20 Hz and 100 Hz are shown in Fig. 1.", "The signal corresponding to the (400) peak from the YAG substrate at ~ 29.76°, allows us to correctly determine the location of the (400) YGG and (400) YAG 2θ peaks from the grown samples, by making sure the (400) YAG 2θ peak from the substrate lines up to its database value of 29.76° [15].", "Since the erbium dopant concentration is only 1% of the yttrium atoms in Er(1%):YGG, we use the (400) YGG peak as a point of reference for simplicity.", "The FWHM values of the peaks are similar when grown at 20 Hz compared with 100 Hz for both YAG (0.080° and 0.065°, respectively) and Er(1%):YGG (0.036° and 0.031°, respectively) films.", "For both materials, the 100-Hz XRD peak has a smaller 2θ value than the 20-Hz peak.", "This is attributed to the reduced Al and Ga content of the respective films, as already noted in [16], which occurs as a result of evaporation from the hot substrate.", "However, we do not think that the trend observed here for a reduced FWHM value for films grown at 100 Hz is systematic, and at this stage do not have any reason to suspect improved crystal quality for such a higher repetition rate growth.", "Measurements using a Zescope optical profiler to determine the number of particles of height > 20 nm over a 100-μm × 100-μm area were taken along with roughness measurements to analyze surface quality, while the surface profiler enables the thickness of the materials to be determined.", "As an example of the surface morphology measurements taken of the films, Fig. 2 displays a 1-dimensional slice of the 2-dimensional optical profile data taken of the surface of YAG films grown at 20 Hz and 100 Hz, showing the greater surface roughness of the film grown at 20 Hz compared with 100 Hz.", "Table 1 displays a comparison of the particulate count, roughness and thickness of YAG and Er(1%):YGG films grown at 20 Hz and 100 Hz.", "As seen from the table, for both targets, the number of particulates is fewer and the roughness is lower for films grown at 100 Hz, compared with the films grown at 20 Hz.", "The thickness of the samples is ~ 2 μm for all samples, indicating that (for the same total number of pulses) thickness is repetition rate independent.", "It can also be seen that growth rates of ~ 20 μm per hour are achievable at 100 Hz.", "To confirm the trend in higher deposition rate leading to a systematic decrease in 2θ, depositions were carried out at 20, 40, 60, 80 and 100 Hz.", "In this instance, we used an undoped YGG target, but with all other experimental conditions kept the same.", "XRD measurements were taken of 5 grown films, and the (400) YGG 2θ peak position value is displayed in Fig. 3 as a function of repetition rate.", "The black dashed-line at 2θ = 29.06° corresponds to the 2θ database value of (400) YGG [15].", "As seen from the plot, as the repetition rate increases, the XRD 2θ peak position decreases, with a total difference of ~ 0.04° in 2θ from the 20 Hz to 100 Hz peak.", "From previous work [16], the shift in 2θ peak position has been observed to occur due to an increase in the CO2 laser-heated substrate temperature, which can cause gallium to be lost in the grown film through evaporation.", "By direct comparison, this difference in 2θ of ~ 0.04° can easily be equated to an equivalent substrate-heating CO2 laser power of ~ 2.75 W.", "This proposed increase in heating due to increased repetition rate can be corroborated by examining the additional power burden transferred to the substrate as a result of the increased number of incident plumes per second when depositing at 100 Hz compared with 20 Hz.", "Since the plume takes some hundreds of μs to travel from the target to the substrate [17], which is shorter than the 10-ms time separation between each pulse at the maximum repetition rate of 100 Hz, subsequent pulses do not affect the plume dynamics of the previous pulse and each pulse can be considered as carrying equal kinetic energy to that at 20 Hz.", "Since the excimer laser is running in constant energy mode, i.e. the energy in each pulse remains the same, the kinetic energy in each plume is the same.", "Thus the systematic change observed in the XRD peak position value, and therefore lattice spacing, must be due to an increasing number of pulses per second affecting the stoichiometry of the crystal.", "An extra 80 pulses incident on the target (at a pulse energy of 100 mJ), and thus an extra 80 plumes incident on the substrate per second, equates to a maximum of 8 W potentially transferred to the substrate.", "Under the simplistic assumption that each particle within the plume has the same kinetic energy and that, the energy within the plume is converted to heat on deposition, the relative heating power delivered by the plume to the substrate can be estimated.", "Considering the ratio of the mass deposited on the 1-cm × 1-cm substrate compared with the total mass carried in the full extent of the plume, allows us to determine the additional level of heating that is generated by plume incidence on the substrate alone.", "By depositing YGG over an unheated 75-mm × 50-mm glass substrate, the plume profile incident at the substrate plane was determined.", "A surface profile measurement of the deposited film, determined by measuring the interference fringes at a wavelength of 530 nm, is displayed in Fig. 4.", "Calculating the volume in the area of the sample (black dashed-line in a 1-cm × 1-cm square) versus the equivalent outside the sample region, the fraction of plume volume incident on the substrate is ~ 0.23.", "Since an additional 80 pulses per second is equivalent to a maximum additional 8 W incident on the target, if we assume all the incident laser energy is transferred into the plume, the 0.23 ratio gives an estimated additional heating of ~ 1.8 W at 100 Hz, compared with 20 Hz deposition.", "This estimate along with the estimation from the shift in the XRD spectra as a function of substrate-heating CO2 laser power, does indicate that the substrate is being heated by at an estimated additional ~ 1.8 W.", "This ability to controllably change the temperature of the substrate, and thus the exact composition of the material, potentially allows control of the refractive index of the material via the repetition rate, which is an interesting new concept in our view, since a change in composition clearly leads to a corresponding change in the refractive index of the grown material.", "Although we examined a range of repetition rates from 20 to 100 Hz, it is possible for the laser to run at any integer value between 1 Hz and 100 Hz, thus allowing an even broader range and smaller incremental values of refractive indices to be contemplated.", "The repetition rate alone could thus be used to develop bespoke waveguides with tailored refractive index profile fabricated in a single PLD growth run.", "We have documented the growth of garnet thin films on 〈100〉-orientated YAG substrates using PLD at an ablation repetition rate of 100 Hz, producing crystalline quality as good as, or even superior, to that obtained at 20 Hz.", "Analysis of the thin film under the appropriate deposition conditions, shows that it is possible to grow these films at a rate of 20 μm per hour.", "Our results demonstrate the possibility of either growing tens of films per day, or even > 100-μm-thick single-crystal films in a single growth run of ~ 5 h.", "In addition, the increase in heating as a result of increase in the number of plumes incident on the sample per second at 100 Hz can cause an increase in lattice size as a result of evaporation of volatile elements from the film.", "There is therefore a direct correlation between the laser repetition rate used in PLD and the resultant refractive index via plume-induced substrate heating, which is a novel approach for sophisticated in-situ control of the material properties of PLD-grown films." ]	[ "High-quality single-crystal 20 μm per hour growth rates.", "PLD at 100 Hz produced fewer particulates on the film's surface than at 20 Hz.", "Systematic changes in repetition rate lead to systematic changes in film properties." ]
Biochemical and immunochemical characterization of venoms from snakes of the genus Agkistrodon	In the present work, venoms from five species of the genus Agkistrodon were evaluated in terms of their enzymatic (Phospholipase A2 and caseinolytic) and biological (edema forming, hemorrhagic, procoagulant and lethal) effects. Horses were used to produce monovalent hyperimmune sera against each of three venoms (A. bilineatus, A. contortrix and A. piscivorus) and their neutralizing potency, expressed as Median Effective Dose (ED50), was determined against the venoms of all five species. In terms of PLA2 and caseinolytic activities, all venoms are extremely homogeneous. PLA2 activity is high, while caseinolytic activity is low when in contrast with that of the rattlesnake Crotalus simus. On the other hand, biological activities showed marked interspecific differences, particularly between the species from Mexico and those from the United States. Mexican species displayed higher edema-forming, hemorrhagic and lethal effects than US species, while none of the species studied presented procoagulant activity. All three monovalent hyperimmune sera showed good neutralizing potency against the analyzed venoms. Nonetheless, we observed relevant immunochemical differences among the venoms using ELISA and Western Blot assays. We conclude that the venoms of A. piscivorus (USA) and A. bilineatus would be ideal to use as immunogens for the production of a polyvalent antivenom with good neutralizing potency against the venoms of all the species of the genus.	[ "PHAR" ]	[ "Agkistrodon venoms", "Antivenom neutralization", "Edema", "Hemorrhage", "Immunochemistry" ]	[ "3005" ]	[ "Subfamily Crotalinae is a group of snakes, within the family Viperidae, containing about 242 species grouped in 21 genera (Uetz et al., 2019).", "Snakes of this subfamily, also known as pit vipers, include some Asian genera as well as all the American Vipers; among the latter is the genus Agkistrodon.", "Campbell and Lamar, in 2004, reported 4 species in the genus: A. bilineatus (3 subspecies), A. contortrix (5 subspecies), A. piscivorus (3 subspecies) and A. taylori.", "Later, in 2013, Porras and collaborators elevated the subspecies of Agkistrodon bilineatus to species level: A. bilineatus, A. russeolus and A. howardgloydi, leaving the genus with six species distributed in North and Central America (Fig. 1) (Campbell and Lamar, 2004; Porras et al., 2013).", "The equine hyperimmunization protocols described in the present study were performed in 2014, and therefore this classification is used throughout the work.", "Nonetheless, it is important to note that in 2015, some of the subspecies within A. piscivorus and A. contortrix were elevated to species, leaving four species for the North American Agkistrodon: A. contortrix, A. laticinctus, A. piscivorus and A. conanti.", "For details on the new taxonomy see (Burbrink and Guiher, 2015).", "In the United States, there are approximately 45,000 snakebites in humans every year.", "Among these, about 8,000 result in envenomations.", "Almost 2,000 are caused by snakes of the genus Agkistrodon (Dart and Gomez, 1996), making it one of the most medically relevant in the country.", "Accurate records regarding snakebite accidents in Mexico are very scarce, but it has been reported that about 4,000 envenomations occur per year, with the genus Agkistrodon also being one of the most medically significant (Chippaux, 2017).", "The clinical syndrome caused in the USA by the copperhead (A. contortrix) is characterized by local symptoms, mainly pain, edema and ecchymosis.", "Permanent loss of function, necrosis and systemic symptoms are unusual (Scharman and Noffsinger, 2001).", "These envenomations are thus considered of low risk and antivenom is not always indicated (Mazer-Amirshahi et al., 2014; Walker and Morrison, 2011).", "Other species of the genus have been reported to cause much more severe envenomations, however.", "Venom of the Mexican cantil (A. bilineatus), for example, in addition to local edema and pain, can cause severe hemorrhages in experimental envenomations (Ownby et al., 1990).", "In experimental envenomations, the main activities described for A. bilineatus are the generation of hemorrhage and edema and various hemorrhagic toxins have been isolated from Agkistrodon venoms (Imai et al., 1989; Ownby et al., 1990).", "The edema-forming activity of the venoms has been attributed to protein families including phospholipases A2 (PLA2s), snake venom metalloproteases (SVMPs) and snake venom serine proteases (SVSPs) (de Freitas Oliveira et al., 2009; Hati et al., 1999; Serrano, 2013).", "Like most viper venoms, Agkistrodon venoms are composed mainly of proteins and peptides while non-proteic components are in lower proportion and include citrate, as well as various ions.", "Lomonte and collaborators (Lomonte et al., 2014), performed a proteomic analysis of the venoms from four species of the genus Agkistrodon and some of their subspecies: A. contortrix (five subspecies), A. piscivorus (three subspecies), A. bilineatus (two subspecies) and A. taylori.", "In that work, they reported that all the venoms have a high proportion of PLA2s (31.5–46.0%) and SVMPs (21.0–33.1%), followed by a lower but still important percentage of SVSPs (8.9–22.5%).", "Together, these three families account for over 60% of the venoms’ components, the rest is composed of other enzymes like L-amino acid oxidases (LAAOs) and non-enzymatic proteins like desintegrins, Cysteine-rich secretory proteins (CRISP) and C-type lectins (CTLs).", "The proportion of protein families present in the venoms was observed to be very similar, which suggests that they are very homogeneous in terms of composition.", "Still, the great diversity present within each protein family, complicates the prediction of biological activities or clinical syndromes, even when the proportion of protein families in a venom is known (Castro et al., 2013).", "Also, the abovementioned differences in severity of the clinical syndromes developed by species of this genus suggest differences in toxicity of individual proteins that are relevant during envenomation and possibly also for antivenom neutralization.", "The aim of this work was to characterize the biochemical and biological activities of the venoms of five species of the genus Agkistrodon, as well as their immunochemical characteristics.", "This knowledge can be of importance in the development of antivenoms and in clinical management of envenomation.", "All animal experiments were performed in compliance with the EU Directive 2010/63/EU for animal experiments (European Parliament, 2010), under the procedures and with the approval of the Institutional Bioethics Committee of the Biotechnology Institute of the National Autonomous University of Mexico (IBt-UNAM) project number 254: “Functional characterization of Agkistrodon venoms and equine immune response against them” (Caracterización funcional del veneno de Agkistrodon, así como la respuesta inmune en caballos contra los mismos).", "ICR mice used in all experiments were obtained from the laboratory animal facility of IBt-UNAM.", "Animal facility staff as well as research staff was trained in the correct and humane handling of mice before the start of any procedure.", "The use of human blood from a single healthy donor was also approved by the Institutional Bioethics Committee of IBt-UNAM, as a part of project 254.", "All residues that had been in contact with the blood were discarded in accordance to Institutional regulations.", "Some individuals from the species A. bilineatus were collected under license number SGPA/DGVS/03459/15 and kept for successive venom extractions at the vivarium “Cantil: Herpetario del IBt” with registration number MOR-IN-166-07-04.", "All housing and handling procedures have been revised and approved by Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT), Mexico in strict accordance with the regulations of the National General Law of Wildlife (Congreso, 2018).", "Horse housing and handling protocols were in strict accordance with Mexican and international animal welfare regulations and were approved by SAGARPA (Secretaría de Desarrollo Agrícola y Desarrollo Rural, Pesca y Alimentación; SENASICA –Dirección General de Salud Animal-) with permit number: DU00411.", "All handling of horses was performed by previously trained staff from ranch Ojo de Agua in Puebla, Mexico and submitted to monthly medical check by an in-house specialized veterinarian.", "Some venoms were from the venom bank of our laboratory at IBt-UNAM while others were obtained through collaborations with the following herpetariums, who kindly lent their snakes for venom extraction: Reptiles Fergo (license number: DGVS-PIMVS-EA-0084-MOR/08), UMA TSÁAB KAAN (license number UMA-IN-0183-YUC-10), Herpetario de la Facultad de Ciencias, UNAM and DeVal Animal (license number DGVS-CR-IN-0957-D.F./07).", "Pools from 2 to 5 individuals were used for each species, except in the case of A. taylori, where only one specimen was available for venom extraction.", "Table 1 and Fig. 1 detail the source of all the venoms analyzed in the present study.", "A pool from 18 adult specimens of Crotalus simus from Veracruz, Mexico, and a venom pool from the scorpion Centruroides limpidus (both from the venom bank at IBt-UNAM) were used for comparison or external controls when needed.", "All venoms were obtained through manual extraction and were then washed from the extraction cup using low volumes of 20 mM ammonium acetate pH 4.7 (maximum proportion of ammonium acetate to venom was 1:10 v/v).", "They were subsequently centrifuged at 16,800 g for 3 min and the supernatant was stored at −70 °C for lyophilization.", "Finally, lyophilized venoms were stored at 4 °C until their use.", "Some of the venoms from A. p. conanti and all A. c. contortrix and A. p. leucostoma were pools purchased from the National Natural Toxin Research Center (NNTRC) in Texas, U.S.", "Protein concentration of the pooled and individual venoms was determined using a Pierce® Bicinchoninic Acid (BCA) Protein Assay (Thermo Scientific), with bovine serum albumin (BSA) as a standard, according to the manufacturer's protocols.", "Twenty-five μg of each venom were loaded on 12.5% SDS-PAGE gels under reducing conditions.", "Samples were diluted using Sample buffer 5X (10% Glycerol, 2.5% SDS, Tris-HCl 50 mM pH 6.8, 5% 2-mercaptoethanol, 0.002% bromophenol blue) to a final volume of 20 μL and boiled for 5 min.", "Electrophoresis was performed with a constant voltage of 80 V for 15 min and then 100 V for approximately 60 min.", "Gels were stained with G-250 Coomassie Brilliant Blue.", "Apparent molecular weights were determined comparing migration distance with 5 μL of molecular weight markers (Precision Plus Protein Dual Xtra Standards, Bio-Rad) using ImageJ software version 1.50i.", "Venom samples (1 mg of dry weight) were dissolved in 1 mL of water containing 0.1% trifluoroacetic acid (TFA), centrifuged to remove debris and fractionated through RP-HPLC on a C18 column (4.6 × 250 mm, 5 μm particle size; Vydac®) using an Agilent 1100 chromatograph.", "Elution was performed at 1 mL/min by applying a gradient to solution B (acetonitrile, containing 0.1% TFA), as follows: 0% B for 5 min, 0–15% B over 15 min, 15–45% B over 60 min, 45–70% B over 12 min, and 70% B for 10 min.", "PLA2 enzymatic activity of pooled venoms was determined using a titrimetric assay with a 10% egg yolk solution (0.1 M NaCl, 0.01 M CaCl2, 0.1% Triton-100 and 10% egg yolk) as substrate (Shiloah et al., 1973).", "The assay was performed on 500 μL of the previously described solution, stabilized in pH 8.05 with 50 mM NaOH.", "The solution was under constant stirring and mild N2 bubbling.", "50 mM NaOH was also used for titration.", "Units of enzymatic activity (U) were defined as μmoles of NaOH consumed per minute and the results were reported in units per milligram of venom (U/mg).", "Proteolytic activity of the venoms was evaluated using a further modification of the method described by (Chen et al., 2004; Yang et al., 2015) and modified by (Gutiérrez et al., 2008).", "Briefly, azocasein was dissolved in a standard solution (50 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl2) to a final concentration of 10 mg/mL.", "Afterwards, 20 μg of venom, dissolved in 20 μL of 150 mM NaCl, were added to 100 μL of the azocasein solution and incubated for 30 min at 37 °C.", "After incubation, the reaction was stopped by adding 200 μL of 5% trichloroacetic acid.", "Then, samples were centrifuged at 16,800 g for 5 min and 150 μL of the supernatant of each sample were added to 150 μL of 500 mM NaOH in a 96 well plate (NUNC).", "Finally, sample absorbance at 450 nm was determined.", "Units of enzymatic activity (U) are defined as the change of 0.2 in the absorbance of the sample per minute.", "In order to verify the linearity of the observed reaction, proteolytic activity was determined through varying incubation times (30, 60 and 90 min).", "The selected incubation time was 30 min, because substrate was still in excess.", "The median lethal dose (LD50) of each venom pool was determined intravenously (i.v.).", "Different venom doses in a total volume of 0.5 mL were inoculated through the tail vein to groups of five ICR mice between 18 and 20 g of body weight (4 groups per venom were used in average).", "The percentage of dead mice was measured 24 h after venom injection with monitoring intervals of approximately 3 h; mice that were evidently moribund were euthanized through cervical dislocation to minimize animal suffering.", "The obtained data were analyzed through a non-linear regression (variable slope, dose-response curve) using the software GraphPad Prism 6.01.", "LD50 was defined as the amount of venom that causes death to 50% of the mice population (Casasola et al., 2009).", "The edema-forming activity of the venom pools was analyzed through the determination of a Minimum Edema-forming Dose (MED), using the method described by (Gutiérrez et al., 1986).", "Throughout these experiments, groups of three ICR mice were subcutaneously (s.c.) injected in the left hind paw with different amounts of venom (4 groups per venom were used in average).", "Concentrations were calculated in order to always inoculate them with a volume of 50 μL of venom, resuspended in PBS.", "The right hind paw of every mouse was injected with 50 μL of PBS to use as individual control.", "After venom inoculation, the diameter of both paws was measured every 10 min for the first hour and every 30 min for the next 2 h using a manual Vernier caliper.", "The increase in limb volume caused by the venom for each measured time was determined using the percentage of diameter increase of the envenomated paw compared to the control paw.", "In order to minimize animal suffering, all mice were euthanized immediately after conclusion of the experiment.", "The procoagulant activity of the venom pools was analyzed through the determination of a Minimum Procoagulant Dose in Plasma (MPD-P), using the method described by (Theakston and Reid, 1983).", "Different amounts of venom were added to glass tubes with 200 μL of citrated (sodium citrate, 3.8 g/dL) human plasma.", "Time was measured between venom addition and evident clot formation while gently moving the glass tube.", "Obtained data was processed with a linear regression, selecting only the initial section of the dose-response curve and verifying linearity (R2 > 0.9).", "The venom dose that generates a clot in 1 min was interpolated using the software GraphPad Prism 6.01.", "The MPD-P was defined as the amount of venom that induces the generation of an evident clot in 60 s.", "In order to minimize variation, blood from the same human donor was used for all the experiments and the time measurements were always taken by the same observer.", "Three adult, male, crossbred horses kept in the farm “Ojo de Agua” in the community Venustiano Carranza (Puebla, Mexico), were inoculated with increasing amounts of venom, using the immunization scheme detailed in Supplementary Table 1.", "Each horse was identified with a number and inoculated with the venom from only one Agkistrodon species as follows: Horse 201 with A. c. contortrix, horse 202 with a pool of the three subspecies of A. piscivorus and horse 203 with A. bilineatus.", "The hyperimmunization pools used are detailed in Table 1.", "Maxisorp (Nunc Inc, USA) plates were coated with 100 μL/well of 5 μg/mL of the venoms, diluted in 100 mM carbonate/bicarbonate buffer, with pH 9.5 and incubated overnight at 4 °C.", "The plates were then washed 3 times with 250 μL/well of washing buffer (50 mM Tris/HCl, 150 mM NaCl, 0.05% Tween 20 and pH 8) in a microplate washer (BIO-RAD Immuno wash 1575).", "The remaining binding sites were blocked with 200 μL/well of blocking buffer (50 mM Tris/HCL, 5 mg/mL gelatin, 0.2% Tween 20 and pH 8) and incubated for 2 h at 37 °C.", "The plates were then washed 3 times as described before.", "Samples of anti-Agkistrodon horse serum were initially mixed with vehicle buffer (50 mM Tris–HCl, 0.5 M NaCl, 1 mg/mL gelatin and 0.05% Tween 20, pH 8.0), at a dilution of 1:300.", "This solution was serially diluted 1:3, with the same buffer on the ELISA plates.", "Plates were incubated for 1 h at 37 °C and, after washing them 3 times, plates were incubated for 1 h at 37 °C with 100 μL/well of peroxidase-conjugated goat anti horse IgG antibody (1:3000 dilution, Gene Tex).", "After washing them 5 times, 100 μL/well ABTS solution (Roche) were added and incubated for 10 min at 25 °C.", "When this timespan concluded, the reaction was stopped with 20 μL/well of 20% sodium dodecyl sulfate (SDS) and absorbances of wells were measured at 405 nm in a Microplate Reader.", "Sigmoidal dose-response curves were generated using non-linear regression with a variable slope with the software GraphPad Prism 4.", "Antibody titer is defined as the serum dilution at which 50% of the colorimetric response is obtained.", "We used two different plates, incubation and assay plates.", "Incubation plates (Maxisorp Nunc Inc, USA) were blocked with 200 μL/well of blocking buffer and incubated for 2 h at 37 °C.", "Samples of Agkistrodon venoms were prepared in vehicle buffer with a concentration of 300 μg/mL or 1000 μg/mL and serially diluted 1:3.", "Also, 100 μL/well of horse serum anti Agkistrodon were added to the plate using a dilution equivalent to the titer (EC50) of each serum for its homologous venom and incubated for 1 h at 37 °C.", "Assay plates were coated with 100 μL/well of 5 μg/mL of venoms from A. bilineatus, A. c. contortrix or a mixture of A. piscivorus subspecies and incubated overnight at 4 °C.", "The remaining binding sites were blocked with 200 μL/well of blocking buffer, incubated for 2 h at 37 °C.", "The mixture from the incubation plates was then added to the assay plates and these were incubated for 1 h at 37 °C.", "Finally, plates were incubated for 1 h at 37 °C with 100 μL/well of peroxidase-conjugated goat anti-horse IgG antibody (1.5 10−3 μg/mL, Gene Tex) and developed using 100 μL/well of ABTS solution (Roche) incubated for 10 min at 25 °C.", "After this timespan concluded, the reaction was stopped with 20 μL/well of 20% SDS.", "Each samples’ absorbance was determined at 405 nm in a Microplate Reader.", "The serum titer for the homologous venom was used to define the serum dilution to add as primary antibody to each ELISA plate.", "The absorbance of the maximum venom dilution of the homologous venom for each plate was defined as 0% competition and that of the highest venom dilution, as the percent of maximum inhibition.", "Data analysis was performed using the software GraphPad Prism 4.0.", "Neutralizing potency of the horse hyperimmune sera was evaluated through the determination of a Median effective Dose (ED50) (Casasola et al., 2009).", "To this end, different serum volumes were incubated for 30 min at 37 °C with 3LD50s of Agkistrodon venom.", "Afterwards, groups of 5 mice between 18 and 20 g of body weight were inoculated i.v. with the samples (4 groups per venom were used in average).", "The survival rate was determined 24 h after venom injection with monitoring intervals of approximately 3 h; mice that were evidently moribund were euthanized through cervical dislocation to minimize animal suffering.", "Obtained data was processed through non-linear regression (variable slope, dose-response curve) using the software GraphPad Prism 6.01.", "The ED50 was defined as the serum volume capable of preventing death in 50% of envenomated mice.", "In order to evaluate differences between treatments, a one way ANOVA and a post hoc Tukey test were performed for PLA2 and caseinolytic activities.", "Results were considered statistically different when P < 0.05.", "Electrophoretic profiles of Agkistrodon venoms are shown in Fig. 2.", "All analyzed venoms showed several conspicuous protein bands of 13.2 kDa–15.3 kDa.", "Venoms from the Mexican species (lanes 2 to 4) showed very similar patterns among them, with two more abundant protein bands of approximately 35.2 and 45.8 kDa and less abundant bands of around 27.1, 45.8 and 58.3 kDa.", "On the other hand, electrophoretic pattern of venoms from A. piscivorus and A. c. contortrix differed from the Mexican venom pattern and were also relatively different from each other.", "These venoms lacked the abundant high molecular weight proteins, or had them in very small proportion, and were instead rich in medium molecular weight protein bands, ranging from 24.9 to 35 kDa (three bands in A. piscivorus and four in A. c. contortrix).", "RP-HPLC profiles also showed the Mexican species to be very similar to each other, with the most abundant components eluting between 60 and 70 min of retention time (RT) and a single, also very abundant peak at approximately 85 min.", "Profiles from A. c. contortrix and the subspecies of A. piscivorus were similar, but not identical, with some marked differences before 50 min RT; both species differed significantly from the profiles of the Mexican species.", "Some of the observed differences were a greater diversity of components around 85 min RT and the presence of one to three abundant components between 50 and 60 min RT (Fig. 3).", "In Table 2 we summarized the results obtained for the biochemical and biological activities of Agkistrodon venoms.", "The venom of C. simus was used for comparison where relevant.", "LD50s of the analyzed venoms ranged from 0.2 to 11.3 μg/g and are shown in Table 2.", "In terms of PLA2 activity, most Agkistrodon venoms showed no statistically significant difference between them (P ≥ 0.05), ranging between 231.9 and 383.2 U/mg; A. taylori was the only one with a significantly higher activity (720.2 U/mg).", "Proteolytic activity on azocasein substrate was also statistically the same for all the Agkistrodon venoms tested (P ≥ 0.05), ranging between 5.2 and 7 U/mg (Table 2).", "In the current work, we observed that edema-forming activity was greatest in the venoms from A. bilineatus, A. taylori and A. russeolus, with MED ranging between 0.1 and 0.2 μg.", "In contrast, the venoms from A. c. contortrix and A. piscivorus showed MEDs of 1.8 and 3.6 μg respectively; between 7.3 and 36.0 times higher than those of the Mexican species (Table 2).", "Also, all venoms tested caused hemorrhage to some extent, yet once again, the venoms from A. bilineatus, A. taylori and A. russeolus had the highest activities with MHDs of 0.8, 0.7 and 0.3 μg respectively.", "The venoms from A. piscivorus and A. c. contortrix had comparatively less activity, with MHDs of 7.4 and 33.8 μg respectively (Table 2).", "To analyze the recognition of the produced hyperimmune sera, we determined ELISA titers using homologous and heterologous Agkistrodon venoms.", "Highest titers were obtained for serum 203 (anti-A.", "bilineatus) against the two other Mexican species (A. russeolus −121,043- and A. taylori −122,525-) while titers against A. c. contortrix and the subspecies of A. piscivorus ranged between 33,522 and 67,872.", "Interestingly, the titer against the homologous venom was relatively low (37,003) (Table 3).", "Serum 201 (anti–A. c. contortrix) had the lowest titers, ranging between 18,467 and 34,457 against the Mexican species and between 23,278 and 29,829 against species from the U.S., including the homologous venom.", "Finally, titers for serum 202 (anti-A.", "piscivorus) were high against the homologous subspecies (83,123 to 110,262) and relatively low against both A. c. contortrix and the Mexican species (15,821 to 49,880) (Table 3).", "Titers for the negative control venom, Centruroides limpidus, were 0 against all sera.", "In the plates with A. bilineatus venom and serum against A. bilineatus, we obtained highest percentages of competition with Mexican species of A. bilineatus, A. russeolus and A. taylori, while less was observed for species from USA, showing that Mexican species are immunochemically similar to each other.", "On the other hand, on the plate with A. piscivorus fixed venom, we obtained the maximum inhibition with homologous A. piscivorus venom and a moderate inhibition with A. c. contortrix venom (70.1%).", "The species of A. bilineatus, A. russeolus and A. taylori were bad competitors.", "Finally, venom from A. c. contortrix was the one that showed lowest competition values with all other species.", "Still, Mexican species share fewer epitopes with it than do the subspecies of A. piscivorus (Fig. 4).", "There was no competition with the venom of the negative control, C. limpidus, in any of the analyzed sera, indicating that there is no non-specific recognition by equine immunoglobulins.", "Monovalent sera, in general, presented a higher neutralization potency against the homologous venom than against the heterologous venoms.", "Serum 201 (anti-A.", "c. contortrix), showed an EC50 of 2.2 mgV/mLS (milligrams of venom per milliliter of serum) against the homologous venom, while this same value ranged between 0.6 and 1.5 mgV/mLS when tested against heterologous venoms.", "Serum 202 (anti-A.", "piscivorus) presented a neutralization potency between 2.6 and 5.7 mgV/mLS for the three subspecies of A. piscivorus and between 0.3 and 2.5 mgV/mLS for other species of the genus.", "On the other hand, serum 203 (anti-A.", "bilineatus) was an exception because it neutralized the venoms from A. russeolus, A. c. contortrix, A. p. leucostoma and A. p. conanti just as well or better than the homologous venom (5.1, 2.0, 2.1 and 2.0 mgV/mLS, respectively) (Table 3).", "General composition in viper venoms is broadly conserved in terms of protein families (Calvete et al., 2009; Lomonte et al., 2014), where the most abundant are usually SVMPs, PLA2s and SVSPs (Tasoulis and Isbister, 2017).", "Differences among the species of a genus such as Agkistrodon are generally given by variation in the proportions of the mentioned protein families and by the presence or absence of individual proteins which can have a strong effect in a particular venom activity (Borja et al., 2018; Castro et al., 2013; Glenn et al., 1994; Saldarriaga et al., 2003).", "Venom variation can be immunochemical, biochemical or in terms of its toxic activities and it can significantly affect neutralization by antivenoms (de Roodt et al., 2011; Gutiérrez et al., 2010; Neri-Castro et al., 2019).", "It has been shown that it can exist between species and within a single species, generated by ontogenetic changes or from one geographic location to another, among other factors (Borja et al., 2018, 2014; 2013; Castro et al., 2013; Durban et al., 2017; Glenn et al., 1994, 1982; Mackessy et al., 2018; Rivas et al., 2017; Wilkinson et al., 2018; Zancolli et al., 2018; Zelanis et al., 2010).", "Only interspecific variation was considered in the present study, while the possibility of the existence of intraspecific variation in Agkistrodon venoms was not evaluated, mainly due to sample availability.", "However, samples from a wide distribution range were available for the case of A. bilineatus (Fig. 1), so biochemical activities as well as SDS-PAGE were performed with 12 individual venoms.", "We observed that there is no evident intraspecific variation across the distribution range of this species (Supplementary Figure 1).", "Also, a pool of 9 neonate individuals from A. bilineatus (born in captivity) was analyzed using the same RP-HPLC method used for the pooled venoms and it proved to be almost identical to the adult pool (Fig. 3).", "Intraspecific variation has been also analyzed using the venom of A. contortrix by (Lagesse and Ford, 1996) and found the venom of this species to have only small variations across its distribution.", "In the SDS-PAGEs performed in the present work, we observed that the Mexican species have similar electrophoretic profiles among them and are different from the species native to the U.S. (Fig. 2).", "This grouping can be also observed in the RP-HPLC profiles (Fig. 3).", "Except for a few small differences, the RP-HPLC profiles were extremely similar to the ones previously obtained by Lomonte and collaborators (Lomonte et al., 2014), during their proteomic characterization of these species (Fig. 3).", "Proteins of the PLA2 families were very abundant in all the venoms studied in the present work.", "Within the PLA2 proteins that have been described in viper venoms there are two distinct groups: those with enzymatic activity, which have an aspartic acid in canonical position 49 (D49), and those with a substitution of this residue to lysine or serine (K49 or S49) and that are devoid of enzymatic activity (Fernández et al., 2013; Kini, 2003; Lomonte et al., 2003).", "Many among the second group are commonly referred to as true myotoxins because they act on cellular membranes of muscle cells causing lysis through direct damage (Lomonte and Rangel, 2012; Ward et al., 2002).", "D49 PLA2s have extremely variable pharmacological activities, often interfering in cellular signaling cascades or causing cell lysis through breakdown of membrane phospholipids (de Freitas Oliveira et al., 2009).", "In the particular case of Agkistrodon venoms, no relationship was observed between the presence of D49 or K49 PLA2s and the edema-forming activity of the complete venoms (Table 4).", "All venoms showed a similar proteolytic activity when using azocasein as substrate.", "This activity is due both by SVMPs and SVSPs (Serrano, 2013).", "SVMPs are found in similarly high proportions in all the species of the genus Agkistrodon as shown in the present work and in (Lomonte et al., 2014).", "Nonetheless, when performing a deeper analysis of the available proteomes, we observe than the Mexican species have a much higher amount of type P-II SVMPs while the ones present in A. c. contortrix and A. piscivorus are mainly of the type P–I (Table 5).", "These differences can also be observed in the electrophoretic profiles (Fig. 2).", "One of the main pharmacological activities that can be caused by SVMPs is hemorrhage (Fox and Serrano, 2005).", "In 1989, Imai et al. (Imai et al., 1989; Nikai et al., 2000) isolated and described a protein they called Bilitoxin-I (PII-SVMP, MW ≈ 48 kDa) from the venom of A. bilineatus.", "Although P-III SVMP have been described generally to be the most potent hemorrhagic SVMPs (Fox and Serrano, 2005), Bilitoxin-I has an extremely potent hemorrhagic activity, MHD of 0.008 μg, and it is likely responsible for the high hemorrhagic activity observed for the Mexican species (Table 2).", "For example, A. taylori, had a MHD of 0.7 μg and this protein and similar isoforms account for about 28% of total venom (Lomonte et al., 2014).", "A. russeolus has been minimally studied but its low MHD (0.3 μg), an abundant RP-HPLC peak with retention time of 83.6 min (consistent with Bilitoxin-I in A. bilineatus and A. taylori), and our observation of severe hemorrhages in envenomated mice, indicate Bilitoxin-I could also be present in high proportions in this venom.", "On the other hand, based on A. c. contortrix or A. piscivorus venom proteomes (Lomonte et al., 2014), these species contain less than 2% of Bilitoxin-I or similar proteins.", "These venoms are much less hemorrhagic, with MHDs of 33.8 and 7.4 μg respectively.", "Hemorrhages observed in these cases may therefore be caused by other, less potent, SVMPs.", "Regarding the minimum procoagulant dose in human plasma (MPD-HP), no clot formation was observed for 10 min when adding 100 μg of any venom to 200 μL of citrated human plasma.", "Procoagulant activity is usually observed in viper venoms that have a high proportion of SVSPs with “thrombin-like” activity as well as other, less common, SVSPs and SVMP that act on different coagulation factors.", "Examples of these include prothrombin and factor X activators within the SVMP family (Ramos and Selistre-de-Araujo, 2006) and activators of factors V, VIII and also factor X and prothrombin within the SVSP family (Serrano, 2013).", "One thrombin-like SVSP, named Bilineobin, has been described in the venom of A. bilineatus (Komori et al., 1993), though it has low procoagulant activity when compared to that of other viper venoms.", "In our experiments, the presence of this protein does not appear to be relevant when the whole venom is tested (Table 2), but the formation of some fibrin fibers was observed.", "Previous reports have also shown a lack of procoagulant activity of various Agkistrodon venoms (Arce et al., 2003; Lomonte et al., 2014).", "Contrasting with this result, procoagulant activity has been previously reported for the venom of A. bilineatus (de Roodt et al., 2005), and a Protein C activator protein has been previously described in the venom of the same species (Nakagaki et al., 1990).", "Some procoagulant enzymes have also been described in the venoms of closely related genus of snakes (Li et al., 2018; Yukelson et al., 1991).", "On the other hand, a fibrinolytic SVMP without hemorrhagic activity has been described in the venom of A. contortrix laticinctus (Selistre De Araujo, 2013).", "One of the most clinically conspicuous signs during Agkistrodon envenomation is edema and inflammation (Scharman and Noffsinger, 2001).", "Edema is the extravasation of fluid to the interstitial space, and even though it is often related to an inflammatory process, this is not always the case (Trigo and Valero, 2004).", "Inflammation is a complex cascade of biochemical and cellular processes initiated and regulated by the immune system.", "The classic manifestations of an inflammatory event are pain, heat, blushing, and local volume increase.", "Inflammation can be classified in acute and chronic, primarily based on duration and the presence of healing at the site of inflammation.", "The first is characterized by being short in duration (generally less than 4 h), usually generated by spontaneous damage, while the second is long in duration and characterized by the initiation of healing (Owen et al., 2014; Trigo and Valero, 2004).", "The venom of several snakes has been reported to start a complex inflammatory process, associated with the liberation or synthesis of mediator molecules that recruit different cells, as well as pain and edema (Gutiérrez, 2002).", "The main protein families that have been described to cause inflammation are SVMPs and PLA2s (de Freitas Oliveira et al., 2009; Gutiérrez and Lomonte, 2013; Mackessy, 2009); both groups possess different mechanisms of activation of the immune response.", "Bothrops asper has one of the most studied venoms in this matter.", "Here, SVMPs favor edema by causing the rupture of blood vessels and therefore the extravasation of liquid to the interstitial space without the previous initiation of an inflammatory process (Rucavado et al., 2002, 1995); inflammation will start later in response to tissue damage.", "On the other hand, some PLA2s from this venom generate damage of muscle tissue (Zuliani et al., 2005), promoting an inflammatory response without an initial edema.", "In the present work, the progression of edema/inflammation was different in both groups of venoms.", "Injection of venom from the Mexican species resulted in a slow increase in limb volume, with a maximum around 50 min, after which it started decreasing and returned to 0% about 3 h after subcutaneous venom injection (Fig. 5).", "This behavior could be caused by an initial rupture of the vascular endothelium by SVMPs generating tissue damage and starting an inflammatory response.", "Also, the venom of A. bilineatus has been described to have bradykinin potentiating factors (BPF) which directly increase vascular permeability (Munawar et al., 2016).", "On the other hand, both species from the U.S. showed a rapid increase in limb diameter, with a maximum at 10 min and a relatively fast decrease, returning to 0% around 2 h after inoculation (Fig. 5).", "This suggests the extravasation of liquid without the initiation of an intense inflammatory process.", "More detailed studies, including histology of envenomated tissue and/or quantification of inflammation mediators, should be done in order to obtain a proper description of this process.", "Another important factor to discuss is the individual variation in humoral immune response of horses used for hyperimmunization.", "It has been previously observed that horses submitted to the same immunization protocol and adjuvants can have different responses in terms of antibody titers as well as neutralization potencies against homologous venoms (Angulo et al., 1997; Calderón Corona, 2011; Estrada et al., 1989).", "In this work, only one horse per venom was used for the production of hyperimmune sera and therefore we have no way of verifying that the differences observed are due to the venom's properties as antigens and not to individual variation between horses.", "Further studies must be done in order to address this issue.", "The venoms from Mexican species of this genus were more lethal to mice than those of both species from the U.S. (Table 2).", "Similar lethal potencies, of 4.6 and 9.1 μg/g, have been previously determined for A. piscivorus and A. c. contortrix, respectively (Arce et al., 2003).", "The high lethality of the Mexican species could also be explained by the presence of the aforementioned Bilitoxin-I, whose reported LD50 is 0.5 μg/g (Kuniko et al., 1989).", "In order to further analyze this, we fractionated the venom from A. bilineatus through gel filtration using a Sephadex G75 column (Supplementary Figure 2) and studied the neutralization of fraction A (mainly SVMPs) by sera 203 and 201.", "We observed that serum 203 (anti-A.", "bilineatus) was much better at neutralizing this fraction, which has an LD50 of 0.96 μg/g (ED50 = 15.6 mgV/mLS), than serum 201 (anti-A.", "c. contortrix) (ED50 < 0.9 mgV/mLS).", "Given that this fraction is mainly composed of Bilitoxin-I (data not shown), the neutralization difference provides strong evidence that the SVMPs in A. c. contortrix are immunochemically different from Bilitoxin-I, a finding that explains both the differences in neutralization potency of the tested sera against whole venoms and, at least in part, the differences in lethality.", "It is important to note that a high ELISA titer does not necessarily reflect better neutralization.", "In fact, the sera with the highest titers were different from the ones with highest neutralization effective doses (Table 3).", "The competitive ELISA experiments have also shown the presence of at least two groups with different immunochemical characteristics in Agkistrodon venoms, with the Mexican species in one and those from the U.S. in another.", "Additionally, the Mexican species appear to be more closely related to each other than A. c. contortrix and A. piscivorus, since these last two species show significantly lower competition percentage between them (Fig. 4).", "Further observations can be made when neutralization potencies are studied in terms of the number of LD50s neutralized per mL of serum.", "When analyzed in that way, we can consider the variations in lethality of each venom.", "Interestingly, here, the venom of A. russeolus was better neutralized with all the analyzed sera.", "Previously, we mentioned that Bilitoxin-I is likely the main protein responsible for lethality of Mexican Agkistrodon venoms.", "Given that A. russeolus has approximately the same proportion of this toxin than A. bilineatus and A. taylori (16.8%, 15.3% and 17.5%, respectively), the higher lethal potency of the first could also be due to other toxins, which are less abundant in the latter.", "A good neutralization of these proteins by all the analyzed sera can be assumed, given their low effective dose against A. russeolus venom.", "However, a lot more studies should be performed to confirm this hypothesis.", "The neutralizing potency of the equine monovalent sera generated in this work was equal to or higher than 1 mg of venom per milliliter of serum (1 mgV/mLS) for the heterologous venoms and higher than 2 mgV/mLS for the homologous venoms (Table 3).", "Judging from the experience of the authors, all sera have good neutralization potencies for antivenom production.", "Given that serum 203 (anti-A.", "bilineatus) was best at neutralizing the venoms from Mexican species and serum 202 (anti-A.", "piscivorus) was best at neutralizing all the analyzed venoms from the U.S., we consider that genus-wide neutralization can be achieved by the inclusion of venoms from A. bilineatus and A. piscivorus in equine immunization protocols.", "Although proteomic analysis suggests that venoms are very similar in the composition of their protein families, our results demonstrate the presence of two groups in terms of immunochemistry and pharmacological activities.", "These differences have a significant influence on the cross neutralization of the produced sera.", "Therefore, the inclusion of one species from each group is important to generate a genus-wide neutralizing serum, efficient for treatment of envenomation by Agkistrodon species from both Mexico and the U.S." ]	[ "Venoms from Agkistrodon species from the US and Mexico are significantly different in terms of biological activities.", "Edema, hemorrhage and procoagulant, as well as enzymatic activities are determined for five species of the genus.", "Neutralization by three monospecific hyperimmune sera show there is significant cross reactivity between species.", "Venoms from A. bilineatus and A. piscivorus are proposed as immunogens to generate a genus-wide neutralizing serum." ]
Genome-editing technologies for gene and cell therapy	Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.	[ "BIOC", "PHAR" ]	[]	[ "1311", "1312", "1313", "3002", "3004" ]	[ "Foundational to the field of gene editing was the discovery that targeted DNA double strand breaks (DSBs) could be used to stimulate the endogenous cellular repair machinery.", "Breaks in the DNA are typically repaired through one of two major pathways—homology-directed repair (HDR) or nonhomologous end-joining (NHEJ) (Figure 1).4", "HDR relies on strand invasion of the broken end into a homologous sequence and subsequent repair of the break in a template-dependent manner.5", "Seminal work from the lab of Maria Jasin demonstrated that the efficiency of gene targeting through homologous recombination in mammalian cells could be stimulated by several orders of magnitude by introducing a DSB at the target site.6,7,8", "Alternatively, NHEJ functions to repair DSBs without a template through direct religation of the cleaved ends.9", "This repair pathway is error-prone and often results in insertions and/or deletions (indels) at the site of the break.", "Stimulation of NHEJ by site-specific DSBs has been used to disrupt target genes in a wide variety of cell types and organisms by taking advantage of these indels to shift the reading frame of a gene.10,11,12,13,14", "Armed with the ability to harness the cell's endogenous DNA repair machinery, it is now possible to engineer a wide variety of genomic alterations in a site-specific manner.", "CRISPR-Cas RNA-guided nucleases are derived from an adaptive immune system that evolved in bacteria to defend against invading plasmids and viruses.", "Decades of work investigating CRISPR systems in various microbial species has elucidated a mechanism by which short sequences of invading nucleic acids are incorporated into CRISPR loci.157", "They are then transcribed and processed into CRISPR RNAs (crRNAs) which, together with a trans-activating crRNAs (tracrRNAs), complex with CRISPR-associated (Cas) proteins to dictate specificity of DNA cleavage by Cas nucleases through Watson-Crick base pairing between nucleic acids.158,159,160,161", "Building off of two studies showing that the three components required for the type II CRISPR nuclease system are the Cas9 protein, the mature crRNA and the tracrRNA,162,163 Doudna, Charpentier and colleagues showed through in vitro DNA cleavage experiments that this system could be reduced to two components by fusion of the crRNA and tracrRNA into a single guide RNA (gRNA).", "Furthermore, they showed that re-targeting of the Cas9/gRNA complex to new sites could be accomplished by altering the sequence of a short portion of the gRNA.164", "Thereafter, a series of publications demonstrated that the CRISPR/Cas9 system could be engineered for efficient genetic modification in mammalian cells.165,166,167,168", "Collectively these studies have propelled the CRISPR/Cas9 technology into the spotlight of the genome-editing field.", "The only sequence limitation of the CRISPR/Cas system derives from the necessity of a protospacer-adjacent motif (PAM) located immediately 3' to the target site.", "The PAM sequence is specific to the species of Cas9.", "For example, the PAM sequence 5'-NGG-3' is necessary for binding and cleavage of DNA by the commonly used Cas9 from Streptococcus pyogenes.169,170,171", "However, Cas9 variants with novel PAMs may be engineered by directed evolution, thus dramatically expanding the number of potential target sequences.172,173", "Cas9 complexed with the crRNA and tracrRNA undergoes a conformational change and associates with PAM motifs throughout the genome interrogating the sequence directly upstream to determine sequence complementarity with the gRNA.171,174,175,176,177", "The formation of a DNA-RNA heteroduplex at a matched target site allows for cleavage of the target DNA by the Cas9-RNA complex.171", "Unlike the three nuclease systems discussed above, CRISPR/Cas nucleases do not require the engineering of novel proteins for each DNA target site.", "The relative ease with which new sites can be targeted, simply by altering the short region of the gRNA that dictates specificity, makes this system a highly attractive method for introducing site-specific DSBs.", "Additionally, because the Cas9 protein is not directly coupled to the gRNA, this system is highly amenable to multiplexing through the concurrent use of multiple gRNAs to induce DSBs at several loci.", "Because the rich diversity of natural CRISPR systems has been largely understudied, it is reasonable to expect many new CRISPR-based gene-editing technologies to emerge, including non-Cas9 based type II systems such as the recently described RNA-guided endonuclease Cpf1 and others.178,179", "The efficacy of targeted gene editing relies on cleaving the DNA in a site-specific manner while mitigating, or ideally preventing, collateral damage to the rest of the genome.", "For this reason, the specificity of targeted nucleases is a major focus of the gene-editing field.", "Modifications to the FokI dimerization domain dramatically increased the specificity of ZFNs and TALENs by requiring two obligate heterodimers to bind the target DNA in a specific orientation and spacing.180,181,182,183", "Reminiscent of the architecture of ZFNs and TALENs, the inactivation of Cas9 nuclease domains to create Cas9 nickases or Cas9-FokI fusions has increased specificity by requiring two gRNA/Cas9 complexes, each cleaving a single strand of DNA, to come together at a precise distance and orientation in order to generate a DSB.184,185,186,187", "Additionally, reducing the length of complementarity between the gRNA and the target site from 20 to 17 nucleotides increases the specificity of DNA cleavage by Cas9 from S. pyogenes.188", "Recently, structure-guided protein engineering has been used to develop novel Cas9 variants with increased specificity properties.189,190", "These improvements have significantly alleviated initial concerns over the specificity of CRISPR/Cas nucleases.191,192,193", "However, regardless of the nuclease technology, it is difficult to determine the full spectrum of off-target cleavage in a complex genome.", "Until recently, specificity studies were largely limited to a priori, in silico identification of potential off-target sites that could be informed by surrogate assays with purified proteins or viral integrations at double-strand breaks.194,195,196", "Whole-genome sequencing of a small number of clones derived from single cells has verified the lack of off-target effects in these select populations, but cannot identify sites that are cleaved at low frequencies in bulk cell populations.197,198,199", "Interrogation of DNA-binding specificity by ChIP-seq was greatly informative for understanding target site recognition, but the vast majority of the off-target binding sites were not predictive of nuclease activity.200,201,202", "Recent development of methods for unbiased, genome-wide assays to determine specificity have significantly advanced the ability to characterize nuclease specificity with a degree of sensitivity that was not previously possible.195,203,204,205,206", "These new methods will likely be critical to advancing targeted gene-editing nucleases as therapeutics.", "Efficient and safe delivery to target cells and tissues has been the long-standing challenge to successful gene therapy strategies (Figure 3).", "This challenge extends to genome-editing methods as well, where the nucleases, and in the case of the CRISPR/Cas9 system, a gRNA, must be efficiently delivered.", "Moreover, the dose of the donor template DNA is important to ensuring efficient homologous recombination.", "The duration and magnitude of nuclease expression is a critical parameter for the level of both on-target and off-target nuclease activity.", "Maximizing the efficiency of delivery is particularly important since gene editing is an inherently stochastic event occurring in only a fraction of the cells in which the nuclease is expressed.", "The most widely reported method for introducing nucleases into cells in proof-of-principle studies is transfection of plasmid DNA carrying nuclease and gRNA expression cassettes.", "Although simple and straightforward, this method is not ideal for most gene and cell therapies due to low efficiency of transfection of primary cells, DNA-related cytotoxicity, the presence of bacterial DNA sequences in plasmid backbones, and the possibility of random integration of plasmid fragments into the genome.", "Consequently, electroporation of mRNA encoding the nucleases and gRNAs generated through in vitro transcription has become a preferred method for ex vivo gene editing of primary cells relevant to gene therapy, such as T cells and hematopoietic stem cells (HSCs).168,207", "Alternatively, the direct delivery of purified nuclease proteins or Cas9 protein-gRNA complexes has also been very successful in achieving high levels of gene editing, either by electroporation208,209 or fusion to cell-penetrating peptides, which obviates electroporation-mediated toxicity.210,211,212", "Chemical modification of the gRNAs can further increase the robustness of gene editing in primary cells by increasing stability and/or decreasing innate immune responses.207", "These studies have collectively shown that by restricting the duration of nuclease activity with short-lived mRNA or proteins, off-target effects can be minimized compared to plasmid-based delivery.", "Future efforts will likely take advantage of emerging nanoparticle formulations for efficient and nontoxic delivery.213", "For many applications, viral vectors are still the optimal vehicle to maximize the efficiency of delivery while minimizing cytotoxicity.214", "In particular, lentiviral vectors have been optimized for highly efficient transduction of T cells and HSCs; however these vectors also integrate into the genome and stably express their transgene cargo.", "In order to take advantage of the efficiency of lentiviral transduction while limiting the duration of nuclease expression in target cells, integrase-deficient lentiviral vectors have been used to transiently deliver genome-editing tools to target cells.57,111", "Similarly, adenoviral systems can also achieve high levels of transduction of a variety of cell types ex vivo while providing only transient nuclease expression.20,137,139", "Both lentiviral and adenoviral vectors also have the advantage of sufficient packaging capacity to carry multiple nucleases or gRNA expression cassettes for multiplex editing of several loci.215", "In vivo gene editing presents additional challenges of tissue-specific targeting, distribution of the vector, and immunogenicity and biocompatibility of the carrier.", "Although several examples of plasmid delivery to the liver have shown important proof-of-principle of in vivo gene editing in animal models,216,217,218 translating these strategies to human therapy is not yet feasible.", "However, in vivo gene delivery with AAV to the liver, eye, nervous system, and skeletal and cardiac muscle has shown impressive efficacy in both preclinical models and clinical trials.219", "Consequently, AAV is also a promising system for delivery of gene-editing nucleases to target tissues.220", "Furthermore, the natural recombinogenic properties of AAV make it a desirable vector for delivery of DNA repair templates.56,61,62,221,222,223,224", "Although some studies have shown targeted recombination of genomic loci with AAV vectors in the absence of nucleases,58,71 the efficiencies are significantly lower than reports that include nucleases.", "Further studies are required to understand which disease indications can be robustly addressed at lower efficiencies of gene editing.", "Although AAV has shown considerable promise for in vivo gene delivery, its packaging capacity is limited to less than ∼4.8 kb of DNA.", "This has posed a challenge for the delivery of large nucleases such as TALENs, that require two monomers each encoded by cDNAs greater than four kb in size, and the commonly used S. pyogenes Cas9 nuclease that is encoded by a ∼4.2 kb cDNA.", "Trans-splicing vectors have been designed to recombine within cells to expand the size of transgenes delivered by AAV,225 but the efficiency of expression is significantly lower than genes delivered by a single AAV.", "A number of smaller Cas9 orthologs exist, and the ∼3.1 kb Cas9 from S. aureus has been thoroughly characterized and shown to mediate highly efficient gene editing in vivo following AAV delivery.35,36,226,227", "This important advance is critical to enabling facile and robust in vivo gene editing with the CRISPR/Cas9 system.", "It is particularly advantageous for developing a translatable gene therapy product that can be packaged in a single vector.", "The ability to manipulate any genomic sequence by gene editing has created diverse opportunities to treating many different diseases and disorders (Figure 4).", "Here, we discuss the major categories of disease indications that have been pursued in preclinical models (Table 1), as well as highlight the ongoing or planned clinical trials using gene-editing strategies (Table 2).", "The most straightforward application of gene editing is to use the relatively efficient NHEJ mechanism to knockout genes in an ex vivo autologous cell therapy, where somatic cells can be isolated, modified, and delivered back to the patient.", "Moreover, one of the most compelling applications of gene editing is the prevention of viral infection or replication.", "Thus the most advanced gene-editing strategy to date is the ex vivo modification of T cells to knock out the CCR5 coreceptor used for primary HIV infection.20", "This early study demonstrated decreased viral loads and increased CD4+ T-cell counts in HIV-infected mice engrafted with T cells in which the CCR5 gene had been knocked out by zinc finger nucleases.20", "This was later followed by demonstration of similar results following gene editing and transplantation of CD34+ HSCs into irradiated mice, allowing for protection of all blood cell lineages from CCR5-tropic HIV infection.21,228", "These studies have led to a series of clinical trials (Table 2) evaluating this approach in HIV-positive human patients.", "Thus far the studies show safe engraftment and survival of CCR5-modified T cells and control of viral load in some patients, providing promising proof-of-principle of a gene-editing approach in humans.22", "Interestingly, data from this study showed a greater clinical efficacy in a patient that was already heterozygous for the naturally-occurring ▵32 mutation, suggesting that gene-editing efficiency may be a critical factor for success.", "Building on these promising studies with ZFNs, several other efforts have developed similar gene-editing strategies to knockout CCR5 with TALENs,134,229 CRISPR/Cas9 (refs. 229, 230) and meganucleases.231", "Other work has expanded beyond targeting only CCR5 to enhance resistance to HIV infection.", "This includes targeting the CXCR4 coreceptor232 or PSIP1 gene encoding the LEDGF/p75 protein required for HIV integration.233,234", "Some studies have used targeted gene integration into the CCR5 gene by HDR to simultaneously knockout CCR5 and introduce anti-HIV factors.235", "Finally, complete excision of the HIV genome from infected cells using nucleases that target sequences in the long terminal repeats (LTRs) flanking the viral genome has also been reported.236", "Thus, a variety of next-generation gene-editing strategies for preventing HIV infection and replication are on the horizon.", "Beyond addressing HIV infection, all of the gene-editing platforms have also been applied to various other viral pathogens23 including hepatitis B virus,217,218,237,238,239,240,241,242 herpes simplex virus,243,244,245 and human papilloma virus.246", "These strategies typically involve removing viral genomes by degradation following nuclease cleavage and by targeting genes essential for genome stability, maintenance, and replication.", "While many of these early studies focused on proof-of-principle reduction of viral load in cell culture or following hydrodynamic plasmid DNA delivery to mice, recent studies using AAV delivery of gene-editing tools directly to the mouse liver provides a plausible path for scalability and clinical translation.238", "A general challenge of antiviral therapies is the high mutability of viral targets.", "This is a compelling argument in favor of targeting host genes, such as CCR5, but may also be addressed by simultaneous targeting of multiple critical sites in the viral genome.", "Cancer immunotherapy has been widely recognized as one of the greatest advances in biomedical research in recent years.247", "In particular, adoptive T-cell immunotherapy, in which autologous T cells are engineered to attack cancer antigens ex vivo and transferred back to the patient, has been impressively successful at treating some cases of lymphoma, leukemia, and melanoma.248", "Despite these successes and promising ongoing clinical trials, there are several areas in which T-cell immunotherapy could be potentially improved by gene editing.", "Here, both the efficacy against diverse tumor types and the ability to manufacture cell products that can be applied to a broad patient population could be enhanced through gene-editing techniques.", "For example, a promising strategy for immunotherapy involves engineering T cells to express synthetic receptors known as chimeric antigen receptors, or CARs, that recognize epitopes on cancer cells.", "Such CAR T cells have been particularly successful in treating B-cell lymphoma by targeting the CD19 cell surface antigen.247,248", "However, one limitation of this approach is that these modified T cells express both the endogenous T-cell receptor as well as the engineered CAR.", "Because these receptors function as dimers, the natural and engineered receptors can dimerize and interact, resulting in unpredictable epitope specificity and potentially reducing therapeutic potency.", "To address this limitation, several studies have focused on knocking out the endogenous T-cell receptors with engineered nucleases.154,249,250,251", "A major challenge to the development of broadly translatable T-cell immunotherapies is the need to use autologous cells to avoid immune rejection.", "To address this, gene editing has been used to knockout the human leukocyte antigen (HLA) by which the immune system discriminates self and foreign cells.252", "Importantly, this approach may be broadly useful for allogeneic cell therapy beyond T-cell immunotherapy.", "For example, similar approaches have been applied in human pluripotent cells potentially having diverse uses in regenerative medicine252 as well as in endothelial cells that could be used for allogeneic vascular grafts.253", "Another major obstacle to successful T-cell immunotherapy is the inhibition of T-cell effector functions by the expression of checkpoint inhibitors on the surface of tumor cells.", "For example, the binding of such inhibitors to the PD-1 receptor on T cells is well documented to block T-cell effector function and induce apoptosis and exhaustion.", "PD-1 receptor inhibition thus provides a mechanism by which cancer cells successfully evade the immune system.", "As a strategy to overcome this, gene editing has been used to knockout PD-1 in T cells,254 leading to increased T-cell effector function.209,254", "The success of this gene-editing strategy is likely extendable to other checkpoint inhibitor pathways that cancer cells exploit to circumvent immunosurveillance, and thus may be a critical technology for broadly enabling immunotherapy for diverse cancer types.", "Finally, for indications such as glioblastoma, the apoptosis of the engineered T cells resulting from post-surgery anti-inflammatory glucocorticoid steroid treatment severely limits the efficacy of T-cell immunotherapy.", "In order to create a glucocorticoid-resistant T-cell source, gene editing was used to knockout the endogenous T-cell receptor.255", "This led to successful anti-glioma T-cell therapy in mouse models255 and was the basis of a subsequent clinical trial (Table 2).", "The first gene therapy clinical trials involved the ex vivo retroviral delivery of a therapeutic adenosine deaminase (ADA) transgene to T cells to treat children with severe combined immunodeficiency (ADA-SCID),256 and later the treatment of X-linked SCID (X-SCID) by retroviral gene delivery to CD34+ hematopoietic stem cells (HSCs).257", "This early focus on ex vivo gene therapy for immunodeficiency was based on the desperate need to develop treatment for these otherwise fatal disorders as well as the availability of methods for efficient retroviral gene delivery to cells in culture.", "The subsequent observation that this early protocol can lead to insertional mutagenesis was a primary catalyst for the gene-editing field and demonstrated the need for correction of gene mutations in these cells, in contrast to transgene delivery.63", "In fact, the first example of endogenous gene correction in human cells focused on the IL2 receptor common gamma chain that is mutated in X-SCID,40 and this approach was more recently extended to gene correction in CD34+ HSCs.57", "Gene-editing tools have also been developed to correct gene mutations associated with ADA-SCID258 and radiosensitive SCID, caused by impaired DNA-dependent protein kinase (DNA-PK) activity.259", "Thus, the ability to efficiently alter gene sequences in T cells, CD34+ HSCs, and human pluripotent cells can provide therapeutic gene-editing strategies for a broad range of different human immunodeficiencies.", "Similarly, the establishment of gene editing in CD34+ HSCs and human pluripotent cells capable of differentiating into erythroid progenitors has provided new options for treating other hematologic disorders, including sickle cell disease, caused by a specific E6V point mutation in the β-globin gene, and β-thalassemia, caused by other types of mutations to β-globin.", "These globin mutations have been corrected by gene editing both in human iPSCs that can be differentiated into functional erythrocytes42,48,260 and directly in CD34+ HSCs.261", "Similar approaches have been developed for targeted integration of therapeutic transgenes into safe harbor sites in human iPSCs for α-thalassemia69 and Fanconi anemia.262", "Sickle cell disease and β-thalassemia are unique in that deficiencies in β-globin function or expression can be compensated for by inducing upregulation of γ-globin, which is expressed during fetal development but silenced after birth.", "BCL11A is a transcriptional regulator that suppresses the expression of γ-globin, and thus the knockout of BCL11A has been proposed as an approach to treat both sickle cell disease and β-thalassemia.", "However, the absence of BCL11A in all hematopoietic lineages was observed to be detrimental in nonerythroid cells.", "Interestingly, an enhancer element was discovered that specifically coordinates BCL11A in erythroid cells and inactivation of this enhancer by gene editing leads to suppression of BCL11A and upregulation of γ-globin only in cells of the erythroid lineage.30,31,263", "Thus, this approach provides both a mechanism of gene-editing therapy for sickle cell disease and β-thalassemia, but more broadly suggests a general strategy of therapeutic modulation of gene expression through the targeted editing of cell type-specific enhancers.", "Beyond the ex vivo gene editing of blood and immune cells, there is intense interest in gene editing in vivo for gene correction and targeted gene addition to tissues for which cell transplantation is challenging or impractical.", "This requires the efficient delivery of gene-editing nucleases and donor vectors to target tissues.", "The first demonstration of highly efficient, nuclease-mediated gene editing in vivo used AAV vectors to deliver ZFNs and a factor IX cDNA, without a promoter, to the liver of a mouse model of hemophilia B.70 Cleavage of the first intron of a mutated human factor IX gene by ZFNs catalyzed the efficient integration of the factor IX cDNA into the locus, leading to correction of the hemophilic phenotype.", "This first study was performed in neonates in which the hepatocytes are actively dividing and thus homology-directed repair pathways are active.", "Notably, a subsequent study demonstrated efficacy in adult mice in which the hepatocytes have presumably exited the cell cycle, although the integrations resulted from a combination of HDR- and NHEJ-mediated events.264", "Additional studies are necessary to determine the role of cell cycle and gene editing with AAV and other delivery vectors in various tissue types.", "Targeted gene correction in the liver has the potential to treat many different diseases, including clotting disorders such as hemophilia A and hemophilia B, as well as lysosomal storage disorders including Fabry disease, Gaucher disease, Pompe disease, von Gierke disease, and Hurler and Hunter syndromes.", "However, each of these patient populations is relatively small and the types of mutations to each gene involved in these diseases are diverse.", "Therefore, the cost of clinical development and regulatory approval to develop safe and efficacious gene-editing tools for each of these diseases may be prohibitive.", "Moreover, it is unclear whether sufficient levels of targeted transgene integration or gene correction could be achieved to reach therapeutic efficacy if driven by the corresponding natural endogenous promoter for each gene.", "A clever approach to address each of these challenges is the targeted integration of therapeutic genes into the albumin locus downstream of the endogenous albumin promoter.71,265", "Because albumin is very highly expressed, even low levels of targeted gene integration to this site are likely to lead to therapeutic levels of gene expression.", "Moreover, this genomic “safe harbor” can be used for diverse diseases, including those listed above, such that a single validated gene-editing reagent can be used for a significantly larger patient population.", "This approach has been used effectively in mouse models with AAV-based homologous donor templates to treat hemophilia without nucleases71 and with ZFNs that are likely to dramatically enhance targeting efficiency.265", "The advent of the CRISPR/Cas9 system has made in vivo gene-editing tools more broadly available to the scientific community and thus many recent studies have used this approach for both developing disease models and strategies for gene therapy.", "The first example of in vivo gene editing with CRISPR/Cas9 involved the correction of a mouse model of hereditary tyrosinemia type I following hydrodynamic tail vein injection of plasmid DNA into mice.216", "Although overall gene-editing efficiencies were relatively low (∼0.4%), this model allows for selection of corrected cells to repopulate the liver and thus it was possible to demonstrate correction of the disease phenotype.", "Although the method of naked DNA delivery to the liver is likely not translatable to humans, this study was a landmark in demonstrating in vivo gene editing with CRISPR/Cas9 in adult tissues.", "Beyond gene correction, the disruption of particular genes in the liver may also have a beneficial effect.", "For example, the PCSK9gene encodes a proteinase that induces degradation of the low density lipoprotein receptor (LDLR).", "Decreased LDLR levels lead to lower metabolism of LDL cholesterol (LDL-C), increased LDL-C levels, and increased risk for cardiovascular disease.", "The discovery of natural genetic variation leading to high or low PCSK9 activity and corresponding cholesterol levels has led to intense interest in PCSK9-blocking drugs for lowering cholesterol.266,267", "In contrast to continuous drug administration, two different studies have shown that a single treatment of Cas9 and a PCSK9-targeted gRNA delivered to the liver can lead to efficient gene knockout and lowered cholesterol levels.226,268", "Finally, in addition to gene editing in the liver, new methods for culture and differentiation of human pluripotent cells into functional hepatocytes are providing options for ex vivo cell correction and engraftment into the liver.", "For example, α-1-antitrypsin mutations were seamlessly corrected by gene editing in human iPSCs and subsequently differentiated into liver cells that expressed the restored gene.45", "Although this type of cell-based product may be significantly more complex than a virus-based drug, the strategy described in this study allows for a comprehensive genomic analysis of the modified cells.", "Advances in gene delivery and cell transplantation to the central nervous system and skeletal and cardiac muscle have created new opportunities for gene and cell therapy for many neuromuscular disorders, including DMD, the limb girdle muscular dystrophies, spinal muscular atrophy, Friedreich's ataxia, Huntington's disease, and amyotrophic lateral sclerosis (ALS).", "Amongst this class of diseases, genome editing has thus far advanced most prominently for DMD, although possible strategies to apply genome editing to other conditions could be envisioned.", "DMD is caused by mutations to the dystrophin gene, most commonly large deletions that shift the downstream gene fragment out of frame and render the protein product nonfunctional.", "Because the coding sequence of the dystrophin gene is exceptionally large (14 kb), it cannot be packaged into size-restricted viral delivery vectors.", "Although truncated minigenes have been developed that do fit into viral vectors, they are only partially functional compared to the full-length gene and their ability to reverse the human disease remains to be determined.", "For these reasons, and because there is currently no available approved therapy for DMD, gene editing to repair the endogenous gene is particularly compelling.", "Early reports suggested a mechanism to repair the dystrophin gene with gene editing,269 which was followed by proof-of-principle experiments in cultured cells from DMD patients demonstrating dystrophin gene repair by targeted integration of the deleted exons270 or restoration of dystrophin protein expression by targeted shifting of the reading frame by NHEJ-mediated indels.17", "However, these two strategies suffer from addressing only a limited patient population with any particular gene-editing strategy or lacking predictable and reliable editing outcomes due to the reliance on stochastic NHEJ-mediated DNA repair, respectively.", "Therefore, more recent studies have focused on deleting one or more exons with a combination of nucleases to generate precisely restored protein products and address larger fractions of the DMD patient population.32,33,34", "This includes a single strategy of deleting >300 kb of genomic DNA comprising exons 45–55 that could be applicable to restoring dystrophin expression in 62% of DMD patients.33", "In order to develop this into an approach that could potentially be applied clinically to DMD patients, recent work has incorporated the CRISPR/Cas9 system into AAV vectors with tropism for skeletal and cardiac muscle.35,36,37", "When applied locally via intramuscular injection or systemically via intravenous injection to a mouse model of DMD, gene editing by CRISPR/Cas9 restored expression of the dystrophin protein and improved muscle pathology and strength.", "Notably, one study showed relatively efficient in vivo gene editing of Pax7-positive muscle progenitor cells that may act as a renewable source of cells in which the dystrophin gene has been repaired.36", "This translational approach builds on demonstration of in vivo gene editing in skeletal muscle with adenoviral delivery271 and correction of dystrophin mutations in single-cell mouse embryos41 to reverse disease symptoms.", "In the future, these efforts may be extended to cell therapies by using patient-derived cell types, such as iPS cells, that could be modified by gene correction or targeted dystrophin transgene insertion272 and expanded to large numbers and efficiently engrafted into muscle tissue.34,273", "The development of engineered skin grafts from autologous and allogeneic cells, including iPS cells, is creating new opportunities for treating genetic diseases that affect the skin.", "For example, recessive dystrophic epidermolysis bullosa is a disease caused by mutations to the gene encoding type VII collagen.", "This disruption of type VII collagen expression results in extensive skin blistering.", "This may be treatable by correcting patient cells with genome editing and using those cells to engineer autologous skin grafts.274", "In one study, the mutations to the type VII collagen gene were corrected in primary patient fibroblasts that were then reprogrammed to iPS cells which could be used to form skin structures in vivo.275", "Another study also corrected the disease-causing mutation in patient iPS cells, and used these cells to generate epithelial keratinocyte sheets, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice.276", "This simplest form of gene editing utilizes the error-prone nature of NHEJ to introduce small indels at the target site.", "Classical NHEJ directly religates unprocessed DNA ends whereas alternative-NHEJ (also known as microhomology-mediated end joining, or MMEJ) requires end-resection followed by annealing of short single-stranded regions of microhomology and subsequent DNA end ligation.15", "Active during all stages of the cell cycle, both of these NHEJ pathways repair DNA with a high frequency of mutagenesis resulting in the formation of indels at the site of the break.15,16", "When the nuclease target site is placed in the coding region of a gene, the resulting indels will often cause frameshifts.", "In diseases such as Duchenne muscular dystrophy (DMD), where gene deletions result in frameshifts and subsequent loss of protein function, targeted NHEJ-induced indels can be used to restore the correct reading frame of the gene.17", "However, the most common application of targeted mutagenesis involves inducing frameshift mutations for the purpose of gene knockout.", "In contrast to traditional gene therapy, which is limited to the addition of exogenous sequence into the genome, the ability to knockout endogenous genes opens a new avenue of therapeutic treatment in which gene function can be permanently disrupted.", "One application of this approach is to target dominant gain-of-function mutations, such as those found in Huntington's disease.", "This disease is caused by a repeat expansion on one allele of the huntingtin (HTT) gene, leading to the production of a toxic mutant HTT protein.", "Eliminating this mutant allele by NHEJ-based gene editing could provide clinical benefit to Huntington's patients.18,19", "In other diseases, it may sometimes be therapeutic to remove the normal function of a gene.", "The most prominent example of this is the gene-editing approach currently in clinical trials for the treatment of HIV, in which knockout of CCR5, the major HIV coreceptor, prohibits viral infection of modified T cells.20,21,22", "Finally, rather than directly targeting the human genome, knockout of critical genes in invading bacteria or DNA-based viruses could serve as effective anti-microbial treatments.23,24", "Recent successes in clinical trials for the treatment of Leber Congenital Amaurosis type 2 (LCA2) have propelled retinal disorders into the spotlight of the gene therapy field.", "Using a gene augmentation approach, subretinal injection of AAV encoding the full RPE65 gene was found to be both safe and efficacious in several concurrent trials.277,278,279,280", "LCA is the leading cause of childhood blindness and is caused by mutations in at least 18 different genes.281", "LCA10, the most common form of LCA, is caused by mutations in the approximately 7.5kb CEP290 gene, and is therefore not amenable to the standard gene therapy approach employed for LCA2 due to the large size of the disease-causing gene.", "While an in vitro proof-of-concept study used lentivirus to deliver the full transgene to iPSC-derived photoreceptor precursor cells,282 the proven safety of subretinal AAV delivery makes a gene-editing strategy, in which the nuclease components are delivered via AAV, particularly attractive.", "As proof-of-principle of gene editing for this disease, S. aureus Cas9 was used to delete an intronic region in the CEP290 gene containing a frequent mutation that creates an aberrant splice site which disrupts the gene coding sequence.283", "Deletion of this intronic region restored proper CEP290 expression.", "Gene editing is also uniquely positioned to address autosomal dominant disorders, such as forms of primary open angle glaucoma and retinitis pigmentosa, which could potentially be treated by targeted knockout of the MYOC and RHO genes, respectively.", "Cystic fibrosis is caused by mutations to the CFTR chloride channel.", "Loss of function of this chloride channel results in dysregulation of epithelial fluid transport in several organs.", "In particular, loss of proper fluid transport in the lung results in thickening of the mucus and thus frequent infection and complications breathing.", "Gene editing has been used to repair the CFTR mutations in cultured patient intestinal stem cells50 and iPS cells that could be subsequently differentiated into epithelial cells.284,285", "Although a long-standing challenge for gene therapy and gene editing for cystic fibrosis has been achieving efficient gene delivery to the lung epithelium, a recent study demonstrating functional correction of mice with cystic fibrosis following intranasal delivery of nanoparticles carrying triplex-forming peptide nucleic acid molecules is a very promising advance in this regard.286", "Beyond altering genes in the human genome, there are a variety of ways in which genome editing can be used to address human disease and improve human health by targeting the genomes of other organisms.", "A primary example of this is the recent application of genome editing to attack pathogenic bacterial infections.24", "For example, gene-editing nucleases can be designed to target genes conferring virulence or antibiotic resistance.", "Additionally, targeting genome sequences specific to pathogenic strains may facilitate their selective removal from a mixed population.", "Two recent studies demonstrated proof-of-principle of this approach using the CRISPR/Cas9 system to eliminate bacteria in a mouse skin colonization model287 and a moth larvae infection model,288 as well as selectively eliminating plasmids and bacterial populations.", "An intriguing alternative strategy to delivering complete CRISPR systems is to turn native CRISPR systems against themselves by delivery of self-targeted crRNAs, as was recently done to selectively remove bacterial strains with type I CRISPR systems.289", "The choice of type I systems is notable given that the Cas3 enzyme of type I systems has exonuclease activity that may facilitate DNA disruption and removal,290 in contrast to the type II CRISPR/Cas9 system which only cuts DNA.", "Furthermore, the majority of native CRISPR systems are type I, with only a minor fraction belonging to the type II category.291", "As with all gene therapies, a primary challenge in moving these platforms forward is the development of a suitable delivery vehicle for clinical translation, and there is promising work in the area of bacteriophage engineering for this purpose.292", "Collectively, these studies suggest a possible approach to address the rising incidence of antibiotic resistance.", "Tremendous progress has been made in addressing the challenges of conventional gene therapy by developing new technologies for precise modification of the human genome.", "This has helped to overcome some of the obstacles that have plagued the field of gene therapy for decades.", "Nevertheless, many challenges still remain to fully realize the potential of genome editing for gene and cell therapy.", "Central to these challenges are the persistent issues of safety and delivery.", "In this regard, rapid advances are being made both for increasing the specificity of genome-editing tools and increasing the sensitivity of methods for assessing this specificity genome-wide.189,190,195,203,204,205,206", "However, it remains unclear whether all off-target effects can be accounted for in a therapy that targets one site within billions of DNA base pairs, involves modification of millions of cells, and is custom prepared for each patient.", "Moreover, many questions remain about how the human immune system will respond to genetically modified cells or the in vivo administration of genome-editing tools.", "Remarkable advances in delivery technologies are also creating many more opportunities for genome editing, including ex vivo delivery to cells with DNA-free components207,208,209,210,212 and in vivo delivery with efficient and tissue-specific vectors.220", "The many successes of the preclinical studies reviewed here, as well as the current progression of genome editing in clinical trials, is a source of significant optimism for the future of this field.", "The rapid progress in the field is likely to continue to lead to new technologies that will expand the scope of genome editing.", "Alternative genome-editing technologies, such as targetable site-specific recombinases293 that do not rely on the creation of double-strand breaks, alternative CRISPR systems with unique properties,178,179 and DNA-guided nuclease systems294 will continue to change what is possible with these tools.", "Epigenome editing, in which DNA-targeting platforms are used to specifically change gene regulation or chromatin structure, is also creating new ways to manipulate the genome for gene and cell therapy.295,296,297", "Inducible or self-regulating systems that enable the control of the expression, activity, and/or stability of genome-editing tools may play an important role in ensuring their precision and safety.", "In summary, genome editing has changed the definition of gene and cell therapy and has been a key factor in the recent resurgence of this field, but there is still significant fundamental and translational work to realize the full promise of these technologies for widely treating human disease.", "In addition to the relatively minor indels resulting from NHEJ, it is possible to delete large segments of DNA by flanking the sequence with two DSBs.", "Indeed, it has been shown that simultaneous introduction of two targeted breaks can give rise to genomic deletions up to several megabases in size.25,26,27,28,29", "This approach is useful for therapeutic strategies that may require the removal of an entire genomic element, such as an enhancer region, as has been proposed for the treatment of hemoglobinopathies by deletion of the BCL11A erythroid-specific enhancer region.30,31", "Additionally, in diseases such as DMD where different internal gene deletions can shift the gene out of frame, the intentional deletion of one or more exons can correct the reading frame and restore the expression of truncated, but partially functional, protein.32,33,34,35,36,37", "As opposed to the unpredictable mutations resulting from NHEJ, targeted DSBs can induce precise gene editing by stimulating HDR with an exogenously supplied donor template.", "Active mainly during the S and G2 phases of the cell cycle, HDR naturally utilizes the sister chromatid as a template for DNA repair.15,16,38", "However, an exogenously supplied donor sequence may also be used as a repair template.39", "Thus the codelivery of targeted nucleases along with a targeting vector containing DNA homologous to the break site enables high-efficiency HDR-based gene editing.6,7,8", "Any sequence differences present in the donor template can thus be incorporated into the endogenous locus to correct disease-causing mutations, as has been demonstrated in many proof-of-concept studies.40,41,42,43,44,45,46,47,48,49,50", "While plasmids have traditionally been the most commonly used source of donor DNA, recent studies have shown that single stranded oligonucleotides (ssODNs), with as little as 80 base pairs of homology, can serve as efficient donor templates for HDR.51,52,53", "For cells that are difficult to transfect, viral vectors such as integrase-deficient lentivirus or adeno-associated virus (AAV) can also be used as a source of donor DNA.54,55,56,57", "In fact, the naturally recombinogenic nature of AAV, especially when combined with the particularly efficient hybrid serotypes such as AAV-DJ, makes them attractive vectors for delivery of the donor template.54,56,58,59,60,61,62", "Although traditional gene therapy has successfully used viral vectors to introduce exogenous genes into the genome, the inability to control the integration site of these viruses raises serious concerns of insertional mutagenesis, as was underscored in the early clinical trials that used murine retroviral vectors.63,64,65", "The use of a donor template, in which the desired genetic insert is flanked by homology arms including sequences identical to the nuclease cut site, enables site-specific DNA insertion through DSB-induced HDR.66", "Targeted insertion of therapeutic transgenes into predetermined sites in the genome, such as “safe harbor” loci, alleviates risks of insertional mutagenesis and enables high levels of ubiquitous gene expression.67,68,69", "To maintain control of gene expression by natural regulatory elements, a wild type copy of the disease-causing gene may be inserted into the corresponding endogenous locus and thus be under the control of its own promoter.70,71", "An alternative mechanism for targeted transgene insertion is to use nuclease-induced DSBs to create compatible overhangs on the donor DNA and the endogenous site, leading to NHEJ-mediated ligation of the insert DNA sequence directly into the target locus.72", "Because DSB-induced gene editing relies on the endogenous repair mechanisms of the cell, it is universally applicable to any cell type or organism that employs these methods for DNA repair.", "The critical element for implementing any of these gene-editing methods is the precise introduction of a targeted DSB.", "Four major platforms currently exist for inducing these site-specific DSBs: zinc finger nucleases (ZFNs), transcription activator-like effector (TALE)-nucleases (TALENs), meganucleases, and most recently the CRISPR/Cas system (Figure 2).", "Zinc finger (ZF) proteins are the most abundant class of transcription factors and the Cys2-His2 zinc finger domain is one of the most common DNA-binding domains encoded in the human genome.73", "The crystal structure of Zif268 has served as the basis for understanding DNA recognition by zinc fingers.74,75,76", "In the presence of a zinc atom, the zinc finger domain forms a compact ββα structure with the α-helical portion of each finger making contact with 3 or 4 bp in the major groove of the DNA.74,77,78", "Tandem fingers in a zinc finger array wrap around the DNA to bind extended target sequences such that a three-finger protein binds a 9 bp target site.", "The modular structure of Zif268 suggested that these proteins might provide an attractive framework for engineering novel DNA-binding motifs.79", "Initial attempts to design ZFs with unique specificities based on a simple set of rules had some success80,81; however, combinatorial libraries combined with selection-based methods proved to be a more robust approach for generating individual fingers with novel DNA-binding specificities.82,83,84,85,86,87", "Following this success, the field was faced with the challenge of engineering multi-finger arrays with novel target sites long enough to be unique in a complex genome.", "The “modular assembly” approach relies on collections of single-finger modules, either identified in naturally occurring proteins88 or selected to bind specific three base pair target sites,89,90,91,92 which are then linked in tandem to generate novel proteins.93,94,95,96,97", "Alternatively, selection-based methods, such as OPEN, may be used to select new proteins from randomized libraries.98", "While significantly more labor intensive, this method takes into account context-dependent interactions between neighboring fingers within a multi-finger array.76,99,100,101", "Several methods, including those used by Sangamo Biosciences and the Sigma-Aldrich CompoZr platform, combine these two approaches to assemble novel arrays using archives of multi-finger units that have been preselected to work well together.13,102,103,104", "The zinc finger nuclease (ZFN) technology was made possible by the discovery that the DNA-binding domain and the cleavage domain of the FokI restriction endonuclease function independently of each other.105", "By replacing the FokI DNA-binding domain with a zinc finger domain, it is possible to generate chimeric nucleases with novel binding specificities.106,107", "Because the FokI nuclease functions as a dimer, two ZFNs binding opposite strands of DNA are required for induction of a DSB.108", "Initial experiments showed that ZFN-induced DSBs could be used to modify the genome through either NHEJ or HDR10,109,110 and this technology has subsequently been used to successfully modify genes in human somatic40,66,98 and pluripotent stem cells.42,44,111,112,113", "The discovery of a simple one-to-one code dictating the DNA-binding specificity of TALE proteins from the plant pathogen Xanthomonas again raised the exciting possibility for modular design of novel DNA-binding proteins.114,115", "Highly conserved 33–35 amino acid TALE repeats each bind a single base pair of DNA with specificity dictated by two hypervariable residues.", "Crystal structures of TALEs bound to DNA revealed that each repeat forms a two-helix structure connected by a loop which presents the hypervariable residue into the major groove as the protein wraps around the DNA in a superhelical structure.116,117", "These modular TALE repeats can be linked together to build long arrays with custom DNA-binding specificities.118,119,120,121,122", "Many platforms exist for engineering TALE arrays.", "The simplest methods use standard cloning techniques to assemble TALEs from archives of plasmids, each consisting of single TALE repeats.123,124", "Several medium-throughput methods rely on the Golden Gate cloning system to assemble multiple pieces simultaneously in a single reaction.120,122,125,126,127,128,129", "The highest-throughput methods utilize solid phase assembly130,131,132 or ligation-independent cloning techniques.133", "Building off the foundation laid by a decade of ZFN-induced genome editing, the discovery of TALEs as a programmable DNA-binding domain was rapidly followed by the engineering of TALENs.", "Like ZFNs, TALEs were fused to the catalytic domain of the FokI endonuclease and shown to function as dimers to cleave their intended DNA target site.119,121,134,135", "Also similar to ZFNs, TALENs have been shown to efficiently induce both NHEJ and HDR in human somatic119,132,134 and pluripotent stem cells.53,136", "TALENs can be engineered to target virtually any sequence given that their only targeting restraint is the requirement for a 5' T, specified by the constant N-terminal domain, for each array.", "This unlimited targeting range, in addition to the ease of engineering new proteins, makes TALENs an attractive platform for targeted gene editing.", "Conversely, the large size and repetitive nature of TALE arrays presents a hurdle for in vivo delivery of these proteins.", "As opposed to a 30 amino acid zinc finger, which binds three bases of DNA, TALENs require 34 amino acids to specify a single base pair and this size difference can prohibit delivery of both TALEN monomers in a single viral vector with limited packaging capacity.", "Additionally, the unstable nature of tandem repeats, such as those present in TALENs, makes it challenging to package repetitive sequences in viral systems.", "Indeed, TALENs delivered by lentivirus have been shown to be susceptible to rearrangements,137 although this phenomenon may be mitigated by codon diversification between the repeats.138", "Adenoviral systems have also been used to successfully deliver TALENs.139", "Meganuclease technology involves re-engineering the DNA-binding specificity of naturally occurring homing endonucleases.", "The largest class of homing endonucleases is the LAGLIDADG family, which includes the well-characterized and commonly used I-CreI and I-SceI enzymes.140", "Through a combination of rational design and selection, these homing endonucleases can be re-engineered to target novel sequences.141,142,143,144,145,146,147,148", "While many studies show promise for the use of meganucleases in genome editing,149,150,151,152 the DNA-binding and cleavage domains of homing endonucleases are difficult to separate, and the relative difficulty of engineering proteins with novel specificities has traditionally limited the use of this platform.", "To address this limitation, chimeric proteins comprising fusions of meganucleases, ZFs, and TALEs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFs and TALEs and the cleavage specificity of meganucleases.153,154,155,156", "One potential advantage associated with meganuclease technology is that DSB-formation by these enzymes results in a 3' overhang, which may be more recombinogenic for HDR than the 5' overhang generated by FokI cleavage.", "Additionally, meganucleases are the smallest class of engineered nucleases, making them potentially amenable to all standard gene delivery methods.", "In fact, multiple meganuclease monomers could be readily packaged into single viral vectors to simultaneously create multiple DSBs." ]	[]
Prediction of Reach Goals in Depth and Direction from the Parietal Cortex	The posterior parietal cortex is well known to mediate sensorimotor transformations during the generation of movement plans, but its ability to control prosthetic limbs in 3D environments has not yet been fully demonstrated. With this aim, we trained monkeys to perform reaches to targets located at various depths and directions and tested whether the reach goal position can be extracted from parietal signals. The reach goal location was reliably decoded with accuracy close to optimal (>90%), and this occurred also well before movement onset. These results, together with recent work showing a reliable decoding of hand grip in the same area, suggest that this is a suitable site to decode the entire prehension action, to be considered in the development of brain-computer interfaces. Filippini et al. show that it is possible to use parietal cortex activity to predict in which direction the arm will move and how far it will reach. This opens up the possibility of neural prostheses that can accurately guide reach and grasp using signals from this part of the brain.	[ "BIOC" ]	[ "V6A", "hand guidance", "machine learning", "monkey", "neuroprosthetics", "offline neural decoding", "prehension", "reaching in depth", "robotics", "visuomotor transformations" ]	[ "1300" ]	[ "When a spinal cord injury or other diseases do not allow motor commands to reach the muscles, the patient is unable to perform voluntary actions, despite an intact brain.", "In cases like these, the advent of brain-computer interfaces (BCIs) has offered the possibility to gain control of external devices (neural prostheses) by using the patient’s own brain activity (Brandman et al., 2017).", "Although in the past decade several technical advances provided impressive examples of successful human applications, the performances achieved are still far from enabling widespread clinical application (Cui, 2016).", "So far, the majority of studies have used primary motor and premotor cortex signals to reconstruct reach trajectories in order to guide robotic limbs in monkeys (Velliste et al., 2008; Wessberg et al., 2000) and humans (Collinger et al., 2013; Hochberg et al., 2012).", "Although decoding of trajectories is still essential in order to provide the user with natural interfaces, progress in computer vision and robotics is leading to prostheses that do not require trajectory information, as simple algorithms can reconstruct this information from reach endpoint goals (Andersen et al., 2014; Hotson et al., 2016; Katyal et al., 2014).", "The posterior parietal cortex (PPC) in humans and monkeys is involved in the sensorimotor transformations required to generate action plans (Andersen et al., 2014; Cui, 2016; Gardner, 2017), so it is a good source for retrieving movement intentions and goals.", "Pioneering studies demonstrated that reach endpoints (Musallam et al., 2004; Serruya et al., 2003), trajectories (Mulliken et al., 2008; Figure 1A), and grips (Schaffelhofer et al., 2015; Figure 1A) can be extracted from monkey PPC.", "Translational work in humans produced similar results (Aflalo et al., 2015; Figure 1B), together with reliable hand shape decoding (Klaes et al., 2015; Figure 1B).", "In the aforementioned studies, reaches were performed on a single plane.", "To the best of our knowledge, only one study in monkey PPC performed decoding of reach goal and trajectory information in a virtual three-dimensional (3D) environment (Hauschild et al., 2012).", "A medial PPC area termed V6A (Figure 1A) is known to encode not only goals and reach movement directions (Bosco et al., 2010, 2016; Breveglieri et al., 2014; Hadjidimitrakis et al., 2014) but also several grasping parameters (Fattori et al., 2017).", "Previous research suggested that V6A could integrate the arm transport and hand grip components of a reach-to-grasp action.", "(Galletti and Fattori, 2018).", "Although a reliable decoding of hand grip from V6A signals has recently been shown (Filippini et al., 2017), decoding of reach-related information has not yet been performed (Figure 1A).", "We addressed this issue, with the aim of finding a parietal region where both grasping and reaching signals can be decoded.", "In a different way to most previous related studies, we varied reaches not only on a frontoparallel plane but using a naturalistic environment also involving depth (distance from the body).", "Data were recorded from two monkeys while they performed a fixation-to-reach task toward nine spatial positions with three different direction angles and three different depth levels (Figure 1C), covering a wide range of positions in peripersonal space.", "Target elevation was kept constant, at eye level.", "We sequentially recorded 264 V6A cells, 181 neurons in monkey 1 (M1) and 83 in monkey 2 (M2).", "Parts of this dataset have already been published in previous studies aimed at exploring the encoding of depth and direction in V6A activity (Hadjidimitrakis et al., 2014, 2017).", "The population discharge of the whole dataset is shown in Figure S1A.", "The plot shows a clear distinction among the activations during the early vision of the target, then during the preparation, and finally during the execution of reaching action.", "Moreover, Figure S1A shows that the V6A neural population starts discriminating among different targets as soon as the LED is illuminated.", "The discrimination power of the population increases slightly when the monkey is preparing the action (epoch delay, from 450 ms after the fixation onset to the arm movement onset), and has a second peak when the action is executed (reach).", "Population tuning properties were confirmed using a sliding-window ANOVA (Figure S1B).", "The activity of each neuron was quantified in the three main epochs depicted in Figure 1D: fix, delay, and reach, corresponding to the period of early fixation of the target, the planning phase of the subsequent reach action, and the execution phase, respectively.", "Subsequently, population decoding analysis was performed using a naive Bayes classifier (see Experimental Procedures).", "The results are presented separately for the two monkeys.", "In each monkey, all recorded cells were included in the analysis, irrespectively of whether they responded differently depending on the position of the target or not.", "Our decoder correctly classified target positions well before movement onset: we found a high correlation between the actual and the decoded spatial positions during fix (Figure 2A).", "The mean accuracies, obtained using a “leave-p-out” 5-fold cross-validation (p value 20% of trials), were excellent in both monkeys (91%–92%) and well above chance level (11%, the conditions being nine).", "Misclassifications were very few and occurred between adjacent targets.", "The decoding accuracies during both reach planning (delay) and execution (reach) were even higher than during fix, again in both monkeys.", "Figure 2B depicts the decoding accuracy as a function of the population size.", "Results varied across epochs and monkeys: in fix (Figure 2B, left), a sample of 20–40 neurons (median 40) was sufficient to achieve 70% accuracy, whereas in reach (Figure 2B, right), 20–30 neurons (median 26) were required, and in delay (Figure 2B, center), between 10 and 20 neurons (median 15) were required.", "In all cases, a small number of neurons was enough to obtain accurate decoding.", "To evaluate the temporal evolution of decoding accuracy, we estimated the decoding performance using activity in smaller time intervals compared with the whole epochs.", "A rapid increase of the decoding performance, occurring around the time of the LED onset (LED on), is evident in Figure 3.", "At fixation onset (fix epoch), the recognition rate reached its peak and remained constant in the subsequent delay interval (delay) and in the reaching execution (reach).", "Interestingly, after the reaching, the gaze and the hand still remained on the target, whereas the decoding accuracy decreased.", "This suggests that the decoding performance is strictly linked to the preparation and execution of reaching, instead of being linked to the gaze fixation of the target, as documented by decoding results shown from a control experiment in Figure S2.", "In the task used in the main text (Figures 1C and 1D), gaze position and reach goal were coincident.", "Rather than related to reach goals, one could argue that the predictions of our classifier were related to gaze position-related and/or reach preparation-related activity (Breveglieri et al., 2012, 2014; Hadjidimitrakis et al., 2011, 2012).", "To uncouple the decoding of gaze and reach goals, 67 neurons out of 83 of the original population were recorded while M2 performed a delayed reaching task toward the same nine targets of the original task with the gaze fixed on the central position (constant-gaze task).", "A yellow flash (cue), in the early phase of the delay, instructed the monkey as to which target should be reached for.", "In the constant-gaze task, the increase of tuned cells occurs at cue onset (i.e., when the monkey receives instruction about the location of the target to be subsequently reached for).", "On the contrary, in the same neurons (n = 67) tested in the fixation-to-reach task, the increase of tuned cells occurred at the fixation onset, because in this task the fixation LED per se instructed the monkey about the reach goal location.", "The same trend was also observed in the decoding performance: the accuracy was very low during fixation before the cue and increased immediately after the cue was given.", "This rules out the possibility that gaze fixation per se is responsible for the high decoding performance achieved in the fixation-to-reach task.", "The accuracy shown in the confusion matrices from the constant-gaze task is not significantly different from the results of the same population of cells when tested for the fixation-to-reach task (results for the fixation-to-reach task for the 67 cells tested for both tasks: 90% [SD 4.1%] for fix epoch, 98% [SD 2.2%] for delay epoch, and 88% [SD 4.9%] for reach epoch, t-test p > 0.05).", "Neuron-dropping and sliding-window analyses (Figures S2D and S2E) support the evidence that as soon as the visual cue was provided, the decoding performance reached optimal values for both constant-gaze and fixation-to-reach tasks.", "The data of the control experiment highlight that gaze information is not necessary to obtain high decoding accuracy from area V6A.", "To evaluate whether the neural code used during the early fixation period was retained or changed during the subsequent planning interval before the reach movement, we performed a generalization analysis by training decoders in either the fix or the reach epoch, then we applied both codes on these epochs and portions of the delay epoch.", "Figure 4 shows the results of this analysis for the two monkeys.", "The code learned during the early fixation period (fix, blue line) was gradually lost in the delay intervals; the accuracy then dropped during movement execution (∼20%).", "This suggests that the neural code used during the earliest fixation phase became progressively weaker as soon as the animal began to prepare the movement.", "The time course of the accuracy obtained by training the algorithm with the movement neural activity (reach, red line in Figure 4), and testing the algorithm with the delay activity demonstrated that the neural code used during the action execution was partially preserved also during the last part of the planning period, but not in the earlier planning phases and initial fixation.", "In summary, by looking at the activity during early fixation, it was not possible to predict the spatial position during reach execution, and vice versa.", "When the accuracy of the classifier trained in the different fractions of the delay was analyzed (Figures 4C and 4D, gray lines), progressive code transformations were present.", "Both monkeys depicted a smooth transition between an earlier code, possibly related to the gaze location information, and a later code correlated with the movement preparation.", "In this study, we demonstrated that neural signals from area V6A can be successfully used for the offline decoding of reach goals located at different depths and directions, in conditions similar to everyday life, in which reaching movements are performed not only on a single plane but also in three dimensions.", "In most cases, just a few neurons (∼20) were sufficient to achieve a correct prediction.", "The accuracy of decoding was optimal from early target fixation to the end of reaching.", "We used a task configuration in which the monkeys fixated the goal of reaching movement, which is the most physiological condition (Hayhoe et al., 2003; Neggers and Bekkering, 2001).", "However, this setup cannot distinguish whether decoding uses gaze signals or arm movement-related activity.", "To exclude gaze-related activity from decoded signals, we performed decoding in another experiment in which the monkey performed a task in which gaze and reaching targets were not coincident (Figure S2).", "In this case too, decoding performance was very high.", "This result is in line with the strong spatial tuning in V6A reach-related activity when gaze is dissociated from the reach target position (Bosco et al., 2016).", "However, in our study, we did not test decoding in a free-gaze condition, in which gaze was truly independent.", "Thus, we cannot exclude the possibility that eye movements could potentially disturb the decoding from V6A.", "However, the very similar results obtained between tasks (Figures 3 and S2E) suggest that free gaze should not interfere with decoding reliability from V6A.", "Nevertheless, these results suggest V6A as a source for BCIs, not only when the patient can move his or her eyes to the reaching target but also in the absence of ocular motility.", "Several monkey studies performed decoding of reach goals (Musallam et al., 2004; Scherberger et al., 2005; Shenoy et al., 2003) and trajectories (Mulliken et al., 2008) in two-dimensional (2D) space from activity in PPC (specifically, from the parietal reach region [PRR]).", "Here, we decoded reach goal from another part of PPC, while also considering the depth dimension.", "In V6A, target location was decoded from neural responses occurring not only during reaching execution but also well before movement onset.", "This is similar to the neighboring PRR area, where neural signals during reach planning were used to online decode up to six reach goals on a screen and to guide a cursor (Musallam et al., 2004).", "Accuracy obtained in PRR was lower than in V6A (from 25% to 60% in PRR [Musallam et al., 2004] versus about 90%–100% in V6A [present results]).", "However, differences in the experimental design may account for these discrepancies.", "Here, the trajectory of the reaching movement could not be extracted, because only information on the reach goal location was available.", "Nevertheless, it was demonstrated that goal specificity is advantageous for ballistic operations (Musallam et al., 2004) and that by incorporating information about the reach goal (target position), the decoding accuracy of the trajectory estimation from PRR signals improved by 17% (goal-based Kalman filter; Mulliken et al., 2008).", "Alternatively, the optimal reconstruction of movement trajectories could be performed by computer vision (Andersen et al., 2014; Katyal et al., 2014).", "Looking at current state-of-the-art neural prosthesis technology, in order to increase prosthesis reliability, we need to increment the number of neurons sampled.", "This involves overcoming several technical limitations and using more invasive implants.", "Intuitively, a mixed neural signals-computer vision-driven BCI looks more feasible.", "From the PPC region, we can retrieve intention of movements, and this information could aid computer vision systems to be “mind controlled” or classic motor BCIs (i.e., BCIs driven by motor cortex) to reconstruct the movement smoothly, knowing movement goals in advance.", "Exploiting higher order, multidimensional information for decoding purposes could allow the development of more natural and user-friendly brain-machine interfaces to achieve fully integrated prehensile actions.", "This study shows the decoding of reaching goals from signals in PPC, also taking into account the depth dimension.", "Several studies demonstrated the feasibility of retrieving instantaneous movement attributes, such as position, velocity, and acceleration useful to drive artificial limbs in 3D space (Brandman et al., 2017).", "This has been achieved using activity from motor and premotor regions in monkeys (Carmena et al., 2003; Jarosiewicz et al., 2008; Taylor et al., 2002; Velliste et al., 2008) and in humans (Collinger et al., 2013; Hochberg et al., 2012).", "In monkey PPC (areas PRR and 5d), continuous trajectory reconstruction of cursor movements in a 3D virtual space was demonstrated by Hauschild et al. (2012).", "In that study, a good decoding performance (R2 ∼ 40%) was obtained using ensembles of about sixty neurons.", "BCI applications that restore basic interaction with objects in tetraplegic patients have recently been reported (Aflalo et al., 2015; Collinger et al., 2013; Hochberg et al., 2012).", "These studies demonstrated the feasibility of BCIs in humans, but there is much work still to be done.", "When the depth information was added, movements became reasonably slower and clumsier (Collinger et al., 2013).", "Thus, our results showing reliable decoding not only in two dimensions but also in depth are of particular importance.", "V6A has recently been suggested as a site of convergence of arm signals for reaching and grip signals for grasping to direct our hands toward efficient prehensile actions (Galletti and Fattori, 2018; Gardner, 2017).", "In humans, fMRI signals from a region that is a likely homolog of monkey V6A (Pitzalis et al., 2013) were used to successfully predict the direction of an upcoming reach, but not of a saccade (Gallivan et al., 2011).", "In a recent study, Nelissen et al. (2018) decoded grasping-related information from fMRI signals in monkey area V6A.", "This finding complements the decoding of the type of grasp (Filippini et al., 2017) and reach goals (present results) and suggests that V6A could be a useful site for the neuroprosthetic control of the entire prehension action.", "Despite the tremendous advances in neural prosthetics on the basis of signals from the motor cortex, the future of BCIs relies on the acquisition of neural signals that also reflect the cognitive state of the patient (i.e., intentions and movement goals) (Andersen et al., 2014).", "These cognitive prostheses may be implemented by decoding neural signals from parietal regions, such as V6A, so as to have signals related to movement intention and execution from the same area.", "V6A incorporates signals typical of parietal regions (intentions of movement) but also signals coding for some useful details of the movement, such as depth and direction of reaching, and even grip type (Filippini et al., 2017).", "These intelligent prosthetics are one potential application of the results presented here.", "Another potential and promising application of decoding arm actions from V6A is in the emerging field of soft robotics, a technology born mimicking natural beings, to replace classical rigid-bodied robots with limbs that are more comfortable and easy to handle (Rus and Tolley, 2015).", "Although soft robotics is becoming more and more popular, the potential of soft machines in the clinical field is still greatly under-exploited, mainly because of limited functionality and versatility caused by the lack of intelligent, natural control systems.", "Indeed, so far soft robots have relied on classic control approaches that reduce the advantages of “soft” robotics in terms of flexible interaction with a variable environment.", "A direction for the very near future is to design more intelligent soft robots taking advantage of bio-inspired controllers that will be developed thanks to advances in artificial intelligence and inspired by the neurophysiology of our bodies (Fani et al., 2016; Santello et al., 2016).", "For a new generation of user-friendly prostheses such as these biomorphic robots, natural signals with multiple neural information such as those from V6A might be exploited for a more dexterous control of artificial limbs.", "The study was performed in accordance with the guidelines of European Union (EU) directives (86/609/EEC and 2010/63/EU) and Italian national laws (D.L. 116-92 and D.L. 26-2014) on the protection of animals used for scientific purposes.", "Protocols were approved by the Animal-Welfare Body of the University of Bologna.", "During training and recording sessions, particular attention was paid to any behavioral and clinical sign of pain or distress.", "For surgical and electrophysiological procedures, see Hadjidimitrakis et al. (2014).", "Two male monkeys (M1 and M2, aged 5 and 8 years) were involved in the study.", "Electrophysiological data were collected while monkeys were performing a fixation-to-reach task with the contralateral limb (with respect to the recording hemisphere), with the head restrained, in darkness, while maintaining steady fixation of the target.", "Reaches were performed to one of nine light-emitting diodes (LEDs; 6 mm in diameter; Figure 1C).", "The LEDs were mounted on a panel located in front of the animal, at different distances and directions with respect to the eyes, but always at eye level.", "Given that the interocular distance for both animals was 30 mm, the nearest targets were located at 10 cm from the eyes, whereas the LEDs placed at intermediate and far positions were at a distance of 15 and 25 cm, respectively.", "Because targets were aligned at eye level, they could potentially obscure each other.", "We solved the problem by masking the nearest LEDs to be visibly thinner than second-line LEDs and the latter thinner than the farthest line.", "Thus, the monkeys were able to easily discriminate them.", "In the task, the monkeys pressed a button located close to their chest (home button [HB]; Figure 1C), fixated one of the targets for a variable period (fix; Figure 1D, left), prepared the movement (delay; Figure 1D, center), and started the reaching movement (reach, Figure 1D, right) toward the foveated target.", "The analyses were performed with customized scripts in MATLAB (The MathWorks; RRID: SCR_001622) and Python (using open-source machine learning toolkit scikit-learn, http://scikit-learn.org; RRID: SCR_002577).", "The neural activity was analyzed by quantifying the discharge in each trial in the following three different epochs (Figure 1D): (1) the early fixation epoch (fix), from 50 ms after the end of the saccade performed to gaze at the LED until 450 ms after it; (2) the preparation epoch (delay), from 450 ms after the end of the saccade to the arm movement onset (given the task structure and the variable reaction time of the monkeys, this epoch had a variable duration from about 1.3 to 2.1 s); and (3) the reach epoch (reach), from arm movement onset (M) until the end of it, signaled by the pressing of the LED target.", "All analyses and modeling were done offline.", "Among the original set of recorded neurons, we considered only cells with at least ten trials for each of the nine targets.", "All recorded neurons, either modulated in the reaching task or not (see Supplemental Experimental Procedures), were used in the decoding analysis.", "For each neuron of the population (181 neurons for M1, 83 for M2, respectively), we computed the mean firing rate (mFR; number of spikes per time unit) over a selected time span using a trial-by-trial approach.", "The decoder outputs were the nine targets.", "Fivefold cross-validation was performed by using 72 samples (8 for each condition) for training and 18 (2 for each condition) for testing for each neuron, to ensure that the classifier was trained and tested on different data.", "Recognition rates and SDs were calculated as means over the five folds’ iterations.", "Not normalized data were used for the decoding procedure.", "We used a naive Bayesian classifier as decoding algorithm.", "Naive Bayes methods are a set of supervised learning algorithms based on applying Bayes’ theorem with the “naive” assumption of independence between every pair of features.", "This technique has been shown to achieve performance closer to optimal compared with other classifiers such as support vector machine (SVM) when analyzing neural data (Carpaneto et al., 2011; Schaffelhofer et al., 2015).", "In our Python custom scripts, we implemented the module of naive Bayes classifiers proposed by scikit-learn libraries (the statistical formulation can be found at http://scikit-learn.org/stable/modules/naive_bayes.html; Zhang, 2004).", "Under the assumption of Poisson distribution of features, we reinforced the model as suggested at the following site: http://github.com/scikit-learn/scikit-learn/pull/3708/files (Ma et al., 2006).", "We performed three types of analysis, computing the following feature vectors over different epochs and time spans: whole-epoch, sliding-window, and generalization analysis.", "The same kinds of analyses have been performed in area V6A from different sets of neurons recorded in a grasping task (Filippini et al., 2017)." ]	[ "Depths and directions of reaching actions are reliably decoded by the parietal cortex", "Goal locations are discriminated well before movement onset", "The entire prehension action could be decoded from a single cortical site", "Neuroprosthetics from the medial parietal cortex can restore lost reach/grasp functions" ]
Manufacture of fine cellulose powder from chemically crosslinked kraft pulp sheets using dry milling	The present study concerns the preparation of cellulosic powders with two-stage dry milling of chemically crosslinked birch kraft pulp sheets. Chemical crosslinking of kraft pulp sheets using glyoxal with and without a catalyst (aluminium sulphate) made the pliable, tenacious kraft pulp sheets brittle. Due to the brittleness, the crosslinked pulp sheets could be disintegrated easily and rapidly using a Wiley mill. The length-weighted average fibre length of the crosslinked pulp powders (0.31–0.33 mm) was shorter than the Wiley-milled reference powder (0.44 mm). The notably higher density and less fluffy character of the crosslinked pulp powders enabled their effortless further processing with an air-flow-type ultra-fine microniser. The medium size value (D50) of the micronised crosslinked powders was around 40 μm. The study finds that chemical crosslinking pre-treatment enhances the dry milling of kraft pulps to a fine powder. Chemical crosslinking may offer a new tool for industrial cellulosic powder manufacturing.	[ "CENG" ]	[ "Cellulose powder", "Crosslinking", "Dry milling", "Microcrystalline cellulose" ]	[ "1500" ]	[ "Cellulose is an abundant natural material that has many valuable properties, such as non-toxicity, biodegradability, and easy chemical modifiability.", "It has already been utilised in many commercial applications from paper to high-cost medical and cosmetic additives.", "Cellulose powders are used, e.g. in paints as fillers and in drug delivery as a tablet former.", "In those applications, the cellulose powder is typically in the micrometer size (1–100 μm).", "These cellulose powders are manufactured by using dry milling, acid hydrolysis, or a combination of those techniques.", "Acid hydrolysis is a common industrial method to produce microcrystalline cellulose powder.", "It is based on a sequence of acid-catalysed hydrolysis of amorphous regions of cellulosic fibres followed by neutralisation, washing, and mechanical process steps.", "The treated material is then evacuated from water with spray drying that produces separated fine microcrystalline cellulose (MCC) particles.", "The medium particle size of commercial MCC products is typically in the range of 20–100 μm.", "Due to the acid-catalysed hydrolysis degree of polymerisation (DP) of the remainder, MCC is typically reduced to a level of 200–300 DP.", "A characteristic property of MCC is its ability to form stable dispersion and gels by vigorously mixing with water.", "Because of this property, MCCs are used as a thickener, a stabiliser, or an emulsifier in a variety of products, such as foodstuffs, cosmetics, and water-based coatings [1,2].", "Dry MCC powders are commonly used as an excipient in pharmaceutical products.", "From MCC, it is possible to compress tablets that are hard but disperse readily in water and during digestion.", "The main problems associated with the industrial manufacturing of MCC are the high acid consumption and high chemical oxygen demand (COD) of process effluents.", "In a study by Vanhatalo and Dahl, the acid hydrolysis of bleached softwood kraft pulp caused around a 15–30% yield loss, depending on hydrolysis conditions, due to the removal of the amorphous regions of the fibre [3].", "Dry milling is based on the milling of dry kraft pulp fibres by using one of several types of mill, e.g. a knife mill, a hammer mill, or a ball mill [4].", "The dry milling of wood fibres is energy-intensive due to the toughness and flexibility of kraft pulp fibres [5,6].", "According to a review article by Mayer-Laigle et al., the high energy consumption associated with the manufacture of micron-scale biomass powders using dry milling can be partly tackled by rendering the biomass more brittle with the use of a mild pyrolysis treatment called torrefaction [6].", "However, torrefaction-induced changes in the chemical composition of cellulosic fibres are not desirable in most cases.", "Another way to render biomass more brittle is cooling.", "In so-called cryogenic milling, the biomass is cooled by using liquid nitrogen.", "By using cryogenic milling, Hemery et al. were able to reduce the dry milling (impact mill) steps of wheat bran from 3 to 1 to reach the targeted medium 50 μm particle size [5].", "Due to the high costs of cryogenic milling, the processed materials are used in the high-added value products, such as adhesives, explosives, and spices [6].", "Therefore there is a need to study new pre-treatment strategies to ease the dry milling procedure of pulp fibres.", "The treatment of cellulosic paper with chemical crosslinking agents with relatively small molecular sizes is reported to cause fibre embrittlement and a decrease in the folding endurance of paper [7–10].", "Chemical crosslinking agents of cellulose are molecules that are capable of forming covalent bridges between hydroxyl groups of adjacent cellulose molecules.", "Chemicals capable of crosslinking cellulose are numerous, including formaldehyde, dialdehydes, such as glyoxal and glutardialdehyde, and tricarboxylic acids such as citric acid and butanetetracarboxylic acid [11–13].", "Glyoxal is also capable of crosslinking cellulose fibres, and it is a relatively safe cellulose crosslinking agent [14] that cures at a lower temperature than most other formaldehyde-free crosslinking agents, such as polycarboxylic acids.", "Both hemiacetal and acetal linkages can be formed in the crosslinking of cellulose with glyoxal.", "Hemiacetal formation is a reversible reaction, and the crosslinks are hydrolysed in water.", "The increase in the curing temperature and the use of catalysts favour the formation of acetal linkages, which are more water-resistant than hemiacetal bonds [7,11,15].", "Due to the differing water resistance of hemiacetal and acetal bonds, glyoxal can then be used to produce either transient or more permanent effects on cellulosic material properties [7].", "The most significant group of crosslinking agents are DMDHEU (1.3-dimethylol-4.5-dihydroxyethylene urea) based agents in cotton fabric finishing.", "The crosslinks formed in cotton fibres reduce the shrinking and wrinkling of cotton textiles in use, and in the washing and drying of textiles.", "Cotton fabric crosslinking is conventionally performed using the so-called pad-dry-cure method, which begins with the immersion on the fabric in a water solution of the crosslinking agent and its catalyst.", "After immersion, the excess solution is squeezed from the fabric and subsequently the fabric is smoothed and dried.", "Finally the fabric carrying the crosslinking agent is cured for a few minutes at an elevated temperature (around 140–170 °C).", "The actual crosslinking reactions in the process take place in the curing stage [12,16].", "Paper made from cellulosic fibres can be crosslinked by using chemicals following the pad-dry-cure method.", "Via crosslinking, it is possible to improve the stiffness and wet strength of paper by forming water-resistant inter-fibre covalent crosslinks.", "An unwanted consequence of chemical crosslinking in paper manufacture is an increase in the brittleness of the paper, resulting in reduced fold strength [15,17–19].", "As chemical crosslinking is performed on the papermaking pulp fibres, the water retention value (WRV) of the fibres decreases and the stiffness of the fibres increases [20,21].", "The decrease in WRV, which is a measure of pulp's capability to maintain water, is probably due to the chemical crosslinks that prevent the swelling of fibre walls caused by water.", "Crosslinked pulps with both low WRV and high wet bulk are used as moisture distribution layers in absorption products, such as napkins [20].", "In the present study we investigated if the sequential glyoxal crosslinking and dry milling technique can be utilised in the production of micrometer size cellulose powder (Scheme 1).", "For comparison, crosslinking of pulp sheets were also performed in the presence of aluminium sulphate, which is a known catalyst of crosslinking reactions [14,22,23].", "For the crosslinking procedure, bleached birch kraft pulp sheets were first immersed in a crosslinking-agent water solution followed by drying and curing of the sheets in elevated temperature.", "Cured pulp sheets were dry-milled using a Wiley mill, which is a rotating blade-type cutting mill.", "This step was followed by further dry-grinding of the powders using a decompression air-type microniser.", "Finally, properties, such as particle size distribution, specific surface area (BET), and bulk and tap densities of the powders were measured.", "The crosslinking reaction was also studied by using CPMAS 13C solid state NMR.", "The idea of using chemical crosslinking in order to reduce the energy consumption of dry milling cellulosic fibres to fine particles has been presented in patent application JP09392001 [24].", "To the best of our knowledge, the method has not been discussed previously in scientific literature.", "The pulp used in the study was dried, bleached birch (Betula bendula) kraft pulp originating from a Finnish pulp mill.", "The pulp was obtained as A4-sized pulp sheets (grammage 830 g/m2, air-dry, dry content 96%).", "The used glyoxal was obtained as a 40% water solution from Merck KGaA (USA).", "Aluminium sulphate tetradecahydrate(Al2(SO4)3·14H2O) was obtained from Kemira OyJ (Finland).", "The water used in the study was deionised water produced with a MilliQ-device.", "All other chemicals were laboratory grade.", "Crosslinking of the birch kraft pulp sheets was carried out following the pad-dry-cure crosslinking method.", "A 1-kg stack of pulp sheets (dry content 96%) was immersed in a water solution containing 4.0 wt% glyoxal for 60 min.", "Aluminium sulphate hydrate (1.33 wt%) was added in the glyoxal water solution, which was optional.", "After impregnation, the excess solution was drained from the pulp sheets.", "The dry content of the treated pulp sheets after the draining was 40%.", "The amount of absorbed glyoxal was 6.0%, and the aluminium sulphate hydrate was 2.0% of the sheet dry content (calculated by assuming that the percentage proportions of crosslinking chemicals in the absorbed solutions were the same as the percentage proportions of the immersion solution).", "The impregnated pulp sheet were first drained and then dried (the curing takes place when the water is evaporated) in a ventilated oven at 110 °C for 5 h.", "The sheets were set apart in an oven in order to ensure even and efficient drying and curing.", "After the drying and curing process, the dry content of the sheets was approximately 97%.", "The chemically crosslinked pulp sheets were stored at 22–23 °C in 25–35% RH.", "The crosslinked pulp sheets were manually cut into 3 × 3 cm2 pieces, which were dry-milled using a Wiley mill (Standard model no. 3, Arthur H. Thomas Co.) The pieces were manually fed into the Wiley mill where revolving knives worked against stationary knives and crushed the material until the resulting particle size was small enough to pass through a 1 mm hole screen.", "Pulp powder made from the untreated reference pulp sheets was fluffy, whereas the pulp made from a crosslinked pulp sheet was denser and showed better flow properties, enabling trouble-free further processing with a decompressed air-flow-type microniser (Ultra fine mill Ceren Miller Dau MKCL8-15J DAU, Masuko Sangyo).", "According to the manufacturer (Masuko Sangyo), the grinding effect is comparable to the effect of jet milling.", "The Wiley-milled pulp was fed once through the microniser.", "The microniser reduced the particle size by way of strong impacts caused by air flow and a rotating impeller.", "The rotation speed of the impeller was kept constant at 7600 rpm, milling time was 90 s, sample feeding time 4–6 s, flushing time was 35 s, and the screw speed was 60 rpm.", "The sample codes of prepared powders are shown in Fig. 1.", "To determine lignin and carbohydrate composition, the samples were hydrolysed using sulphuric acid (2 stages), and the resulting monosaccharides were determined using HPAEC with pulse amperometric detection (Dionex ICS 5000 equipped with CarboPac PA20 column [25].", "The acid-insoluble lignin content (Klason lignin) in the kraft pulp was determined according to the TAPPI T 222 om-02 method.", "The acid-soluble lignin was determined using the spectrophotometric method based on the absorption of ultraviolet radiation at 215 and 280 nm and using the equation described by Sarkanen and Ludwig [26].", "The ash content was determined according to ISO 1762:15.", "The amount of acid-insoluble lignin (Klason lignin) in the pulp was 1.3% and the amount of acid-soluble lignin was <0.1%.", "According to monosaccaride analysis, the carbohydrate fraction was composed of glucose (73%), xylose (25%), and mannose (0.7%) units.", "The percentage of ash (525 °C) in the pulp was 0.5%.", "The fibre-length distribution of the pulp before and after dry milling via glyoxal crosslinking was measured using a Valmet F5 Fiber Image Analyzer (Valmet, Finland) according to ISO 16065-2.", "All measurements were replicated.", "The WRV(ml/g) of the pulp before and after dry milling via glyoxal crosslinking was determined using the centrifugal method according to ISO 23714.", "All measurements were at least duplicated.", "The bulk density of the powders was determined by pouring a 5 g sample into a graduated 50 ml test tube and by measuring the volume of the powder.", "The tap density of the powders was determined by dropping (tapping) the test tube from a height of 2.5 cm onto a hard surface until no more settling of powder occurred.", "The Hausner ratio (H) was determined from the relation of tapping density ρT and bulk density ρB, (H = ρT/ρB).", "All density measurements were triplicated.", "Particle size distributions of micronised powders dispersed in water were measured using the laser light diffraction method with a Beckman Coulter LS 230 (Beckman Coulter, Inc., USA).", "For the measurement, 1 g powder samples were dispersed into 40 ml of milli-Q water with mixing for 15 min using a magnetic stirrer.", "The particle size measurements were duplicated.", "For comparison, the particle size distribution of powders in dry form was also measured using a Malvern Mastersizer 3000 (Malvern Panalytical Ltd., UK) laser diffraction particle size analyser equipped with standard sample tray and dispersion unit Aero S.", "The particle size measurements of dry powders were triplicated.", "FTIR spectroscopy was used to characterise the produced materials.", "The measurement was performed with a Thermo Scientific Nicolet iS50 FT-IR spectrometer with an ATR diamond (Thermo Scientific, USA).", "All spectra were obtained from 32 scans with a resolution of 4 cm-1 and absorption mode by using the wavelength range of 400 to 4000 cm−1.", "At least three repetitions were carried out for each specimen.", "Chemical changes in the paper caused by crosslinking with glyoxal were characterised by using a 13C cross polarisation magic angle spinning (CP-MAS) NMR spectrometer (Bruker AVANCE-III 400 MHz, Bruker BioSpin, Germany).", "For all samples, 20,000 scans were collected using an 8 kHz spinning frequency, 2-ms contact time, and a 5-s delay between pulses.", "The unstained samples were visualised using confocal laser scanning microscopy (CLSM) equipment consisting of a Zeiss LSM 710 (Zeiss, Jena, Germany) attached to a Zeiss AxioImager.Z microscope.", "Fibres of each sample were separated in water and were examined on a microscope slide as sealed preparates.", "Samples were imaged utilising a transmitted light detector with a 10× objective (Zeiss EC Epiplan-Neofluar, numerical aperture of 0.16) and a 20× objective (Zeiss EC Epiplan-Neofluar, numerical aperture of 0.30) with a resolution of 1024 × 1024 using ZEN software (Zeiss).", "Representative images were selected for publication.", "Thermal decomposition of the powders was studied using a Netzsch STA 449 F1 Jupiter thermal analyser (Netzsch-Gerätebau GmbH, Germany).", "For TGA measurements, samples of about 10 mg were analysed in air at a heating rate of 10 °C/min from 35 to 700 °C.", "Crosslinking treatments using glyoxal and glyoxal with aluminium sulphate caused a clear visible change in the brittleness of the pulp sheets (Fig. 2a).", "The crosslinked pulp sheet broke the first time it was folded into two pieces, whereas the reference pulp sheet lasted several dozen folds without breaking.", "Both paper sheets were pretreated using Wiley milling since the micronisation technique is not capable of handling large paper sheets.", "The passing time of the reference pulp (Sample B) and the crosslinked pulps (with and without aluminium sulphate, Samples C and D) in the Wiley mill, was around 45–60 s and 5–10 s, respectively.", "The crosslinked pulps milled fluently, whereas the reference pulp formed fluffy compactions in the mill (Fig. 2b).", "Thus, the processability of the crosslinked pulp from the Wiley mill was significantly better than that of the reference pulp.", "Chemically crosslinked Wiley-milled pulps (Samples C and D) were further processed with dry grinding using a decompressed air-type microniser.", "Due to fluffy character and poor flowability, the reference pulp (Sample B) was not processable with the microniser.", "FTIR measurements were undertaken to verify the crosslinking of cellulose with glyoxal with and without aluminium sulphate.", "The FTIR spectra of the reference pulp powder, Sample B, and crosslinked pulp powder, Samples C and D, are shown in Fig. 3a.", "The decrease in absorbance around 3300 1/cm (OH stretching of intramolecular H-bonding) is a likely consequence of reactions of glyoxal with cellulosic hydroxyl groups.", "An absorbance peak of 1735 1/cm may be due to unreacted glyoxal ends (CO stretching peak).", "The formation of acetal and hemiacetal linkages took place within the fingerprint area of cellulose (800–1200), and thus were not visible in the spectra (Samples C and D).", "A quantitative analysis of the amount of acetal and hemiacetal crosslinks is difficult in practice.", "Schramm and Rinderer [23] have introduced a chromatographic method that can be used of quantifying the portion of glyoxal that has reacted with cellulosic material.", "However, the method does not deliver information on whether the crosslinks were achieved by an acetal or a hemiacetal linkage.", "The FTIR spectra suggest that, with the exception of the formation of acetal and/or hemiacetal bonds, no other major modifications of the cellulose and hemicellulose had taken place in the crosslinking treatments.", "Cellulose powder samples characterised with CPMAS 13C solid state NMR (Fig. 3b) were used to study the hemiacetal and acetal formation within the crosslinking of the cellulose fibres.", "As expected, the untreated powder (Sample B) showed a signal for native cellulose with crystalline cellulose I and non-crystalline cellulose [27].", "Glyoxal crosslinking with and without glyoxal (Samples D and C) did not alter the carbon peak assignment or the crystallinity of cellulose compared to Sample B. However, a new broad peak arose at 94–98 ppm, which was identified as (hemi)acetal formation (acetal peak in 90–100 ppm).", "In a 13C NMR spectrum, hemiacetal linkages had a peak at 90–95 ppm and acetal linkages had a peak 95–100 ppm.", "Therefore, based on the measured spectra, it cannot be clearly verified that glyoxal formed with cellulose hemiacetal or acetal bonds.", "Most probably there both bond types were present after glyoxal crosslinking.", "In order to evaluate the thermal stability of crosslinked cellulose powder, thermogravimetric analysis (TGA) was performed (Fig. 4).", "The TGA curves of reference Sample B and glyoxal-crosslinked Sample C are very similar in the figure, except for a slightly higher loss of weight-% in the area of 200 °C — 300 °C.", "A TGA curve of cellulose powder crosslinked with glyoxal and aluminium sulphate differs more significantly from the reference powder in the figure.", "An examination of the reasons for the deviation was not carried out in the present study.", "From a powder potential application point of view, no remarkable difference in the thermal stability of the powders below 170 °C was found.", "This indicates that glyoxal crosslinked cellulose powder can be utilised in the same applications where regular microcrystalline cellulose is currently utilised.", "The fibre lengths of the Wiley-milled crosslinked pulp powders (Samples C and D, without and with aluminium sulphate addition, respectively) were significantly shorter than those of the reference pulp powder (Fig. 5 and Table 1).", "Optical microscopy images (Fig. 6a-d) show that the fibre cut in the Wiley mill occurred without visible fibrillation of the fibres.", "Chemical crosslinking also resulted in a significantly higher amount of fibre fines (length < 0.2 mm) in the milled pulp.", "This is evidently a consequence of the brittleness of the crosslinked fibres.", "The addition of aluminium sulphate as a catalyst in the crosslinking reaction did not have any significant effect on the properties of the fibre length properties of the Wiley-milled samples.", "WRV is an important value for cellulose powders that are utilised in water suspensions and for drug delivery.", "The results in Table 1 show that Wiley milling of the reference pulp (Samples A and B) resulted in decreased WRV, from 1.13 g/g to 0.84 g/g.", "There is very little published information on the effect of dry milling on the WRV of kraft pulps.", "The results presented in US Patent 8,449,720 B2 [28] regarding the effects of Wiley milling on the WRV of softwood kraft pulp fibres are in accordance with the results of the present study.", "According to Ekman et al., the Wiley milling of softwood kraft pulp fibres from 2.25 mm to 0.6 mm (length-weighted averages) decreases the WRV of the pulp from 1.17 g/g to 0.92 g/g.", "The reason for the decrease in WRV remains unclear, but it might have been due to the mechanical impact-induced permanent compactions of fibre walls that took place in the Wiley milling.", "The glyoxal crosslinking (Samples C and D) further decreased the WRV values to 0.6 g/g and 0.51 g/g, respectively.", "This indicates that the crosslinking solidified the cell wall of the fibre fragments and, thus, decreased their ability to absorb water.", "The bulk and tapping densities of the Wiley-milled pulps were significantly higher than those of the reference.", "The Hausner ratio of both crosslinked Wiley-milled pulps decreased.", "The Hausner ratio is generally used in industries as an indication of the flowability of a powder; the lower value indicates better flowability [29,30].", "Wiley-milled cellulose powders were further processed with the micronisation technique.", "Micronisation clearly decreased the length of fibre segments (Fig. 6e and f), and no clear fibrillation was observed.", "However, unlike microcrystalline cellulose particles the appearance of which is typically grainy (Hindi 2017), the particles of both powders (Samples E and F) still look like pieces of fibre.", "Table 2 shows the properties of the micronised crosslinked pulp powders.", "Micronising resulted in powders (Samples E and F), the medium particle size value (D50) of which were close to 40 μm.", "Particle size distributions measured using dry and wet dispersion methods showed no major deviations (Fig. 7).", "The medium particle size value of the prepared powders are comparable to many commercial microcrystalline celluloses, such as Avicel PH-101 (D50 ~50 μm).", "The use of aluminium sulphate as a crosslinking reaction catalyst did not cause any clear visible differences in the appearance of the crosslinked powder particles.", "Apparent specific surfaces of a commercial microcrystalline cellulose, such as Avicel PH 101, determined using the BET method are reported to be in the range of 1.0–1.5 m2/g [31].", "The specific surface area (BET) of powders samples E and F were 1.95 g/m2 and 1.53 g/m2, respectively.", "The lower surface area of Sample F compared to Sample E is possibly a consequence of higher density and a less porous particle structure.", "Both the bulk and tapping density of the pulp powder increased substantially during micronisation.", "Bulk and tapped densities of powders samples E and F were found to be close to those of the microcrystalline celluloses Avicell PH 101, 102, and 103 reported by Rojas et al. [32].", "However, the higher Hausner values for Samples E and F compared to the commercial Avicel products (1.31–1.40) indicate somewhat poorer flow properties.", "A clear difference between the crosslinked pulp powders (Samples E and F) and the microcrystalline cellulose Avicel PH 101 is their dispersability in water.", "Vigorous mixing of the Avicel in water (e.g., 10 wt% in water with Ultra Turrex using 12,000 rpm for 10 min) results in stable milky dispersions, whereas crosslinked powders start to sediment immediately after the mixing.", "The reason for the different behaviour of the Avicel and the crosslinked powder was not examined in the present study, but this behaviour may be related to the firmness and chemical composition of the crosslinked particles.", "Further research should examine the effects of different cellulose crosslinking agents, such as tricarboxylic acids, and different crosslinking conditions on the processability of cellulosic materials in dry milling and on the properties of the dry-milled kraft pulp powders.", "The present study examined dry milling of cellulose fibres into micro-size cellulose powder.", "A chemical crosslinking treatment of bleached birch kraft pulp sheets made the birch sheets more brittle and subsequently enhanced dry milling.", "The increased brittleness of the sheets resulted in a significantly shorter passing time for the dry pulps in the Wiley mill and a shorter average fibre length in the resultant powder.", "Crosslinked Wiley-milled pulp powder also showed higher density and better flow properties, thus enabling the micronisation of this powder with a decompressed airflow-type microniser.", "After fine grinding, the medium particle size value (D50) was around 40 μm.", "These findings strongly indicate that the hypothesis of the present study is correct.", "In addition, both FTIR and NMR spectrum analyses suggest that glyoxal with and without a catalyst (aluminium sulphate) formed hemiacetal and/or acetal linkages in the treated birch kraft pulp sheets.", "Unfortunately, it is not possible to estimate the proportions of hemiacetal- and acetal linkages based on chemical analysis.", "Unlike some commercial microcrystalline celluloses, which have comparable particle size distribution, the crosslinked fine powders do not form stable dispersions when mixed vigorously in water.", "Therefore, the potential industrial uses for crosslinked kraft pulp powders are not necessarily the same as those for MCCs, but the properties of the powders may enable their use as a filler, for example, in plastics and paper products.", "The lower water absorbency of the powders compared to non-crosslinked cellulose powders may be an advantage in those industrial applications.", "Both authors contributed equally to the manuscript, and both authors have given their approval to the final version of the manuscript." ]	[ "Novel method to produce microsize cellulose powder is studied.", "Yield of the dry milling method is 100%.", "Glyoxal crosslinking harder cellulose materials, making it dry milling in room condition possible." ]
Rising to the medication's requirements: The experience of elderly cancer patients receiving palliative chemotherapy in the elective oncogeriatrics field	A new subfield of oncology has emerged in the last twenty years to raise awareness and address the specific needs of elderly cancer patients, a population that was long neglected in oncology. We sought to understand the individual experiences, as well as moral and social implications of considering elderly cancer patients as “treatable”. Following an anthropological critical interpretative approach focusing on practical and symbolic effects of chemotherapy in a rapidly evolving medical field, we conducted 20 semi-structured interviews and observations of medicine storage places at home among elderly cancer patients aged 70 and over in a clearly incurable situation receiving palliative chemotherapy. We used photographs representing paths as triggers in interviews, and compared the patients' views with those of 12 health professionals in oncology during a brief open-ended interview. Elderly cancer patients consider themselves to be survivors and fighters. Their long trajectory is a result of their successful struggle and tolerance of the treatments allowing them to carry on. They continually observe their physical ability and test their resistance, they resist complaining and are grateful to have cancer at a late stage of life. By highlighting their active life rather than the treatment inconveniences, they show they are “young elderly” persons, capable of keeping active physically. They are treated precisely because they demonstrated that they had the physical and moral capacity to take the hit of the chemotherapy to their bodies and had the will to fight. The development of oncogeriatrics has enabled the treatment of the fittest cancer patients over 70, but the ethical debate to treat some elderly patients and not others, and decisions of therapeutic abstention facing frail elderly cancer patients remains an issue rarely discussed. This aspect should not be eluded by the important progress achieved in medicine facing cancer.	[ "ARTS", "SOCI" ]	[ "Chemotherapy", "Decisions", "Elderly", "End of life", "Experience", "Medicines", "Oncogeriatrics", "Palliative", "Switzerland" ]	[ "1207", "3306" ]	[ "As one of the contemporary symbols of severe illness (Sontag, 1983), cancer is considered one of the most feared and unfair illnesses, striking the population indiscriminately, from children to the elderly, with still insufficient understanding of its logic and its causes.", "Perhaps only Alzheimer's disease, with the dreaded loss of memory it causes concomitant with old age, raises similar fears in the collective imagination.", "It is unsurprising then that oncology research is among the most innovative domains in modern medicine, with new medicines and treatments regularly entering the market.", "The 20th century cancer treatment “trio” - surgery, radiation and chemotherapy - is still improving, and scientific breakthroughs are promising: genetic sequencing, targeted and thus less harmful therapies, and immunotherapy, i.e., restoring the immune system response against cancer cells.", "Those important advances have increased the cure rate of many cancers and lengthened survival time for many more (see “Closing in on cancer”, The Economist, 2017).", "With such progress achieved, cancer is increasingly considered among professional caregivers as a chronic condition, with the key notion of “remission” testifying to this change (Ménoret, 2007).", "Nonetheless, survival rates are significantly different between developed and developing countries, and social disparities in access to cancer care in many countries are striking, e.g., in the U.S., where there are important variations in survival rates among patients from different social and cultural backgrounds.", "As Freeman (2004) concludes, there is a disconnect between what we discover and what we deliver in cancer care.", "Good access to care, prevention and early detection of cancer remain salient issues to better tackle cancer and identify underdiagnosed and undertreated persons and groups (Ward et al., 2004).", "Although the size of the gap differs across countries, discrimination due to age is widespread throughout most countries of the world with similar conditions.", "For decades, treating elderly cancer patients older than 70 years was not an option, and this population remained overlooked.", "However, with recent oncology developments, cancer care specialists have been increasingly focusing on survivorship and long-term cancer situations as well as elderly patients.", "These are new areas of interest in cancer care, together with the ability to stay at home as long as possible, even in advanced palliative situations.", "This article focuses on the experience of those patients who are now treated despite their advanced age and the incurable nature of their cancer, as well as the moral and social implications of considering elderly cancer patients as “treatable”.", "We now review two major changes, which led to considering the elderly patient in oncology: the rise of oncogeriatrics, and the reshaping of the limits between cure and care.", "Together with the new therapeutic possibilities for treating cancer patients more safely and for longer, with an increase in patients' lifespan, a new subfield of oncology has emerged in the last twenty years focused on the treatment of elderly cancer patients, a population that was long neglected in oncology.", "Though it used to be commonly accepted to manage cancer up to the age of seventy (Extermann and Balducci, 2003), elderly patients were often excluded from cancer treatment programs.", "The first reason was because of their frailty, since they often experience several comorbidities that weaken them and reduce their chances of being able to receive chemotherapies (Ogle et al., 2000; Satariano and Silliman, 2003; Williams et al., 2016).", "Second, social impediments, such as low income and/or absence of relatives, have also made it difficult to consider elderly patients as able to go to a hospital to receive their treatments and pay for them, even in developed countries (Goodwin et al., 1993).", "Finally, social representations of the elderly, in which death is considered “in the nature of things” for them (Loffeier et al., 2017), do not facilitate care of elderly patients.", "In general, the elderly do not represent a priority target in curative medicine, since their remaining lifespan is limited.", "However, the mobilization of some physicians and medical researchers at clinical and political levels has shed light on the specific question of cancer treatment for the elderly.", "In the 1980s, doctors began raising awareness in the medical oncology community of the necessity of addressing the specific needs of the elderly, emphasizing the growing importance of old age and the increasing demographic ageing of western societies.", "Rosemary Yancik, an epidemiologist at the National Cancer Institute (USA), who is considered one of the pioneer figures in oncogeriatrics, edited in 1983, “Perspectives on Prevention and Treatment of Cancer in the Elderly”, a book that broadly proposes a clinical research program able to face this major demographic challenge.", "The first clinical observations focusing on elderly cancer patients started in the 1970s (Butler and Gastel, 1979) and were continued throughout the 1980s.", "Those studies showed that toxicity-related issues and complications due to cancer treatments differ in the elderly (Begg and Carbone, 1983) and that chemotherapy must thus be adapted to this population and selected carefully (Balducci et al., 1988).", "Several studies showed that elderly patients are given chemotherapy treatments less often than younger patients (Samet et al., 1986).", "Articles in the new field of oncogeriatrics were progressively superseded by more politically engaged papers denouncing forms of unequal treatment towards elderly patients.", "One article published in 1990 entitled, “Cancer in the elderly: why so badly treated?”", "(Fentiman et al., 1990), condemns the lack of consideration for elderly patients' points of view regarding their choice to be treated considering their life expectancy and quality of life.", "It also notes that elderly patients are often excluded from clinical research mainly due to strict restrictions on comorbidities and the requirement of standard lab values in trials.", "This led to a serious lack of knowledge and clinical data regarding elderly cancer patients (Epstein, 2007).", "Calls for mobilization increased.", "For example, BJ Kennedy, the president of the American Society of Clinical Oncology, in the opening speech of the 1988 congress, encouraged the rapid development of oncogeriatrics.", "In the year 2000, the International Society for Geriatric Oncology (SIOG) was founded to improve the care of older cancer patients.", "Beginning in 2005, it organized an annual meeting.", "It also publishes the Journal of Geriatric Oncology, regularly issued since 2010 with thematic taskforces writing position papers on several topics to attract the attention of the medical community.", "In the medical literature on the topic, statistics are often quoted to emphasize that cancer is one of the principal causes of mortality in the elderly and that half of all cancers are diagnosed after the age of 65 years.", "A common statement, quoted in one paper that has since become a powerful line, suggests, “If you're not a pediatric oncologist, you're a geriatric oncologist” (Lichtman et al., 2007, p.1821).", "However, the different mobilizations and associations created in oncogeriatrics did not necessarily aim at creating a new medical discipline per se.", "Rather, they sought a global mobilization of medical oncology around elderly patients, making them a central point of the future development of the discipline.", "Until the interwar period, therapeutic innovation in oncology was exclusively aimed at “curing” patients.", "It gave priority to patients who could heal and the early detection of cancers (Pinell, 2000).", "In the 1970s, the emergence of the palliative care movement and later of palliative medicine, a discipline dedicated to “caring” for incurable patients, delimited different regimes of medicine around cancer.", "On one side, oncology attempted to make live (Memmi and Taïeb, 2009) and help patients survive, and on the other side, palliative care aimed to let die and provide better quality of life for patients through therapeutic abstention and allowing them a time to die (Clark, 2007).", "Nonetheless, the important therapeutic innovations in oncology in the last forty years have affected cancer trajectory patterns drastically.", "Declaring “there's nothing else to do” in cancer care takes place much later than it did before (Muller and Koenig, 1988).", "The phase of metastasis apparition – signaling a generalized cancer and most often entering the incurable phase – can now be better controlled and extended.", "While cancer trajectories used to be characterized by a slow decline towards death, they are increasingly taking on the shape of a long plateau phase with a rapid decline at the end, shortly before death (Lynn and Adamson, 2003), enabling patients to stay at home until a few days before dying.", "If there used to be a clear demarcation between the cure and care of cancer and a “passage” from one to the other, often represented by the transition from oncology to palliative care, such limits are now rather blurred, and the palliative phase has become complex, with increasing overlap between cure and care.", "Now, one can distinguish two palliative stages in cancer care, well described by French sociologist Isabelle Baszanger (2002):", "a “still therapeutical oncological palliative phase”, which corresponds to situations where the ill person is incurable but can benefit from anticancer treatments to slow the growth of the damage caused by the illness, thus possibly lengthening life for months and sometimes years while ameliorating quality of life and controlling the symptoms of an advanced cancer, and", "an “exclusive palliative phase” referring to the interruption of anticancer treatments with an emphasis on symptom and pain control, i.e., what palliative care and hospices traditionally offer.", "This is an important turn for oncology, namely, actually treating an increasing number of people in advanced stages of cancer who are often incurable and thus in oncological palliative phases, many of whom are older than 70 years.", "In this study, we focus on those elderly cancer patients who receive what can be considered palliative chemotherapies aimed at slowing the growth of cancer rather than curing it.", "Those patients were typically in a “still therapeutical oncological palliative phase” as defined above, progressing after at least one initial palliative systemic treatment.", "In medical anthropology, medicines are considered objects with pharmacological efficacy as well as objects invested with meaning by those who conceive them, prescribe them, deliver them or receive them (Van der Geest and Reynolds Whyte, 2003).", "Medicines are important symbolic mediators to oneself, others and the world.", "They can represent important landmarks in illness trajectories of persons experiencing uncertainty in the liminal state of having cancer (Little et al., 1998; Foley, 2016).", "Domestic pharmacies and private spaces are analyzers of the person's way of relating to the illness (Fainzang, 2003).", "This interpretative approach can be combined with a critical perspective: the first approach considers the meaning individuals give to their illness, as well as their representations, subjective experiences and practices.", "The second approach explores contextual and institutional aspects, which influence individuals' narratives (Good, 1993) and regulate their representations and governance of their body (Fassin and Memmi, 2004).", "Although the starting point for our studies is the individual and his/her point of view expressed during an in-depth interview and observations at home, we link those interpretative elements to a critical angle examining the institutional transformations affecting individual experiences and daily life.", "Considered as the “third way” in medical anthropology (Massé, 2007), this critically interpretative approach proved to be well adapted to understand subjective experience in a rapidly evolving professional context—here, the medical field of cancer care and sub-field of oncogeriatrics.", "We conducted a semi-structured interview and observations of medicine storage places among elderly metastatic cancer patients aged 70 and over in a clearly incurable situation receiving oral or intravenous systemic therapy after at least one systemic palliative treatment.", "The patients selected were all treated by the oncology service of a French-speaking public hospital in an alpine region of Switzerland (Sion, Wallis).", "This service, whose chief physician (SA) is on the research team, occupies 3 sites (Sion, Martigny and Sierre) and cares for approx. 1700 patients a year.", "After Japan, Switzerland has the highest life expectancy (81 years for men, 83 for women).", "Cancer care in Switzerland is provided by GPs at their office and by private or public hospitals, based on principles of free demand and supply.", "The Swiss Federal Law on Compulsory Health Care (LAMal) instituted a mandatory healthcare insurance for all persons living in Switzerland.", "The Law ensures that everyone has access to high quality, comprehensive health care.", "It offers the same range of services and benefits to all insured people, with no limitation based on age.", "Surgery, radiotherapy and systemic anticancer treatment, like the vast majority of services, are reimbursed on a fee-for-service system; nevertheless, travel costs are not refunded.", "The chief physician on the team made an initial screening of eligible patients, and two researchers in medical anthropology (RAF) and sociology (AA) contacted the identified patients to ask them to take part in an interview, which was conducted face-to-face between May 2014 and February 2015.", "The selection was more difficult than expected because of our specific criteria (being over 70 years old, having had at least one previous systemic treatment in the palliative stage, and still being treated, i.e., not being in a terminal phase).", "Recruitment was prolonged to obtain the purposive sample of 20 patients, which reflected a sufficient variety of situations in terms of cancer type (colon, uterus, breast, prostate, pancreas, ovaries, gastric, bladder, kidneys) gender, length of trajectory and age.", "We proceeded by conducting semi-structured interviews ranging from 1 h to an hour and a half.", "We used a photographic “trigger” representing the metaphor of a path.", "At the beginning of each interview, we showed patients 4 photographs of standard paths corresponding to the hypotheses we had of one long trajectory, one risky and uncertain path, one crossroad signaling choices and a “protected” tunnel hinting to guidance received from health professionals (see appendix).", "The choice and selection of those photographs is described elsewhere (Anchisi et al., 2018), showing that our hypotheses on illness trajectories were constructed in an ongoing iterative process, emerging from previous research results on elderly cancer patients (Anchisi et al., 2006, 2012; Foley et al., 2015).", "We asked the elderly cancer patients included in this study, which one - if any of them - best represented their illness trajectory since they were diagnosed with cancer.", "We considered the photographs as a useful medium and a trigger (can opener) in qualitative research (Collier, 1986; Maresca and Meyer, 2013), giving access to patients' own trajectory indirectly through the metaphor of the path and helping them view it as a whole, linked to past stages as well as to steps still to come.", "The interview questions sought to capture the different phases of the patients' biographical and illness trajectory (life before illness, cancer diagnosis, major events and stages since, what is still to come, the lived experience of old age), while the observations sought to describe the spatial and temporal organizations of chemotherapy.", "Twenty patients were met at home: 9 women and 11 men (ranging from 70 to 86 years of age, median age 75 years).", "The persons in our purposive sample had been in a palliative situation for 2 years on average.", "Some had cancer for over 12 years, whereas others were diagnosed with cancer only 1 year prior to the interview.", "Eleven of them were incurable upon diagnosis, while 9 of them passed through a substantial curative phase before relapsing and becoming incurable.", "During the recruitment phase, 3 persons refused to participate, 2 persons for linguistic reasons (they were German speakers).", "By the end of the study, 5 of the 20 persons interviewed had died of cancer (see Table 1).", "In a complementary part of this study, we showed the same 4 photographs of paths to 12 health professionals in the oncology ward (7 nurses, 5 oncologists).", "We asked them, in a brief open-ended 15–25 min interview, to choose the path that best corresponded, in their opinion, to the trajectory of the elderly incurable cancer patients they encountered in the oncology ward.", "They were invited to tell us the key words that came to mind, which we reported manually and then coded (see Table 2).", "The research project was approved by the clinical research ethics board of the canton of Valais, where the study was carried out.", "The contents of all 20 interviews and observations were audio recorded and fully transcribed, as well as field notes.", "The research team conducted a preliminary analysis by discussing and identifying emerging themes in an ongoing process during data collection.", "Once all interviews and observations were complete, a computer-assisted analysis was carried out.", "Interviews and observations were coded separately by one researcher, and cross-checked by the other medical anthropologist using the software Atlas.ti (see list of themes and codes in Table 2).", "Following an iterative and flexible process (Beaud and Weber, 1997), key themes appearing throughout the interviews and observations were identified, compared and discussed.", "The medical doctor on the team only accessed the anonymized data at this stage of the analysis.", "The semantic analysis conducted on the basis of the same photographs shown to health professionals in the oncology ward, was detailed in Table 2 and discussed elsewhere (Anchisi et al., 2018).", "It showed striking differences in perceptions and was used in this article as a point of comparison with the elderly cancer patients' discourses on the question of the stages of illness (curative and palliative) and the notion of having the choice (or not) to accept treatment.", "A first set of results shows that the experience of cancer in the elderly is a long moral and physical fight, in many ways similar to the experience of younger cancer patients taking chemotherapy.", "The second set of results, however, hints toward particular constraints and the fragile situation of battling against cancer while being old.", "The third set of results reveals the social load of being elderly and the moral obligation not to complain about having cancer at a late stage in life.", "When they were questioned about the path they followed since they fell ill and its major stages, a majority of patients (17/20) chose the long and winding uphill road to describe their experience without ever referring to it using words such as “curative” or “palliative”, though several of them spoke spontaneously of the moment when the metastases were found.", "Those results contrast with health professionals' views: several of them (6/12) believed the elderly incurable patients' they encountered were following a “palliative”, “flat”, “calm” and “peaceful” path.", "The elderly patients emphasized going beyond the bleak prognosis they had been given that predicted a short life expectancy.", "Most of them feel that they have stood firm and are amazed that they are still alive after several months or years.", "This sentiment was expressed by most persons interviewed, including Catherine Bulliet (fictive name), a 73-year-old woman with kidney cancer:", "“I was operated 7 years ago, in October, and I had been told I'd live until February the next year, so sometimes I annoy the Doctor a bit and tell him, “you see, I'm still here” (laughter)”.", "Everything experienced in daily life takes a positive flavor and is a surprise, as expressed by Mr. René Meyer:", "“It's rare that I am worried, it's annoying because I love life, I would like to keep on going as long as possible.", "With my wife we have many activities, we travel, we take pictures (…), now I'm part of the committee of a photographic association … I have a good appetite, I love life, I love traveling, I still do traditional photographs, I'm a dinosaur.”", "This type of discourse unveils a positive and active way of living with advanced cancer while living at home.", "Mr. Jean Fabri, 72 years old with cancer of the rectum, reflects that his path has remained in many ways the same as before he had cancer:", "“It's a long path but not a hard one.", "Yes, because I told you I was able to do all my activities as I used to, for example, last year I did about 40 days of skiing, you see?”", "Having the same activities as in the past is reassuring, and living life as before is strongly linked to the ability to battle against the medication.", "The patients' road is broad if and as long as they can receive treatment.", "Although the interviewed elderly patients also had to learn to live with painful technical interventions, such as ostomy pouches and port-a-caths, most felt they were facing a silent illness.", "Even when they could feel their tumor (for example, when bending down), most of them did not feel pain.", "Its progression was thus difficult to detect, whereas they directly felt the effects of chemotherapy:", "“First I have to tell you I have never felt ill.", "I have the illness but I never felt my illness, I don't know what it’s like.", "I never suffered from it.", "But I had trouble afterwards with the chemotherapies.", "That yes.”", "(Mr. Jean Fabri, 72 years, cancer of the rectum).", "The representation of their cancer is of an invisible illness that “spreads”, “burns” or “infects” the body or “flourishes as in a vegetable garden”, as two elderly persons put it.", "The elderly patients made no distinction in potency or strength between curative and palliative chemotherapy, whereas the oncologists viewed palliative chemotherapy as an overall less aggressive treatment.", "Chemotherapy, regardless of type, is represented in the patients' discourses as unequivocally the worst, strongest and most toxic medicine, in contrast to hormonotherapy, x-rays, or nuclear medicine, which they refer to as lighter.", "Starting chemotherapy was often associated with moving to a “heavy” stage of the illness.", "Some of the health professionals' discourses reinforce such representations—that the way is wearisome with chemotherapy—as Mrs. Anne Rubin (78 years old, with bladder cancer), explains:", "“The surgeon told me, ‘you're embarking upon the way of the cross'.", "That was the hardest time because I had to accept chemo, I had to accept all those painful treatments, I didn't know where I was going, I didn't know much.", "From the patients' perspective, their path has been a fight against the treatments—to tolerate them, one after the other.", "These representations correspond to how anthropologists and historians describe chemotherapy, i.e., medicines as harmful as the disease itself (Pouchelle, 2002), killing the degenerative cells in the living body (Peter, 2004).", "Looking back, the attempts to find “the right treatment” with the least side effects were important stages.", "Mrs. Simone Lener, a 70-year-old with uterine cancer says significantly:", "“You have to bounce back; it doesn't happen right away, it's one treatment after the other”.", "Some of them thought they were going to die from the major side effects they experienced.", "Mrs. Madeleine Ambiel, 81 years old with metastatic colonic cancer, explains:", "“Now I'm fine, but first they gave me such a strong chemo, I said, I'm going to die, I had no strength whatsoever”.", "Mrs. Catherine Bulliet reflects on the rhythm of taking and pausing the medication and the fact that the medications made her ill:", "“The hard times of that medicine, when I paused the treatment, I had a two-week break, so that was fantastic, I put on weight again and when I started being in good form I had to start it all again” (Mrs. Catherine Bulliet, 73 years, metastatic kidney cancer).", "Some regretted having to interrupt a treatment because their body could not take it:", "“If I had been able to eat well, I think I would have tolerated the Sutent … It's a very good treatment I think, it's a pity I had to stop it” (Mrs. Catherine Bulliet, 73 years, metastatic kidney cancer).", "The fight against the medicine is linked to their body's ability to tolerate the medicine.", "This is at the forefront rather than the fight against the illness itself.", "As shown in studies conducted among younger patients, the treatment makes them ill and leaves social signs and stigmata, whereas the illness itself is invisible (Sarradon-Eck and Pellegrini, 2012).", "Cancer is incomprehensible and escapes the patients' understanding.", "In this study, however, not only was the discovery of the illness silent, but its aggravation was hardly ever felt in the body.", "Most of the persons interviewed delegate knowledge about their illness to health professionals.", "The latter are generally highly esteemed.", "Patients express gratitude for the “excellent doctors” who “listen to you” and “understand you”, even if the most critical admit, as Mrs. Anne Rubin (78 years, bladder cancer) does, that facing doctors, “we are very docile”.", "They have little control over the choice of the treatments they are administered or over the illness itself, which they cannot act on other than by receiving chemotherapy.", "In the absence of knowledge about the illness and its evolution, the patients share a common point of reference: waiting for the blood tests results before receiving chemotherapy.", "This is a crucial event for most of the elderly patients in the sample.", "The platelet or leucocyte levels determine whether the patient will be able to continue receiving chemotherapy and be treated.", "Through those tests, the ill persons have an indication as to whether their body can tolerate the treatment or not during this “follow-up”, which is circumscribed and can be reassuring.", "If the “platelets are good”, this means to the patients that the illness has not invaded the whole body, whereas in the doctors' eyes, this event is not as revealing regarding the progression of the illness as patients assume it to be.", "The extent to which the illness has progressed must be evaluated through other means, such as laboratory tests and scans.", "However, the elderly patients interviewed believe that the primary reason the doctor changes their medication is not progression of their illness but the fact they did not tolerate the previous medication.", "While most health professionals in the oncology ward held the viewpoint that elderly cancer patients in a palliative stage could choose to receive their chemotherapy, not one elderly cancer patient in our sample selected the photograph of a crossroad, signaling their choice to continue taking their medication, whereas 4 health professionals chose that photograph (see Table 2).", "The patients' concern is rather that they are put to the test of climbing a long, steep road.", "The proof they can withstand the treatment is found in the performance of their daily activities and in the maintenance of a healthy lifestyle, as well as in mental resilience.", "The central self-representation of being a “fighter” is highly valorized.", "For example, Mrs. Christine Bellon declared, despite experiencing acute pain due to polyarthritis, that she has mental strength she had underestimated:", "“I must say I took things better than (…) I was more solid than I thought I was.", "I thought to myself “it's over, at Christmas, it's over”, but straight after I reacted, I thought “in any case he [the cancer] isn't the one ruling the world and we'll see what happens, and I'm going to fight (…) Is it worth it to fight, I think that if we have inner strength that helps us, then we have to use it, it's no point ruminating on the illness” (Mrs Christine Bellon, 70 years old, with peritoneal carcinosis).", "Mr. Hans Pistolleti, a 75-year-old with urethral cancer, notes the importance of mental strength in fighting against illness and depression:", "“So then I strengthened my head, my brain, with the idea to fight”.", "The identity as a fighter is clearly linked to a past good health status and for many of the patients corresponds to a way of living in general and of enduring adversity.", "Some describe a current “healthy” old age with few comorbidities, despite their incurable cancer.", "They describe two different areas of health: a general & a cancer state of health.", "The idea that they should conceive of their general health situation as independent of their cancer situation is a conception professionals share and encourage.", "Mrs. Denise Sola, a 72-year-old with pancreatic adenocarcinoma, assesses her situation:", "“Because otherwise I have a very good health, I don't have diabetes, I don't have hypertension, I don't have cholesterol, things like that, my health is rather good.", "So the oncologist said “we're going to battle because you are in good health”, I said “yes, apart from that (laughter).", "So I'm lucky because it's true I was never really sick in my entire life, that's it finally”.", "From an oncological point of view, the patients are in an “in-between” situation: they responded badly or transiently to a prior treatment but have a reasonably good general condition.", "This distinction was clearly observed in the domestic pharmacies of elderly cancer patients in their homes: cancer-associated treatments were always stored in spaces separate from other medications, such as those prescribed for age-associated problems (hypertension, diabetes, heart failure, arthritis).", "Those results are consistent with previous results found among elderly cancer patients taking oral chemotherapy (Anchisi et al., 2012; Foley et al., 2013).", "Although often considered by elderly cancer patients as “poisoned candy”, which showed that cancer could be considered as involving “lighter” and simpler care at home, the medication invaded their domestic space and interfered with their daily family and social life as they tried to maintain hope in the face of death.", "Ethnographic observations in our previous research showed that places in the house where the chemotherapy are stored were different from others.", "They were highly symbolic e.g., in the cellar to distance the medicine from the household, or with their strong alcohol because it is toxic and must not be found by children.", "Still others kept the medicine in the centre of the house among family photographs and altars, to help them see and remember it.", "In this study, we found similar symbolic references stretching to cancer-associated medicines, objects and supplies, such as those needed for ostomy pouches, which were stored in private bathrooms, whereas more common cancer associated medicines were kept in the kitchen for immediate access and practical reasons.", "Most interviewed persons adopt a stance that they have “to move”, keep busy and eat well.", "If they stop, if they stay on their couch, then they are doomed.", "For many, activating one's body can be achieved through sport (hiking, skiing, going to the gym, swimming).", "This stance is supported by the Leagues against cancer, and the notion of keeping hope and spirits up even at an advanced stage of cancer is a central value in oncology (Del Vecchio Good et al., 1990; Saillant, 1990; McMullin and Weiner, 2009; Soum-Pouyalet et al., 2009).", "Mr. Pierre Varga, 80 years old with gastric cancer, did not change much in his daily life:", "“I can still do everything, I can work in the garden, I can do some DIY, I look after the trees a bit, I spend some time everywhere, with really no problem”.", "Moving is about maintaining the same life rhythm, as Mr. Marc Arnold, a 78-year-old with prostate adenocarcinoma who goes to the gym, states:", "“As long as I can do everything as usual, it means it's all fine”.", "Mr. Raymond Imfeld, 72 years old with prostate carcinoma, has noticed changes while walking up his stairs.", "Mr. Jean Fabri goes mountain hiking and observes his breathing and fatigue:", "“So I did some tests while walking up hill”.", "These activities allow them to observe their physical ability and see if the illness has weakened the body much.", "The positive discourses are often strongly linked to the fact of being old, which seems to help participants see cancer as less severe, since their old age also generates frailty and ultimately leads to death.", "They had lived a long life before they were diagnosed with cancer, and being ill is part of old age.", "Furthermore, most of the elderly cancer patients interviewed were not particularly anxious about dying.", "Death was on the horizon whether cancer was part of the picture or not.", "Mr. Pierre Varga explains that cancer is only one of the plausible causes of death he has to face: “At my age, from one day to another I can have a heart attack, I won't necessarily die of cancer, we all agree upon that.", "I take things as they come (…)”", "For others who were interviewed, being old helped them relativize the diagnosis:", "On this matter, Mrs. Catherine Bulliet (73 years, metastatic kidney cancer) says, “But you know, they told me that and it didn't hit me at all (…) I phoned my husband and said, I have cancer, but because of the age I was, I thought to myself “too bad” and that's all.", "Even now, I'm not scared, the other day I even told the doctor “I don't have cancer”.", "Some of the interviewed persons said they were grateful that cancer fell on them because they were “taking the load” from younger persons.", "They have integrated a representation of cancer as a collective scourge that can hit anybody, from children to active and finally retired persons.", "Since it must happen to someone, it is rather a good thing, in their opinion, that it happened to someone as old as them who had been lucky to live a long life as a “healthy person” before being diagnosed with cancer.", "In some ways, they believe it is more of a shame when cancer hits younger people, as their bodies are less “worn out”.", "Several of the persons interviewed explained they were deeply moved when they met younger cancer patients in the oncology ward, as if their situation was different than that of the younger patients.", "For example, Mrs. Christine Bellon (70 years old, with peritoneal carcinosis) said,", "“You know, seeing the others who are younger really shocks me”.", "Being old when having cancer thus seems to be a lesser evil and a way of generating social fairness in the face of this unjust illness.", "Nevertheless, the interviewees' discourses can also reveal how much they have internalized negative representations of old age, i.e., that, in a way, their life counts for less than that of a younger patient.", "The participants believe that their survivor's trajectory has much to do with their positive attitude.", "The recurrence of affirmations such as “one has to battle”, “we have to keep our spirits up”, and “I can't complain”, could however, hint towards some weight on their shoulders, pressure to put on a good face and battle on despite their situation.", "Moreover, the fact that it seems to them a rather good outcome to have cancer in their old age seems to add to the importance to remain positive.", "The elderly patients with advanced cancer we interviewed rarely complained about their situation or expressed their suffering.", "The interview mainly served to strengthen the building of a discourse about one's body and oneself as a fighter, an identity highly valued by them.", "If their illness was silent from the start, its progression was unfelt too.", "The terms “curative” or “palliative” are not useful labels to them.", "These patients are still fighting and are susceptible to the discourse shared among oncology health professionals that a strong active body better fights against cancer.", "The battle to remain treated seems in many ways similar to the hard work younger cancer and chronic patients have to face (May, 2006), with symptoms they feel are predominantly due to chemotherapy and not to their tumors.", "Cancer care is the area of medicine in which military metaphors are most used: cancer is the enemy, you have to fight against it, and chemotherapy is a powerful weapon to do that (Del Vecchio Good et al., 1990; Saillant, 1990; Garrisson, 2007; McMullin and Weiner, 2009; Slobod and Fuks, 2012).", "The history of chemotherapy discovery dates to World War I, when nitrogen gas, used to poison the enemy, was discovered to interrupt white cell reproduction.", "Chemotherapy thus originated as a deadly weapon and encapsulates the military metaphor.", "As raised by Slobod & Fuks, “when medicine is seen as war, patients may believe that becoming healthy is only a matter of “fighting” hard enough.", "But, who loses when disease “triumphs”?”", "(2012, p. 144).", "The risk is that patients feel strongly responsible for their situation and feel the need to endure the treatment no matter the cost.", "In this study, elderly patients have widely internalized moral attitudes and behaviors about staying healthy and having a fighter's attitude in their daily activities.", "As far as elderly cancer patients are concerned, it is interesting to see that the image of being a “fighter” and the need to battle on are in contrast to sociological theories of “déprise” being characteristic of old age trajectories (Caradec, 2007; Membrado and Salord, 2009).", "The elderly cancer patients never mention this progressive letting go of the environing world often experienced by elderly persons.", "Globally, it seems that the cancer experience takes over the old age experience.", "Remaining active to fight against the illness through their treatment seems to overshadow the ideas of decline and of growing old.", "We can assume that this lived experience of advanced cancer, very anchored in the present time and involving an active body, and described in a homogenous way by most of the interviewed elderly persons, has to do with the convergence of old age and chemotherapy.", "Patients tend not feel old age as strongly because they are too busy fighting to tolerate their medications.", "However, it seems crucial to put this assertion in perspective by noting the pressure the elderly cancer patients seem to feel of having to be active and live as younger persons.", "The fact that these patients correspond to a portion of elderly cancer patients who have been selected to receive chemotherapy treatment despite their advanced age and incurable situation must be borne in mind.", "By focusing on palliative cancer situations of persons above 70 years of age, we grew aware that these patients represented a still rare field of oncology practice and are, in some ways, the oncogeriatrics “champions”.", "Their homogenous discourses led us to reflect on what characterizes elderly cancer “treated” patients.", "As reported by some of the patients interviewed and then confirmed by the oncologists in the ward, one of them being in our interdisciplinary research team, the patients treated with palliative chemotherapy have a general good health status with little comorbidities and are treated precisely because they demonstrated that they had the physical and moral capacity, i.e, the will to fight, to take the hit of the chemotherapy treatment to their bodies despite incurability.", "In oncology it is not chronological age that matters but physiological age—the way age has altered the body of each individual.", "In a way, cancer care, by treating elderly persons, is more and more moving to patient-centered care within an interdisciplinary network of professionals, promoting self-empowerment and patient reported outcomes.", "A qualitative study conducted in a French pilot unit of collaborating oncology and geriatrics specialists (Sifer-Rivière et al., 2010; Sifer-Rivière et al., 2011), the creation of which was promoted by successive public health plans (Maraninchi, 2009), shows how geriatric assessments led by collaborating oncology and geriatrics specialists repartition patients according to their level of vulnerability.", "The geriatricians are invested with the responsibility of demonstrating that patients are not too old to be treated.", "On the other hand, the oncologists are concerned with the discourse on equal rights to cancer care access and look after patients qualified as only elderly, i.e., capable of being treated for cancer like younger cancer patients because of their low vulnerability.", "Nevertheless, oncologists tend to defer to the geriatrics specialists and consider that cancer treatment is irrelevant for patients who present strong dependency and impaired cognitive functions.", "If the gained life expectancy with the treatment implies reduced quality of life or more reduced autonomy, then chemotherapy can be undesirable (Fried et al., 2002).", "As shown by Kentish-Barnes (2007), the confrontation of medical progress with moral imperatives place the physician in front of the choice to know what is technically possible to do and what is humanly reasonable to decide.", "Interestingly, if the health professionals in oncology present palliative chemotherapy as a choice to the elderly cancer patients, the elderly patients mostly feel they have to accept the opportunity given to them.", "They have conform attitudes and are grateful to receive treatment despite their age.", "In current medicine, it seems the choice to be treated is increasingly handed off to the patients and their relatives (Mol, 2008), and in this case, the responsibility to choose falls on the elderly patient's body, which will be able or will not be able to rise to the medication's requirements.", "Although it is seldom discussed in the oncology literature, the decision of therapeutic abstention facing frail elderly cancer patients is an important issue.", "The development of oncogeriatrics has enabled the treatment of the fittest cancer patients over 70, but the ethical debate of when to stop and enter an “exclusive palliative phase” results in crucial decision making facing many other patients.", "The moral reasoning at stake, justified by the idea of respecting the natural course of old age while considering the quality of life the treatment will enable, is close to that found in other medical domains where the limits of life and death and medical decisions are important, such as in intensive care (Timmermans, 1998) and general medicine (Muller, 1992).", "Through resuscitative efforts and the use of reanimation codes in patients' files, health professionals select which patients are given full “curative” treatment and which are not.", "Such studies show that there is a strong risk of inequality in death for those with low social value, among whom elderly patients are an important group.", "Although there are more and more social actors who take a position on life and death decisions (assisted suicide and euthanasia movements, organ donors associations, patient organizations), our societies have few answers to such questions of arbitrary reasoning around the limits of life-sustaining treatments and technologies.", "It still mainly remains the responsibility of physicians to invent the norms for when to treat and when to stop treating (Slomka, 1992; Kentish-Barnes, 2007).", "The medical fields relevant to elderly cancer patients “share” and “place” patients according to their physical and mental ability to receive chemotherapy.", "The ethical dilemmas faced are rarely discussed, despite the fact that physicians in such fields decide, on a regular basis, which patients it is ethical to make live and which patients they should let die.", "In this context, elderly cancer patients' ways of presenting themselves are without a doubt linked to the division in place between oncology and geriatrics: highlighting their active life rather than the treatment inconveniences, they show they are “young elderly” persons, fighters, capable of keeping active physically in the same way they lived prior to being diagnosed with cancer.", "They are not only fighting against the treatments, they are also battling against aging.", "By being able to incarnate this identity, they show that they are worthy of being treated.", "More than ever before, cancers are well controlled and their treatments allow people to live at home and remain active even when they are older than 70 and in an incurable stage of their illness.", "The elderly cancer patients treated with palliative chemotherapy are optimistic and have energy to continue with their active lives despite advanced cancer.", "They are living proof of cancer care achievements, and chemotherapy in palliative situations is an emblem of medical progress.", "Nonetheless, a closer look at the experience of elderly persons taking palliative chemotherapy reveals that they also have to constantly evaluate their body, have the moral attitude of a fighter, resist complaining and be grateful to have cancer at a late stage of life.", "Health professionals in oncology are confident for the elderly patients receiving palliative chemotherapy, but they rarely discuss their decisions to treat the fittest elderly patients and not others in oncology settings and oncogeriatrics.", "This aspect should not be eluded by the important progress achieved by medicine facing cancer.", "This study was financed by the Pallium Foundation in Lausanne, Switzerland.", "The previous sets of studies leading to the current research were supported by the Swiss National Science Foundation (FNS, DORE).", "The funding source(s) had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication." ]	[ "Unique focus on elderly cancer patients receiving palliative chemotherapy.", "Retraces the emergence of oncogeriatrics.", "Combines interpretative and critical medical anthropology.", "Reveals life and death medical decisions facing the elderly." ]
Long-term trends in vegetation phenology and productivity over Namaqualand using the GIMMS AVHRR NDVI3g data from 1982 to 2011	Vegetation monitoring of arid and semi-arid environments using remotely sensed vegetation indices over long periods of time is essential to improve the understanding of the processes related to change. In this paper, 30 years of biweekly AVHRR NDVI3g (1982–2011) were used to examine the spatial and temporal patterns of vegetation productivity and phenology across Namaqualand, a winter-rainfall desert of South Africa. We applied a previously developed threshold based method, TIMESAT, to extract phenological metrics such as the start, peak, end and length of the growing season and estimates of productivity such as the small and large integral of NDVI. The results confirm that the vegetation in Namaqualand is strongly influenced by rainfall and that it varies considerably along rainfall gradients. A clear pattern of vegetation seasonality is observed where the growing cycle starts in autumn (March–April) and extends through the wet winter months with peak growth occurring in late-winter (August). The analysis of trends over the 30 years suggests that there has been a shift in vegetation phenology over Namaqualand to an earlier start, later end and consequently an increase in the length of the growing season, especially in the northern parts of the region. Vegetation productivity, as reflected in the small and large integrals, has also increased over many areas in Namaqualand. Although it is difficult to attribute the changes to any one factor, increases in temperature, changes in land use practices and an increase in the concentration of atmospheric CO2 are advised as possible reasons for the observed changes. Continued monitoring using finer spatial scale vegetation indices is essential to track the progression of these trends in the future.	[ "AGRI" ]	[ "AVHRR NDVI3g", "Arid environments", "Global change", "Namaqualand", "Phenology", "Productivity", "Temporal trends", "Vegetation" ]	[ "1110" ]	[ "Considerable attention has been directed towards understanding how climate change is likely to alter ecosystem structure and function across the globe (Zhang et al., 2013) and there is a growing research impetus to detect early signs of change and to identify vulnerable regions.", "Since vegetation productivity and phenology are strongly linked to climate factors, such as rainfall and temperature, studies often utilise remotely sensed vegetation indices to assess and monitor changes in terrestrial ecosystems (Fensholt et al., 2012; Zhu et al., 2016).", "The Normalised Difference Vegetation Index (NDVI) derived from the Advanced Very High Resolution Radiometer (AVHRR) sensor is often used as a proxy for terrestrial vegetation productivity and is the most commonly used tool for detecting long-term vegetation change at global, continental and regional scales (Pinzon and Tucker, 2014).", "In its third generation version, the dataset now has more than 30-years of vegetation data and provides an unprecedented opportunity to examine long-term vegetation dynamics.", "In this study, the 30 year AVHRR NDVI3g data set was used to assess the rate and extent of long-term changes (1982–2011) in vegetation phenology and productivity across Namaqualand, a mega-diverse desert region of north-western South Africa.", "In Namaqualand, vegetation productivity is of great importance since there is a high dependence of people on natural resources, livestock, and agriculture (Benjaminsen et al., 2006; Cousins et al., 2007).", "While overgrazing by livestock has negatively influenced the vegetation and species composition of the region in the past (Hahn et al., 2005; Todd and Hoffman, 2009; Anderson and Hoffman, 2011), future changes in climate are expected to drive further negative impacts (Rutherford et al., 1999; Midgley and Thuiller, 2007).", "Warming trends and increasing temperature extremes have been observed over the region (MacKellar et al., 2014; Davis et al., 2016) and are projected to continue to increase into the future (Engelbrecht et al., 2015).", "There is uncertainty about changes in rainfall but projections suggest that rainfall will decline in future as a result of the poleward retreat of mid-latitude cyclones, which bring rain to Namaqualand (MacKellar et al., 2007; Engelbrecht et al., 2009).", "To date, the majority of evidence for changes in vegetation in Namaqualand has been provided by site specific field-based observations (for example Schmiedel et al., 2012) and repeat photography studies (Hoffman and Rohde, 2010) and there has been little work using satellite imagery to understand large scale vegetation dynamics over the region.", "In one of the only studies, Fox et al. (2005) utilised AVHRR NDVI data to map the average phenological patterns of vegetation in Namaqualand based on mean monthly NDVI values.", "This study builds on this analysis by using more advanced mathematical approaches to detect and extract phenology and productivity metrics including the start, end and length of the growing season from remotely sensed data as well as the inclusion of trend analysis to detect changes in both productivity and phenology metrics over the 30 years.", "This study also investigated the relationship between climate and vegetation productivity and phenology to provide an improved understanding of the potential impacts of climate change in the region.", "Namaqualand is located in the north-west corner of South Africa and extends from the Orange River in the north to the Olifants River in the south (Fig. 1).", "Namaqualand forms part of the Succulent Karoo biome which is globally recognised as one of only two biodiversity hotpots located within a desert environment (Myers et al., 2000).", "The region experiences a complex range of land use (Fig. S2) including conservation and tourism, commercial and communal livestock production and smallholder livestock systems (Hoffman and Rohde, 2007).", "Agriculture, mainly livestock production, is the primary land-use in Namaqualand and is practised on both commercial and communal farmlands (Rohde et al., 2001).", "The communal areas account for more than 30% of the region and are used extensively for livestock production and support 45% of its population (Hoffman et al., 1999).", "Mean annual precipitation ranges from 50 mm in the north-west to 400 mm per annum over the central Kamiesberg Mountains (Fig. 1).", "For the western region of Namaqualand, more than 60% of the rainfall occurs during the winter months between May and September as a result of the cold, westerly fronts from the southern oceans (Tyson and Preston-Whyte, 2000).", "In this paper, we define Namaqualand as the strongly winter-rainfall part of the Succulent Karoo biome (Mucina and Rutherford, 2006) and exclude the area east of the Escarpment commonly known as Bushmanland which falls within the largely summer-rainfall Nama-Karoo biome (Cowling et al., 1999, 2004).", "More detailed aspects of the region's climate, biogeography and ecology have already been described in numerous regional scale reviews (e.g. Cowling and Pierce, 1999; Cowling et al., 1999; Desmet, 2007).", "The 8-km AVHRR NDVI3g (third generation) dataset constructed by the Global Inventory Modelling and Mapping Studies (GIMMS) project for the period 1982–2011 was used.", "In this study, a longer time series of NDVI was prioritised over higher resolution and shorter time-series alternatives in order to assess change over the longest period possible.", "The AVHRR NDVI3g dataset contains two maximum-value NDVI composites per month and is acquired from a number of NOAA (National Oceanic and Atmospheric Administration) satellites over the 30 year period (Pinzon and Tucker, 2014).", "As with the original NDVI GIMMS dataset the NDVI3g dataset takes into account deleterious effects, such as calibration loss, orbit drift and volcanic eruption (Tucker et al., 2005).", "The third generation dataset has an improved calibration and cloud-masking function (Pinzon and Tucker, 2014).", "The monthly evolution of rainfall and NDVI from 1982 to 2011 averaged over Namaqualand is shown in Fig. S1.", "Bare soil and sparsely vegetated areas with NDVI < 0.1 were masked from the analysis.", "Daily weather station data provided by the South African Weather Service for four stations located in the study area (Springbok, Calvinia, Vredendal, and Port Nolloth) were used (Table S1).", "The weather station data were subjected to quality control to remove any erroneous values.", "The AVHRR NDVI3g long-term dataset was analysed using the time-series analysis program TIMESAT version 3.11 (Lund University & Malmo University).", "TIMESAT was developed for the exploration and extraction of productivity and seasonality parameters from time series data (Jönsson and Eklundh, 2004) and has been widely used to calculate phenology metrics from AVHRR NDVI data (Olsson et al., 2005; Heumann et al., 2007; Jönsson et al., 2010; Wessels et al., 2010; Tan et al., 2011).", "In the TIMESAT processing scheme, a smooth annual time-series was created for Namaqualand using the adaptive Savitzky–Golay filter (Savitzky and Golay, 1964).", "This filter was applied as it closely modelled the raw time series data while capturing the sudden rises in the data values.", "A window width of 4 data points was used in the successive curve fitting steps.", "Vegetation in Namaqualand has one growing season per year and thus the number of growth seasons was assumed to be one and the ‘season cut-off value’ was set to one in processing scheme settings.", "From the fitted model functions, a number of phenology and productivity parameters were extracted (Table 1).", "The start and end of the season were identified using a threshold based method.", "A user defined threshold of 20% of the seasonal amplitude, as measured from the left minima of the seasonal curve, was set as the start of the growing season.", "By using a dynamic threshold based method, the growth season was therefore not fixed but variable in timing and length.", "A 20% threshold was chosen as the optimal value based on the visual analysis of the fitted time-series curves for individually selected pixels.", "The end of the growing season was defined as the date at which the right edge had decreased to 20% as measured from the right minima.", "For the 30 year period investigated (1981–2011), 29 full growing seasons were identified.", "Vegetation productivity over the region was estimated using the large and small integral.", "The large integral (LI) is an estimate of the total vegetation productivity whereas the small integral (SI) is a measure of the productivity within the growing season.", "The peak NDVI (MAX) value of the growing season was also used as an indicator of vegetation productivity.", "The long-term mean was calculated for all parameters while the inter-annual variability of the phenology metrics was expressed as standard deviations (SD) in days and that of the productivity metrics as the coefficient of variation (CV).", "Patterns of average phenology and productivity are presented for the greater Namaqualand region including both the winter and summer rainfall regions.", "Temporal trends in each of the phenology and productivity metrics were examined using the non-parametric Mann–Kendall trend test (Kendall, 1938).", "The Mann–Kendall technique is a non-linear trend indicator that measures the degree to which a trend is consistently increasing or decreasing.", "The Kendall's correlation coefficient ranges from − 1 to + 1 where a value of + 1 indicates a trend that consistently increases and never decreases and the opposite is true of a value of − 1.", "A value of zero indicates no consistent trend.", "The associated statistical significance (p < 0.05) was also computed using a Mann–Kendall significance (Kendall, 1938) test.", "Trends presented only consider the winter-rainfall region of Namaqualand.", "In order to compare climatic variables from the weather station data with phenology and productivity metrics derived from AVHRR NDVI3g an annual sample time-series of each metric was extracted for 1982–2011 using a buffer distance of 25 km around each of the weather station locations.", "A multiple linear regression was then performed on the extracted time series in order to assess the relationship between phenology and productivity metrics and climate for the period 1982–2011 (n = 29) at each of the weather stations.", "Since vegetation is influenced by rainfall in the current growing season, the rainfall in the months preceding the growing season as well as the rainfall in the growing season of the preceding year (Wang et al., 2003), a number of different correlation analyses were performed.", "Correlation coefficients between the productivity metrics and rainfall totals in the current growing season, rainfall totals in the previous growing season, pre-season rainfall totals, and the rainfall during the month of maximum NDVI were calculated.", "Similarly, temperatures are important for early season vegetation growth and control several phenological parameters over the growing season (Chambers et al., 2013; Jeong et al., 2011).", "Correlation coefficients between the phenology metrics and pre-season temperatures, growing season temperatures, and late season temperatures were calculated.", "Pre-season rainfall totals and temperatures were based on the average temperature three months before the start of the growing season and late season temperatures were based on the average temperature of the three months at the end of the growing season.", "The main vegetation growing season over the winter rainfall region of Namaqualand extends through winter over a period of seven and a half months (± SD 53 days).", "While noting some spatial variability in the phenology metrics (Fig. S2), the growing season starts in autumn (April), peaks in late-winter (August) and ends in summer (December), (Fig. 2).", "The growing season usually ends before the following growing season starts demonstrating that in the period between December and February there is limited vegetation growth.", "The low rainfall areas to the north-east of the winter-rainfall region of Namaqualand, commonly referred to as Bushmanland, demonstrate the highest inter-annual variability in the start, peak and end of the growing season (Fig. S3).", "The high coefficient of variation recorded for this region is a result of it being a transitional area between winter and summer rainfall regimes.", "On average, the start of growing season (SOS) varies by 43 days, the mid position of the growing season (MID) by 37.5 days and the end of the growing season (EOS) by 45 days over the winter-rainfall region of Namaqualand.", "The spatial patterns in vegetation productivity reflect the physical characteristics of the region where higher productivity is observed over the higher rainfall and higher elevation regions along the Kamiesberg, Bokkeveld and Roggeveld escarpments (Fig. 3).", "Higher vegetation productivity is also observed in association with the Olifants River where extensive irrigation occurs.", "Relatively low vegetation productivity is observed over Bushmanland in the north-eastern parts of the study area.", "As with the phenology metrics the greatest inter-annual variability in vegetation productivity is observed along the central eastern border of the winter-rainfall region extending eastwards into the summer rainfall Bushmanland region.", "The strongest correlations between vegetation and climatic factors were observed between productivity metrics and rainfall over the three months preceding the peak growing month (May–June–July), (Table 2 and Table S2).", "The strongest correlation was observed for the small integral and maximum NDVI value suggesting that higher rainfall totals in May–June–July result in greater productivity within the growing season.", "Positive correlations were found with maximum and minimum temperatures three months before peak vegetation growth and the phenology metrics.", "For the Calvinia weather station, a positive correlation (R2 = 0.36, n = 25, p < 0.05) between maximum temperature and three months before peak growth and the end of the growing season was found (Table S3).", "For the Port Nolloth and Calvinia weather stations, a positive correlation between annual minimum temperatures and the start of the growing season was found (Table S4).", "Furthermore, a positive correlation (R2 = 0.32, n = 25, p < 0.05) was found between minimum temperature three months before peak growth and the length of the growing season for the Springbok weather station (Table S4).", "Considering only the statistically significant pixels in the winter-rainfall region of Namaqualand, there has been a general shift to an earlier start of the growing season as well as a shift to an earlier date for peak growth (MAX) between 1982 and 2011 (Fig. 4).", "These trends are observed in areas inland of Port Nolloth, around Springbok, including the Goegap Nature Reserve, and in areas to the east of the Kamiesberg escarpment (Fig. 5).", "Some areas associated with the Olifants River and Bokkeveld escarpment in the south show an increase in the start date of the season.", "The trends in the end of the season show a clear spatial pattern with a later end date over the northern region of Namaqualand and an earlier end date towards the southern and eastern regions (Fig. 5).", "These changes in the start and end of the season have resulted in a slightly longer growing season across Namaqualand particularly over the central and northern regions.", "Some areas, associated with the Bokkeveld escarpment towards the south have, however, experienced a decrease in the length of the growing season.", "The small integral which provides an estimate of vegetation productivity within the growing season has increased significantly over many different areas in Namaqualand (Figs. 4 and 6).", "Patterns of changes in the large integral, which is a measure of total annual productivity within the growing season, are similar to those associated with the small integral.", "Spatial differences in maximum NDVI values are evident across Namaqualand (Fig. 4).", "Areas to the south, which are associated with the Olifants River and Bokkeveld and Roggeveld escarpments, have experienced an increase in maximum NDVI values while areas in the central and northern regions, especially around Springbok, have experienced a reduction in maximum NDVI (Fig. 6).", "This paper documents the spatial and temporal variability of vegetation phenology and productivity in Namaqualand over the last 30 years using the long-term AVHRR NDVI3g dataset for the period 1982–2011.", "Characterising vegetation productivity patterns across Namaqualand is important since it provides spatially explicit information on the vegetation dynamics across the region as well as the necessary baseline information to effectively understand change.", "The seasonality and productivity metrics defined by TIMESAT were able to capture the spatial patterns of vegetation dynamics across Namaqualand.", "The seasonal cycle presented here is consistent with known plant phenological patterns and regional winter rainfall patterns for the region (Cowling et al., 1999; Fox et al., 2005; Desmet, 2007).", "The spatial patterns of vegetation productivity are strongly associated with altitudinal and rainfall gradients.", "The higher rainfall and higher elevation areas along the Kamiesberg and Bokkeveld escarpments have higher vegetation productivity compared to the lower rainfall and lower lying areas of the eastern parts of Namaqualand.", "These patterns of productivity are similar to those reported by Fox et al. (2005), and Anderson et al. (2010).", "The high vegetation productivity observed south of the Olifants River reflects the intensively irrigated vineyards and citrus orchards in this region (Conradie et al., 2009; Bruwer, 2010).", "These findings highlight the potential use of remotely sensed vegetation indices to characterise vegetation production across the arid and semi-arid Namaqualand region.", "Based on the trends observed in the phenology metrics, there is evidence to suggest long-term changes in the start and end of the growing season which have resulted in an increase in the length of the growing season especially in the northern parts of Namaqualand.", "Studies, mostly in the northern hemisphere, have shown that temperature changes have had a large impact on the start and end of the vegetative growing season (Tucker et al., 2001; Linderholm, 2006; Jeong et al., 2011).", "A directional shift in phenology over long periods is often viewed as an indicator of climate change (Potter et al., 2003; Reed, 2006).", "The correlations between temperature and the phenology metrics recorded for Namaqualand suggest that low temperatures during the winter months may limit the length of the growing season.", "Recent increases in temperature over the region (Davis et al., 2016) may therefore have facilitated the observed trend towards a longer growing season.", "The observed changes in the start, end, and length of the growing season provide evidence of a shift in the phenology of Namaqualand's vegetation in response to climate change and the potential influence that temperature has on Namaqualand's vegetation deserves further investigation.", "As important as large scale regional and intercontinental comparisons are (e.g. Esler and Rundel, 1999), integration using finer scale vegetation data combined with spatially-explicit temperature trends is required.", "Detailed plant ecophysiological research on the phenological responses of different growth forms and photosynthetic pathways to changes in temperature will contribute substantially to understanding the role of increased temperature on phenology in Namaqualand.", "The trends in productivity metrics, specifically the small integral, show many areas in Namaqualand where vegetation productivity has increased within the growing season.", "Considering the aridity of the region and the strong relationship with vegetation productivity and rainfall three months before peak growth, changes in rainfall over the region would be the most likely driver of changes in productivity with the growing season.", "However, trends in rainfall over Namaqualand presented in other studies (MacKellar et al., 2014; Davis et al., 2016) are inconsistent and no directional change, either wetting or drying has been observed.", "Other drivers are therefore likely to be responsible for the change in productivity and although discussed separately below, probably also interact with each other in complex ways to influence the vegetation of Namaqualand over time.", "The first of these drivers relates to the change in temperature discussed earlier.", "Since the primary growing season occurs in the cooler winter months, vegetation productivity is likely to be constrained by temperature.", "In addition, to affecting the start and end date, and therefore the length of the growing season, increases in growing season temperatures may also facilitate increased productivity and growth within the vegetative season.", "A second explanation for the change in productivity could be related to the changes in land-use that have occurred across Namaqualand over the last century.", "An increase in vegetation cover over time has been documented by several field-based observations (Todd and Hoffman, 2009; Schmiedel et al., 2012; Van Rooyen et al., 2015) and repeat photography studies in Namaqualand (Hoffman and Rohde, 2007; Rohde and Hoffman, 2008; Hoffman and Rohde, 2010, 2011).", "The increase in vegetation cover documented by Van Rooyen et al. (2015), for example, at the Goegap Nature Reserve which has been formally protected for 35 years, was attributed to the removal of large numbers of domestic livestock from the area.", "This is also not an isolated case, as a reduction of especially goats and equines over the course of the 20th century in Namaqualand is clearly evident in the agricultural census data for the district (Hoffman, 2014) and has been used in previous studies to explain the increase in vegetation cover in the region.", "The abandonment of cultivation across large parts of the study area including around Springbok and Steinkopf in the northern parts of the Namaqualand (Hoffman and Rohde, 2007) has also contributed to an increase in perennial plant cover at many locations.", "However, the increase in cover and therefore productivity presented in this study has been observed in areas under different land use practices (Fig. S2), including rangelands managed under both communal and private tenure.", "This suggests that, irrespective of land use, other factors including changes in climate and increased atmospheric CO2 might be responsible for the changes observed in vegetation productivity.", "An increase in atmospheric CO2 is likely to cause an increase in vegetation productivity (Zhu et al., 2016) particularly in an arid environment where C3 shrubs and herbaceous plants form a substantial component of the biomass (Cowling et al., 1999).", "This notion provides a third reason for the observed increase in productivity.", "The CO2 fertilisation effect has been documented in a number of arid environments across the globe (Donohue et al., 2013) as well as in terrestrial ecosystems in South Africa (Kgope et al., 2010; Buitenwerf et al., 2012).", "Studies investigating the response of succulent CAM species to CO2 have shown that increases in atmospheric CO2 result in an increase of biomass productivity due to enhanced plant water-use efficiency (Drennan and Nobel, 2000; Housman et al., 2006).", "An understanding of the role that increased atmospheric CO2 has in driving changes in succulent CAM species in South Africa in combination with changes in rainfall and temperature is limited and current Dynamic Vegetation Models do not include this photosynthetic pathway in their analyses (Moncrieff et al., 2015).", "The high temporal resolution of the NDVI dataset has provided new insights into long-term changes in the vegetation of the region and provides evidence of landscape scale shifts in vegetation phenology and productivity over the last three decades.", "The findings presented in this paper provide important baseline information on the characteristics of Namaqualand's vegetation that will provide a benchmark for assessing future change in the region.", "This study has demonstrated the potential to use remotely sensed vegetation indices to characterise and evaluate changes in vegetation over time.", "Such information is essential in supporting local environmental governance and climate change adaptation responses in Namaqualand (Midgley and Thuiller, 2007).", "The lack of long-term weather station records over the region is a key challenge (MacKellar et al., 2014; Davis et al., 2016) in the attribution of the changes observed.", "Finer spatial and temporal scales of analysis may better reveal climatic controls over vegetation growth than broader scales of analysis presented here.", "Understanding the mechanisms behind trends in NDVI is a critical step towards an improved understanding of the role of land-use change and climate change and towards an improved understanding of future projections of vegetation change in the region." ]	[ "Vegetation in Namaqualand is strongly influenced by rainfall which varies considerably over the region.", "The phenology and productivity metrics defined by application TIMESAT were able to reproduce the spatial patterns of vegetation dynamics across Namaqualand.", "Over the last 30 years the growing season has become longer due to a shift in the timing of the start and the end of the season.", "Vegetation productivity within the growing season has increased.", "The shifts in vegetation phenology and productivity could present first evidence of the impacts of changing climate on Namaqualand.", "Continued monitoring using finer spatial scale vegetation indices is essential to track the progression of these trends in the future." ]
Regional hotspots of temperature extremes under 1.5 °C and 2 °C of global mean warming	Local- and regional-scale heat extremes can increase at a significantly greater rate than global mean changes, presenting challenges for human health, infrastructure, industry and ecosystems. We examine changes in regional absolute temperature extremes for a suite of global regions under 1.5 °C and 2 °C of warming above pre-industrial levels, as described by the Paris Agreement. We focus on area-average values of observed monthly averages of daily maximum and minimum temperatures in 12 regions and calculate the most extreme monthly records observed. Next, using a large ensemble (HAPPI; Half a Degree Additional warming, Prognosis, and Projected Impacts) of decade-long simulations both of the present day and stabilised at these higher warming thresholds, we explore how changes in temperature extremes temperatures scale with global mean warming in these timeslice simulations. In the models, we focus on the 99th percentile values of monthly maximum temperatures and the 1st percentile of the monthly minimum temperatures. We define and identify hotspots of warming for various global mean warming levels, where projected changes in regional extremes are greater than global mean temperature changes. We identify overall hotspots of extremes, which are regions where the tail of the temperature distribution (above 99th percentile) warms at a faster rate than the rest of the temperature distribution in response to mean global warming increase. For monthly maximum temperatures, Central Europe, North Asia, West and East North America experience the greatest projected increases in extremes relative to means, and for monthly minimum temperatures, Central, West, East and North Asia, and East North America are identified as extremes hotspots. Although the scaling of increasing extremes with global mean temperatures is regionally variable, all regions benefit from the reduced severity of monthly maximum temperatures under lower global warming thresholds.	[ "EART", "ENVI", "SOCI" ]	[]	[ "1902", "2308", "3305" ]	[ "As extreme weather and climate events pose significant risks to human and natural systems, understanding past changes and future projections of extremes is an important and active research area.", "Studies have adopted various approaches to understanding future extremes and provide valuable information about possible future changes in extremes typically of a magnitude that has already been observed.", "These approaches include, for example, i) quantifying changes in the characteristics of extremes attributable to specific forcings (e.g., King et al., 2017; Nangombe et al., 2018), ii) assessing changes in the frequency or duration of extremes in the future (e.g., Perkins, 2015), iii) process-based (dynamic and land-surface) understandings of changes (Zappa and Shepherd, 2017) and iv) determining the time in the future when current extremes can no longer be considered an outcome of natural climates (e.g., King et al., 2016).", "Fewer studies to date have examined potential changes in the magnitude of future temperature extremes (i.e. those which are unprecedented in the historical climate) under enhanced anthropogenic global mean warming levels such as Paris Agreement levels.", "The 2015 Paris Agreement on Climate Change commits to ‘[h]olding the increase in the global average temperature to less than 2 °C above pre-industrial levels and to pursue efforts to limit the temperature increase to only 1.5 °C above pre-industrial levels, recognizing that this would significantly reduce the risks and impacts of climate change’ (UNFCCC, 2016).", "Projections for Australia show unprecedented temperature extremes are simulated even under the Paris Agreement global mean warming targets, and do not necessarily scale with global mean warming, or with current observational record-breaking temperatures (Lewis et al., 2017).", "Further studies have examined the relationship between regional temperatures and global mean temperatures (GMT) using pattern scaling approaches.", "Pattern scaling provides a means to assess future climate projections using spatial features of an externally forced change.", "Herger et al. (2015) explain that the “simplest traditional pattern scaling approach approximates future changes by the product of a time-evolving GMT change and a pattern that varies spatially but is constant over time, scenario, and model characteristics.”", "Some studies have shown that climate change signals in large-scale temperatures and precipitation patterns are linear as a function of global temperature (Tebaldi and Knutti, 2018).", "Pattern scaling approaches have been successful at emulating forced responses for varying degrees of global warming (King et al., 2018; Tebaldi and Arblaster, 2014) are consistent with model results showing changes in regional extreme temperatures tend to scale with GMT, independent of the emission scenario considered (Seneviratne et al., 2016).", "However, such scaling approaches are not universally applicable (King, 2019), and further analysis demonstrates that for extremes of temperature, this assumption of linearity may not hold in all locations (Lustenberger et al., 2014).", "Understanding changes in the magnitude of future extremes with future GMT increases, in addition to previous work on changes in event frequency, is critical for determining the vulnerability of various systems to future climate change.", "On a global level, an assessment of changes in the severity of future extremes relative to global means has not been conducted, although previous work highlights that global temperature changes are much smaller than the expected changes in regional temperature extremes, making GMT a measure of limited use for overall climatic change (Seneviratne et al., 2016).", "In this current study, we look globally at how the magnitude of extreme temperatures events may differ from current conditions in the future by focusing on the Paris Agreement GMT levels.", "This globally-focused assessment is important, as exposure to future unfamiliar climates and extremes is regionally variable (Diffenbaugh et al., 2018).", "We also define and identify hotspots of future extreme temperatures (defined below), which are areas that demonstrate the strongest response in regional-scale extremes to global-scale mean warming of the land surface.", "We use a combination of observations and models to assess how the magnitude of temperatures extremes may change in the future.", "We focus this analysis on monthly, rather than daily or shorter duration, temperature extremes and examine in observations and models:", "Tmax, which is the monthly average of daily maximum temperatures for all months", "Tmin, which is the monthly average of daily minimum temperatures in all months.", "For observations, we first compare values in two gridded observation-based products (Berkeley Earth Surface Temperature and CRU TS) that provide gridded monthly temperature data.", "The Berkley Earth (BEST) (Rohde and Coauthors, 2013) product merges temperature observations from multiple sources, providing a finer spatial resolution, and greater spatial and temporal coverage than CRU TS (Harris et al., 2014).", "We compare monthly temperature extremes in 25 land-dominated Intergovernmental Panel on Climate Change (IPCC, 2012) regions (excluding Antarctica, ANT) in the observed products, analyzing regions in which there is the closest agreement between products with the same spatial representation.", "For each IPCC region, area-average values of observed average monthly maximum temperatures are calculated and then a maximum record of regional values is determined (see Fig. 1a and b for BEST), rather than an area-average of gridbox maxima.", "Regions are included for further analysis in BEST based on two criteria, 1) the difference in maximum recorded area-average Tmax values between BEST and CRU TS is less than 1 K and 2) less than half the standard deviation of temperature variability for that region.", "The 12 regions highlighted for further exploration in HAPPI models are listed in Table 1 and the similarity between observational data products can be seen in Fig. 1c for Tmax, and Fig. 1d for Tmin.", "Projections of future temperature changes are determined from the HAPPI model dataset, which was designed for examining extreme events in simulated worlds that are 1.5 °C and 2 °C warmer than pre-industrial, in addition to the current (2006–2015) climate.", "Several prior studies have used transient, rather than timeslice, simulations (such as CMIP5) to examine facets of extremes although the difficulties of using such model frameworks have also been noted, including from the scenario dependence of warming (Wang et al., 2017).", "Mitchell et al. (2016) point out that there is a large spread in timing for when transient CMIP models cross global mean warming thresholds and the relative importance of difference forcings at these disparate times.", "Simulations like HAPPI that are tailored to 1.5 °C and 2.0 °C targets provide an avenue for exploring extremes and return times under stabilised climates (Mitchell and Coauthors, 2017).", "For a comprehensive discussion of the different methods used in producing projections of extremes see James et al. (2017).", "Participating HAPPI models (NorESM1, ECHAM63LR, CanAM4, Cam4-degree and MIROC5) contribute large atmosphere-only ensembles for three decade-length timeslices, including 2006–2015 (historical or HAPPI2006-2015) and under 1.5 °C and 2 °C (HAPPI1.5 and HAPPI2) of warming.", "Realisations, or ensemble members, within an experiment differ in their initial weather state.", "Simulations of the current decade (2006–2015) are driven by observed sea surface temperatures (SSTs) and sea ice.", "Boundary conditions for HAPPI1.5 (HAPPI2) are calculated by adding the change between the decadal average of the modelled 2006–2015 period and the decadal average of the modelled 1.5 °C (2 °C) world over 2091–2 100 to the observed 2006–2015 SSTs.", "Changes are determined from the CMIP5 (Taylor et al., 2012) multi-model ensemble from the representative concentration pathway 2.6 (RCP2.6) HAPPI1.5 and RCP4.5 for HAPPI2.", "We use multiple realisations of HAPPI Tmax (monthly average of daily maximum temperatures) and Tmin (monthly average of daily minimum temperatures) data (Table 2).", "A minimum of 500 decade-long realisations was used for each experiment, providing at least 6000 months of temperature data.", "Regional area-mean Tmax and Tmin are calculated in each HAPPI realisation.", "Model Tmax data are compared to observations to evaluate how well observed variability is simulated, in addition to how accurately the multi-model ensemble mean matches observed.", "Following Lewis et al. (2017), variability in observed temperatures is estimated using synthesised timeseries.", "Using a bootstrap resampling method; we generated 2000 timeseries of 80-year length, calculated a suite of standard deviations of observed temperature and obtained a spread of plausible observed standard deviations, based on the BEST data.", "In this calculation, we have not removed an estimate of any trend over this period, and hence a larger variability in observed temperature may occur.", "However, the probability density functions (PDFs) of simulated Tmax and Tmin values in HAPPI2006-2015 are somewhat broader than observed in many regions (Supplementary Figs. 1a and b and Fig. 2a and b).", "This larger simulated variability may indicate that the substantially larger sample size available in model data captures a broader range of temperatures, and hence greater variances.", "It is also possible that discrepancies in simulated and observed temperature distributions relate to observational uncertainties and spatial inhomogeneities in data collection (see Alexander and Coauthors, 2006).", "Alternatively, this may demonstrate key biases in capturing observed means and variability in AMIP runs may result from errors in model sensitivity to surface temperature forcings.", "Previous analyses of temperatures in model simulations demonstrate a sensitivity in simulated climates to modelling approach.", "For example, projected extreme temperatures may be systematically biased towards high temperatures in SST-driven simulations compared to fully coupled runs (Fischer et al., 2018).", "Further studies, however, advocate for the use of SST-driven simulations for assessing regional climate projections over land (He and Soden, 2016).", "Due to these potential biases in SST-driven model runs, we consider only regions in which observed temperature characteristics are accurately simulated.", "We find 12 regions (CAS; CEU; CNA; EAS; ENA; MED; NAS; NAU; SAS; SSA; WAS; WNA as outlined in Table 1) in which simulated variability falls within the observed bootstrapped range and there is consistency in maximum recorded area-average Tmax in observed products.", "In addition to focusing on these 12 best performing regions, we use HAPPI data in combination with observed data as a constraint on simulated extremes (discussed in section 2.3).", "While a greater coverage of regions would be ideal for examining future extremes, we preference analysis of regions where models perform best compared with observed.", "We also note that the regions examined here provide coverage over many global areas (see Fig. 1a) and hence the potential for insight across a variety of areas.", "We also note that the regions examined here provide coverage over a diversity of areas across the globe (see Fig. 1a) and hence the potential for insight across a variety of climates.", "We also limit our analysis to monthly Tmax and Tmin, rather than daily temperatures, which demonstrate notable warm tail biases in many regions compared to observed (Supplementary Figs. 2a and b).", "Prior to calculating temperature values in HAPPI we apply a simple bias correction based on the differential between regional average Tmax values observed over 2006–2015 and the HAPPI2006-2015 ensemble mean value (historical).", "This correction is applied to HAPPI simulated mean and extreme values for all experiments.", "Next Tmax and Tmin temperatures in HAPPI experiments are investigated using several analytical steps.", "First, we calculate the ensemble mean Tmax values for each HAPPI experiment for each region for various percentile levels.", "Percentile values are calculated by first fitting a generalised extreme value (GEV) distribution to the Tmax timeseries for each realisation and calculating percentile values in the fitted distribution.", "Distributions are fitted to the raw time series rather than seasonally-corrected anomalies.", "Values are calculated for 99th, 95th, 90th, 75th percentiles in HAPPI1.5 and HAPPI2 (e.g., HAPPI1.5/99; HAPPI2/99) in each realisation and an ensemble mean value calculated.", "Equivalently, we calculate 1st, 5th, 10th and 25th percentile Tmin values (e.g., HAPPI1.5/1; HAPPI2/1) from the Tmin timeseries.", "A range of percentile values are provided for comparison, although we focus primarily on 99th Tmax and 1st Tmin values.", "This measure ideally provides a comparatively conservative estimate of future extreme temperatures, with this value, for example, exceeded every 1-in-100 months under 1.5 °C level of global mean warming for HAPPI1.5/99 HAPPI2/99.", "As with (Wehrli et al., 2018), we also find values are robust to statistical model fitted, models and number of realisations included, including if percentile values are calculated directly from timeseries without fitting a GEV distribution.", "We also note that the percentile values used are not all inherently extreme, but are rather selected to provide information on changes across a large part of the temperature distribution.", "These steps identify hotspots regions where extremes warm faster than GMT (assuming that global mean warming in the historical simulations from 2006 to 2015 is 1 °C above pre-industrial (Haustein et al., 2017)).", "In summary, these analytical approaches provide various assessments of how extreme Tmax and Tmin in various regions change with background global land surface warming, including how regional extremes scale with GLMT and how the tails of the regional temperature distributions change with warming.", "The simulated regional-average 99th percentile Tmax values exceed the current observed record in all instances for HAPPI1.5 and HAPPI2 (Fig. 2).", "In all regions except for WAS (West Asia) the observed constraint value, determined as the number of anomalous standard deviations above the mean defining the current observed record, is lower than HAPPI1.5/99 or HAPPI2/99.", "Conversely, for WAS, the observed constraint is larger than the 99th percentile simulated, indicating that temperatures at least as large as the HAPPI1.5/99 or HAPPI2/99 values should be expected to occur under these warming thresholds based on both observed and simulated characteristics of temperatures for this region.", "In all other analysed regions, the observed constraint is larger than HAPPI1.5/99 and HAPPI2/99 values, and these values are used hereafter to described future extremes under these global mean warming levels.", "For Tmin, projected changes relative to observed records are less consistent across regions (Fig. 3).", "In certain regions, projected temperatures are cooler than those encountered to date (e.g. North and South Asia, North Australia and Eastern North America).", "These regions may incur other climatological changes that result in less warming in cold extremes than elsewhere, possibly due to precipitation trends or the occurrence of cold outbreaks.", "There is also a larger range of Tmin values for the WAS region determined through model estimates than indicated by observed values, suggesting that the SSTs imposed in the model do not provide a sufficient constraint on WAS temperatures.", "The WAS region, including the Arabian Peninsula, is subjected to strong westerly wind flow, meaning that the SST conditions imposed act as less of a constraint in this region compared to regions with ocean-dominated flow.", "For WAS, it is also possible that conditions occurring during 2006–2015 may not be sufficiently representative of climatic variability.", "We next explore how extreme temperatures scale with mean warming (Figs. 4 and 5).", "For each region, we determine the relationship between ensemble mean and 99th percentile Tmax values in order to assess whether changes in extreme values exceed changes in regional mean Tmax values, as well as the 95th, 90th and 75th percentile values.", "The same approach is applied to the ensemble mean and 1st, 5th, 10th and 25th percentile Tmin values (Fig. 5).", "In many of the analysed regions shown in Fig. 4, HAPPI1.5 or HAPPI2 values fall above the identity line wherein mean and extreme values increase comparably.", "The specific regions vary depending on the percentile value examined.", "Similarly, several regions demonstrate warming of Tmin (1st percentile) at a rate exceeding increases in monthly means of daily mean temperatures (Fig. 5).", "Regions in which extreme-global mean land surface scaling exceeds 1:1 (where increases in extremes are greater than mean values) are designated hotspots and summarised in Table 3.", "These regions are highlighted by hatching for Tmax in Fig. 6 and Tmin in Fig. 7, and are notably different for the Tmax and Tmin parameters.", "At 1.5 °C of global mean warming, Tmax warming hotspots occur for Central Asia, East Asia, North Asia, South Asia, West Asia, Central Europe, Mediterranean and North Australia (Fig. 6a).", "At 2 °C of global mean warming, warming hotspots occur for East Asia, South Asia, Central Asia, Central Europe, Mediterranean, North Australia, Central North America and Eastern North America (Fig. 6b).", "For Tmin, 1.5 °C hotspot regions are Central Europe, West and North Asia, and Western and Central North America (Figs. 7a) and 2 °C hotspot regions are Central Europe, West, Central, South and North Asia, and Western, Central and Eastern North America (Fig. 7b).", "Central Europe is identified as a hotspot of warming for all warming levels and temperature variables and is discussed in further detail in section 4).", "These Tmax extreme-mean relationships are shown in greater detail (see Fig. 8) for the Central Europe (CEU) and Mediterranean (MED) regions, which are hotspots at both warming threshold levels.", "In both regions there are clear increases in extreme Tmax values above the global mean land surface warming for both 1.5 °C and 2 °C of global mean warming.", "The increase in Tmax 99th percentile values in these regions is over twice the increase in global mean land surface warming.", "While these regions demonstrate simulated warming of Tmax extremes occurring more than twice the rate of global mean surface temperature changes, ascertaining the absolute value of extreme temperatures under these warming scenarios remains difficult due to potential biases in models and uncertainties in warming responses.", "Although HAPPI data can be evaluated against observations for the recent period (2006–2015) to determine potential biases in mean model or variability, assessing the validity of HAPP1.5 and HAPPI2 simulations is harder.", "In order to assess absolute temperature extreme values for these regions, we impose a further observational adjustment.", "We apply the observationally constrained simulated extreme values (NσObs_max * σHAPPI1.5) to the recently observed regional mean temperatures (2006–2015) adjusted for future global mean warming levels.", "This aims to provide an observationally derived conservative estimate of future regional temperature extremes.", "Applying this approach to the highlighted hotspots regions gives temperature extremes up to 6 °C above existing records for just 1.5 °C of global mean warming in the Mediterranean, and a 1.8 °C increase in Tmax extremes between 1.5 °C and 2 °C of global mean warming.", "We discuss this very strong warming further later.", "For Tmin extremes we focus on WNA and CEU as hotspot regions of interest (Fig. 9).", "In both these regions, warming of Tmin extremes occur at a rate greater than warming of the global land surface, for both 1.5 °C and 2 °C of warming.", "We summarise these results by identifying extremes hotspots of projected future warming (Fig. 10), where the tail of the temperature distribution expands with global mean warming levels in the HAPPI experiments.", "Hotspot regions for Tmax are CEU, NAS, WNA and ENA (Fig. 10a).", "For Tmin, hotspots are CAS, WAS, EAS, NAS and ENA (Fig. 10b).", "This summary of hotspots (Table 3) provides information about regions where the tail of the temperature distribution is most sensitive to warming between the two global mean thresholds.", "The results presented here are largely consistent with previous studies that have shown extreme local- or regional-scale temperatures increasing at a faster rate than mean temperature in some regions.", "By exploring events of various durations in distinct model projects, studies have identified regions of accelerated warming (e.g. Coumou et al., 2013; Coumou and Robinson, 2013; Diffenbaugh and Giorgi, 2012).", "These highlighted examples typically use CMIP5 coupled models with transient simulations to assess changed in extremes relative to mean conditions for a suite of regions and temperature metrics, including for example warm seasons, rates of record-breaking and percentage of global land area experiencing extremes.", "Despite exploring extremes using diverse measures, prior studies generally demonstrate a consensus of larger increases in threshold exceeding extreme temperature events than of mean temperatures for many regions (Coumou and Robinson, 2013; Hansen et al., 2012).", "This effect of disproportionate increases in regional-scale extreme temperatures is noted even in moving between levels of 1.5 °C and 2 °C of global mean warming.", "In one study examining Australian extremes, including seasonal temperatures, an increase of ~25% in the likelihood of record heat extremes was projected if warming of 2 °C rather than 1.5 °C occurred (King et al., 2017).", "Global warming of 2 °C results in substantially larger simulated changes in the probabilities of the extreme temperatures events over much of the land surface than for 1.5 °C of warming (Kharin et al., 2018).", "This effect is also reported for the contiguous USA (Karmalkar and Bradley, 2017) and for Europe (Dosio et al., 2017).", "More detailed studies of the relationships between local and regional temperature changes with global mean warming of 1.5 °C and 2 °C have been conducted (King et al., 2018).", "This study used transient CMIP5 climate simulation to evaluate how well simulated climates at 1.5 °C predict simulated climates at 2 °C of global mean warming.", "While in many locations, the seasonal average was determined to be a useful predictor of change at higher levels of global warming, accelerated increased in extreme temperatures in the East Asian region were noted.", "In the King et al. (2018) investigation, temperature extremes increased between 1.5 °C and 2 °C levels of global mean warming, in part due to synoptic changes and changes in anthropogenic aerosol concentrations.", "The location of hotspots of monthly temperature extremes determined in our current study also aligns well with previous studies, including the identification of East Asia as ahotspot of Tmax warming at 1.5 and 2 °C levels (Fig. 6).", "We note that the developing world regions around the tropics tended to fail the model evaluation tests we imposed, and we do not provide analysis of these regions, despite their established vulnerability to future climatic changes (Althor et al., 2016).", "The regions analysed in our study and determined to be hotspots of warming at largely mid-latitude areas, including the Mediterranean.", "The Mediterranean region has been previously identified as a regional climate change hotspot (Diffenbaugh and Giorgi, 2012; Seneviratne et al., 2016), and projections for increases in heatwave intensity here are the largest simulated globally (Perkins-Kirkpatrick and Gibson, 2017).", "The Mediterranean region is highlighted as a hotspot of warming due to the strong soil moisture-temperature feedback that likely enhances changes in temperature extremes (Diffenbaugh et al., 2007; Seneviratne et al., 2006).", "Soil moisture conditions and feedbacks also have a notably large impact on the heatwave magnitude of heatwaves in central Europe (Bador et al., 2017; Vogel et al., 2018, 2017).", "At the specific individual extreme event level, such as the 2010 Russian heatwave and 2003 European heatwaves, heatwaves associated with blocking high pressure systems and soil-moisture feedbacks can amplify warming (Quesada et al., 2012; Miralles et al., 2014) and result temperature extremes of extended duration (Coumou et al., 2013; Bador et al., 2017).", "The overall magnitude of warming in 99th percentile regional monthly Tmax values determined in our study is at the higher end of values reported, though direct comparison across studies is difficult given the differing temperature metrics reported and different model experiments used.", "For example, one study found changes in heatwave intensity are 0.5–1.5 °C above a given global warming threshold (1.5 °C/2 °C), though values are higher over the Mediterranean (Perkins-Kirkpatrick and Gibson, 2017).", "The high warming values determined in our study (up to 6 °C above present for 1.5 °C of global mean warming) may result from several factors.", "First, Diffenbaugh et al. (2007) note that increases in temperature are highly sensitive to percentile analysed, and we use a comparatively more extreme temperature metric (99th rather than 95th percentile).", "This sensitivity to percentile is also demonstrated here in Fig. 4 (and Supplementary Figs. 5 and 6).", "In studies focused on record-breaking temperatures (e.g. the absolute highest values recorded), changes in the rates of record-breaking increase 5-fold (Coumou et al., 2013) or greater (King, 2017).", "Second, this very strong result for the Mediterranean may be caused by a combination of genuine sensitivity of extremes to mean temperature changes and additionally indicate that the 2006–2015 period used in the HAPPI modelling framework does not sufficiently constrain the tail of the distribution nor temperature variability in the extratropical Mediterranean location.", "We have attempted to constrain the value of future warming in three case study regions (CEU and MED for Tmax; WNA and CEU for Tmin) using a large time slice-based ensemble, and by applying bias correction and observational constraints (Fig. 8c and d, Fig. 9 c and d).", "However, the values presented are based on a single modelling framework and hence a systematic examination of projections and uncertainties in a greater number and variety of models would assist in providing these projections of possible absolute regional temperature extremes under various levels of global mean warming.", "In particular, the estimation of variability simulated in models requires further examination.", "While we have highlighted the MED and CEU regions, in particular, we note that many other regions analysed are classified as hotspots of warming for extreme monthly Tmax at 1.5 °C (CAS, EAS, NAS, SAS, WAS, CEU, MED, NAU) and/or 2 °C (EAS, SAS, CAS, CEU, MED, NAU, CAN, ENA) of global mean warming.", "In these listed regions, Tmax increases by more than the rate of warming of the global land surface.", "Processes such as land-atmosphere feedbacks (Seneviratne et al., 2010; Vogel et al., 2017) and/or dynamical changes (Wehrli et al., 2018) may be particularly important in these regions.", "For example, previous studies identify that land-atmosphere feedback in CEU amplify extreme temperatures in climate change projections (Vogel et al., 2017; Seneviratne and Coauthors, 2013).", "We also note that regions which are extremes hotspots of future Tmax warming (where the tails of the temperature distribution are most sensitive to global land surface warming) are not identical to Tmin hotspots.", "For example, NAU shows a greater sensitivity of Tmax to mean warming than for Tmin extremes.", "We label CEU, NAS, WNA and ENA as Tmax hotspots, and CAS, WAS, EAS, NAS, and ENA as Tmin hotspots.", "In general, the proportionally large increase in extremes of temperature relative to global or regional temperature means is well established.", "One important result from this HAPPI analysis is that in many regions, the most extreme temperature percentiles analysed warmed at a faster rate with mean global warming level (e.g. 99th compared to 95th percentiles, see Fig. 8).", "We also show that this is regionally variable, and this relationship is also critically influenced by the mean global warming level, the temperature variable and extreme percentile considered.", "We summarise this complexity in mean-extreme relationships by describing overall hotspots of extreme warming, a description of regions that targets changes in the far tail of temperature distributions.", "Extreme temperatures in these regions, shown in Fig. 10 and Table 3, are most impacts by background changes in mean warming of the land surface.", "These results help to identify regions in which simple pattern scaling approaches may not be insightful about changes in regional extremes with increases in GLMT.", "Pattern scaling techniques may assist in approximating forced changes under increasing concentrations of greenhouse gases (Tebaldi and Arblaster, 2014).", "However, such scaling approaches are not universally applicable and can be model dependent.", "Further analysis demonstrates that for extremes of temperature, this assumption of linearity may not hold (Lustenberger et al., 2014).", "The regional variability in responses of extremes to mean temperature changes is likely dependent on many factors, including forced responses in circulation, local- and regional-scale feedbacks and the degree of natural variability in temperatures.", "Exploring these dynamic processes and feedbacks, and their regional and seasonal variations in further detail would be useful.", "In particular decomposing changes in extremes into changes relating to mean warming and dynamically driven components would provide further insight into changes in these hotspot regions (Wehrli et al., 2018).", "Previous studies have also explored the role of antecedent rainfall or soil moisture in driving extreme high temperatures (Donat et al., 2018; Vogel et al., 2018).", "Donat et al. (2018) used the observed precipitation-hot temperature relationship as a constraint on projections of future temperature extremes.", "Such an approach could also be applied to simulations at varying levels of global mean warming, such as in HAPPI.", "In our study, we restricted the analysis to HAPPI and explored temperatures only in multi-model ensembles.", "An exploration of potential inter- (between HAPPI models) and intra-model (between individual HAPPI model realisations) differences may provide insight into process-based understandings of warming of extremes.", "In particular, the relationship between precipitation and temperatures in models may elucidate strong Mediterranean region warming.", "Overall, this study confirms the outcomes of prior studies that investigate the changing probability of current extreme temperatures under 1.5 °C and 2 °C of warming in transient model simulations, and demonstrate the overall benefits of limiting warming for reducing the frequency of future regional extreme temperatures (Ciavarella et al., 2017).", "By estimating absolute temperature extremes under Paris Agreement higher global mean warming levels in stabilised model simulations, we provide additional information about the regions in which changes in extremes are greatest relative to changes in mean temperatures, and the severity of such changes in temperature extremes under the Paris Agreement temperature thresholds.", "We find that under both Paris Agreement levels of warming, increases in 99th percentile monthly Tmax values are projected for all regions, although these projections are regionally variable and values of these Tmax extremes do not increase 1-1 with global mean warming in many regions.", "We find many mid-latitude regions where extremes of Tmax (99th percentile) and Tmin (1st percentile) values increase more than average warming of the global land surface (the 1:1 warming line shown in Figs. 4, 5, 8 and 9), although the designation of these regions as hotspots depends on the temperature variable (Tmin or Tmax), percentile and global mean warming level considered.", "We define hotspots of extremes as regions in which the tail of the temperature distribution (above 99th percentile) increases with mean land surface warming at a faster rate than the rest of the temperature distribution.", "For Tmax, CEU, NAS, WAS and ENA experience the greatest projected increases in extremes relative to means, and for Tmin CAS, WAS, EAS, NAS and ENA are identified as extremes hotspots.", "Prior studies have also found that regional extreme temperatures increase with global mean temperatures, but with a slope greater than 1 (Seneviratne et al., 2016).", "These results using different temperature parameters (99th percentile values) and model frameworks re-iterate that while global mean warming provides a useful and necessary metric for examining large-scale climate changes and for policy discussions, this single metric does not sufficiently reveal changes in regional temperature across the spectrum of the temperature distribution.", "In some regions, changes in extremes are highly sensitive to and far exceed changes in global means.", "For example, we show projections for changes in Tmax extremes in the Mediterranean region that are up to 6 °C above present for 1.5 °C of global mean warming.", "While this specific result requires further process-based exploration and analysis in an expanded set of models, it further demonstrates the necessity for exploring projected changes in regional-scale temperature extremes." ]	[]
Single cell analysis of cancer genomes	Genomic studies have provided key insights into how cancers develop, evolve, metastasize and respond to treatment. Cancers result from an interplay between mutation, selection and clonal expansions. In solid tumours, this Darwinian competition between subclones is also influenced by topological factors. Recent advances have made it possible to study cancers at the single cell level. These methods represent important tools to dissect cancer evolution and provide the potential to considerably change both cancer research and clinical practice. Here we discuss state-of-the-art methods for the isolation of a single cell, whole-genome and whole-transcriptome amplification of the cell's nucleic acids, as well as microarray and massively parallel sequencing analysis of such amplification products. We discuss the strengths and the limitations of the techniques, and explore single-cell methodologies for future cancer research, as well as diagnosis and treatment of the disease. © 2013 The Authors.	[ "BIOC" ]	[]	[ "1309", "1311" ]	[ "Cancer is a disease caused by changes to the DNA, whereby the cancer genome is shaped by the interplay of processes of DNA damage and repair, cellular selection and clonal expansions [1,2].", "Tumour evolution is classically thought of as a series of clonal expansions that are each triggered by new driver mutations conferring a selective advantage [3,4], hence ‘new’ cells undergo Darwinian evolution, very much like how species develop [5,6].", "Over the past decades, we have learnt much about how cancers develop from studying their genomes, most notably through the introduction of massively parallel sequencing.", "Comparison of cancer samples from different sites or different time points is increasingly painting a picture of cancers undergoing branching evolution, resulting in competition between different subclones [7–13].", "In solid tumours, this picture is further complicated by a topological component [8,14], with potentially different selection forces operating at different locations of the tumour.", "Most current large-scale cancer genome sequencing studies use DNA extracted from millions of cells.", "The resulting genome sequences therefore contain intermixed sequences from different tumour clones, as well as from admixed normal cells.", "Computational methods can determine which mutations are clonal (present in all tumour cells) and which are subclonal [15].", "In addition, by analyzing point mutation and copy number data further with bioinformatics algorithms, phylogenetic trees of different tumour subclones can be inferred [12].", "Although these methods provide important information on the genomes of distinct cell populations within the tumour, the number of tumour cell populations they can disentangle is limited, and inferring rare subclonal populations remains difficult.", "Recent advances have made it possible to profile the genomes of single cells.", "The isolation of single cancer cells, followed by amplification of the DNA and array profiling or next-generation sequencing (Figure 1), opens avenues to study tumour subclonal architecture and tumour evolution in unprecedented depth.", "Here, we provide an overview of current methods to profile genomes of single cells.", "We discuss their strengths and limitations and the perspectives they offer for cancer research and therapy monitoring.", "To isolate single cells from solid tumours, two main approaches have been developed.", "The first method exploits the precision of modern flow cytometry to sort nuclei from single cells [16,17••].", "Tissue-cubes of ∼1 mm3, cut off a (frozen) solid tumour, are teased apart in cell lysis buffer, containing DAPI, a fluorescent DNA-intercalator, and the resulting single nuclei are flow-sorted based on DNA content.", "This technique provides the advantage of allowing identification and isolation of tumour subpopulations on the basis of ploidy [16,17••].", "Although the cytoplasm is lost, extensions to analyses of the transcriptome per se are possible [18].", "However, this approach also entails limitations.", "In particular, micronuclei may be lost.", "Micronuclei are not merely by-products from genomic instability but are likely prone to DNA-replication stress and further DNA-mutational processes [19] and therefore may be important players in tumour evolution.", "A second method disperses the tissue from fresh solid tumour biopsies in a single-cell suspension, using enzymatic treatments, including, for example, collagenases [20•].", "Intact individual cells can subsequently be isolated using (mouth-controlled) pipetting, modern cell-sorting or microfluidics systems with or without applying immunocytochemistry.", "Microfluidics devices provide the advantage that in addition to capturing individual cells, they also provide nanoliter reaction chambers to further process the nucleic acids of multiple individual cells in parallel under highly standardized conditions at significantly reduced reagent costs.", "Both of the above technologies have the disadvantage that precise topological information about the cell is lost, which can be overcome by using laser capture microdissection from fixed tissues or cryosections.", "However, capturing a full cell or nucleus can be problematic [21].", "Solid tumours also shed cells in a patient's blood stream (circulating tumour cells or CTCs) and cells disseminating to distant organs (disseminated tumour cells or DTCs) (Figure 1).", "DTCs can remain dormant over a prolonged period of time following resection of the primary tumour, before giving rise to overt metastases [22].", "Investigating CTCs and DTCs is important not only for understanding tumour evolution and progression, but also as liquid biopsies of a solid tumour for guiding diagnosis, prognosis and treatment.", "Although often just a few CTCs in millilitres of peripheral blood of a cancer patient are present, various isolation techniques based on physical and biological properties of CTCs have been described [23–25].", "However, a main difficulty remains that unbiased CTC-isolation requires the definition of suitable biomarkers that are expressed in all blood-borne tumour cells, but not in normal circulating cells.", "Similarly, defined physical and biological properties of DTCs, commonly homing to the bone marrow, can be used for their isolation following needle aspiration through the iliac crest [23,24•].", "Modern genomics technologies require hundreds of nanograms of input material, while a normal diploid human cell contains about 7 pg of DNA.", "Hence, whole-genome amplification (WGA) is required to enable analysis of a single cell.", "WGA of single-cell DNA is based on Multiple Displacement Amplification (MDA), Polymerase Chain Reaction (PCR), or a combination of principles of both displacement amplification and PCR (Figure 2).", "Importantly, all amplification methods suffer from various imperfections that hamper straightforward reliable identification of genetic variation.", "The breadth of genomic coverage, amplification biases (due to local differences in %GC-content or other factors), the prevalence of chimeric DNA molecules, allelic drop outs (ADO), preferential allelic amplifications (PA) and nucleotide copy errors can differ significantly between different WGA approaches.", "As such, some methods are more apt than others to detect specific genetic variants [26••,27••,28,29].", "In theory, massively parallel sequencing allows profiling the full spectrum of genetic variation in a cell's WGA product, from ploidy changes to aneuploidy and (un)balanced structural variants, down to indels and base substitutions.", "However, the various confounding factors of WGA complicate this process (Figure 3).", "A one-fit-all WGA method remains to be established, and a comparative analysis of all WGA methods against a benchmark case is acutely needed, assaying the potency of genetic variation detection, including examining the favourable effects of the reduction of reaction volumes and amplification cycles [30••].", "Despite these dire straits, several groups have proven the efficacy of single-cell microarray and primarily massively parallel sequence analyses to detect multiple classes of mutation in single tumour cells.", "Hybridization of single-cell WGA products to DNA microarrays or SNP arrays allows uncovering copy number changes in the cell.", "SNP arrays provide a distinct advantage as copy number calls can be integrated with B allele fractions of SNPs and with genotype calls [31], thus also allowing the discovery of copy neutral LOH changes in a cell [32,33], or even to haplotype its entire genome [34,35].", "Despite the use of ultra-high resolution array platforms and the development of state-of-the-art computational and statistical methods, the majority of array-based methods can only reliably detect copy number changes encompassing millions of bases in a solitary cell [36,37,38•,39].", "The main difficulty is to distinguish a genuine copy number change from a local allelic WGA artefact due to %GC-bias, ADO or PA events [28].", "In addition, the cell-cycle stage of the isolated cell can complicate the analysis as cells in S phase can have 2, 3 or 4 copies for a diploid locus, leading to false structural DNA-imbalance discoveries [40].", "Remarkably, a recent study reported the detection of copy number alterations as small as 56 kb in single-cell PCR WGA products hybridized to 180 K oligo-arrays [41].", "Array profiling of single cells has been applied to study the biology of CTCs [42••] and DTCs [38•,39].", "Heitzer et al. used the technology to profile genetic relationships between primary colorectal carcinomas, metastases and CTCs derived from the same patients [42••].", "Although CTCs shared a number of gains and losses with the primary tumour and/or the metastasis, interestingly, they also observed private copy number changes in CTCs as well as heterogeneity between CTCs.", "Such results are paving the way for using CTCs as a liquid biopsy to guide clinical decision-making.", "Sequencing of single-cell WGA-products recently improved the resolution of a cell's DNA-copy number profile by algorithmic focal sequence-read depth analyses [16,17••,27••,43] (Figure 3b).", "Ni et al. [44••] demonstrated that copy number aberration patterns of CTCs in different patients with the same lung cancer subtype can be extraordinarily similar, but dissimilar when compared to copy number landscapes of CTCs in patients with different lung cancer subtypes, and thus be of diagnostic significance.", "Furthermore, driven to understand intra-tumour cell population structure and genome evolution in breast cancer, Navin and colleagues [16,17••] developed single-nucleus sequencing for copy number profiling of single cancer cells able to detect alterations with a resolution of 54 kb on average.", "By phylogenetic analyses, they could infer common ancestors, clonal expansions and divergence of subpopulations.", "Genome-wide profiling of structural variation in a single cell is still in its infancy.", "Each WGA method likely fabricates its own characteristic set of chimeric DNA molecules, which have to be discriminated from bona fide structural variants following sequencing, a process that is further complicated by genome coverage issues.", "Therefore, currently, many structural variants are still missed by single-cell genome sequencing.", "Nevertheless, filters have been designed to permit the detection of the structural architecture of copy number alterations following mapping of paired-end sequences (Figure 3c) [27••] and approaches to detect L1-retrotransposition have been developed [45•].", "In a recent study, we were able to discover and fine-map intra-chromosomal as well as inter-chromosomal rearrangements in single cells.", "Furthermore, we performed single-cell genome sequencing of individual breast cancer cells related by one cell cycle, and detected large de novo structural DNA imbalances acquired over one cell division [27••], providing proof of principle that single-cell sequencing can track tumour evolution in real time.", "Sequencing allows discovering single nucleotide mutations (Figure 3d).", "However, genuine base substitutions in the cell have to be discriminated from WGA-polymerase base infidelities and sequencing errors [20•,26••,42••,46••,47,48,49].", "Therefore, reliable single-nucleotide substitution detection in non-haploid loci currently necessitates sequencing of multiple single cells [20•,26••,46••,47,48], or confirmation by deep-sequencing of matched bulk tissue [42••], thus posing problems for the characterization of rare cell populations.", "Targeted sequencing of single-cell WGA products was recently applied to investigate single-nucleotide mutations in the exome, to hunt for heterogeneity in a renal carcinoma [20•], a myeloproliferative neoplasm [46••] and a bladder cancer [47].", "Although variant calls of at least three cells had to be considered to filter WGA and sequencing artefacts from genuine base alterations, subclonal population structure could be profiled at high accuracy, providing insight into progression and selection processes, and understanding of the difficulty of treating cancer.", "Single nucleotide and indel mutational landscapes in CTCs in patients with lung cancer [44••,50] and colorectal cancer [42••,51] were recently also determined by single-cell exome and cancer gene panel re-sequencing, respectively.", "These studies are signalling the promise of CTC sequencing for identifying therapeutic targets and regimens for personalized treatment.", "Using short in vitro cultures, mutation rates have been tracked over a limited amount of cell divisions.", "Whole-genome sequencing of multiple WGAed cells revealed a base mutation rate in a colorectal adenocarcinoma cell line that was 10-fold higher when compared to estimates of germline studies [26••].", "Altogether, despite current limitations, massively parallel sequencing of single cells revealed a dynamic picture of on-going genetic evolution and clonal expansion in cancer, and is delivering genomic snapshots of rare tumour cells, such as CTCs and DTCs, and thus new insight in the disease.", "Following single-cell isolation, the epigenome and the transcriptome may also be studied [21].", "While epigenomics of single cells remains challenging [52–55], methods for single-cell transcriptomics have flourished (Figure 4) and delivered baffling new insight into the (functional) heterogeneity of cell populations [56–58].", "A single cell contains less than 1 pg of mRNA.", "To characterize it via array [59,60] or sequencing [56–58] approaches, whole-transcriptome amplification (WTA) is required.", "Methods for WTA are grounded on PCR-based [61–64,65•,66–69], MDA-based [67] or in vitro transcription (IVT)-based [70] amplification of reverse transcribed single-cell mRNA, whereby IVT likely results in a more linear amplification.", "However, WTA and subsequent analysis methods struggle with reliable amplification and detection of transcripts expressed at less than 10 copies per cell.", "In addition, the majority of the methods only selectively amplify the polyadenylated RNAs of a cell's transcript repertoire [61–64,65•,66,70], and may be biased to the 3′-end [70] or the 5′-end [61,62] of a transcript (Figure 4).", "Full-length mRNA-characterization from a single cell can only be achieved by a few WTA-methods [63,64,65•,66,67,68] (Figure 4).", "To the best of our knowledge, no large-scale single cancer cell transcriptome sequencing studies have been reported, although this is on the horizon [71–73].", "In a recent elegant proof-of-principle experiment, Ramsköld et al. found differences between melanoma CTCs and primary melanocytes, giving insight into the disease [65•].", "Additionally, the technology allowed defining potent plasma membrane CTC biomarkers and discovering expressed coding mutations.", "This and other studies [60,73–75] show that single-cell transcriptomics will illuminate further insights into oncogenesis, tumour subclonal architecture and cell lineage diversity.", "Single-cell sequencing studies currently only process either WGA-products or WTA-products of a cell, although protocols for combined approaches are under development [76•,77].", "The ability to profile both the genome and the transcriptome of the same cell has enormous potential to elucidate heterogeneity at the genome, epigenome and transcriptome level.", "In addition, such techniques would allow mutations of the genome in a single cell to be confirmed in that cell's transcriptome, opening avenues to detect mutations at high confidence, even if they are observed only in one single cell.", "The emerging field of single cell genomics opens new avenues that may have far-reaching implications for cancer research and clinical practice.", "It allows characterization of intra-tumour genetic heterogeneity genome-wide to single-cell resolution, and thereby offers a unique viewpoint into tumour evolution.", "Current single-cell sequencing studies have focused on only tens to hundreds of cells, but already revealed complex heterogeneity at this level.", "We envisage that the scale of these experiments will increase impressively in the coming years.", "Emergence of microfluidics systems, able to generate sequencing-ready libraries for thousands to millions of individual cells in parallel is likely.", "Such methods, as well as massive single-cell genotyping assays [78], combined with clever bioinformatics approaches to infer relationships and life histories of individual cells, will provide detailed insight into the emergence and clonal expansion of each tumour subclone, allowing a truly holistic view on tumour evolution.", "Little is known about the variability in the epigenome and the transcriptome of single cells, as this is masked in current analyses of mixed large cell populations.", "We envisage that future methods that can profile the (epi)genome and the transcriptome of the same single cell will allow detailed insights into the transcriptional and phenotypic consequences of genomic changes in cancer.", "Finally, by sequencing individual CTCs and DTCs together with primary tumour cells and metastases, we will learn more about the mechanisms that trigger single tumour cells to leave the site of their origin, the dormancy of DTCs and their resistance to cancer therapy.", "We anticipate that partial or full cancer genomes of (fine-needle) cancer biopsies, CTCs and/or DTCs will routinely be sequenced as part of the clinical evaluation and likely personalized treatments in the future.", "CTCs may be particularly important in this regard as they represent easily obtainable liquid biopsies allowing real-time monitoring of both metastatic potential and patient-specific suitability of therapy.", "The last few years have seen rapid development of technologies that permit detailed analysis of the genomes and transcriptomes of single cells.", "Single-cell approaches now stand poised to provide an unprecedented view into cancer evolution.", "T.V. is a co-inventor on patent applications involving single-cell analyses.", "Papers of particular interest, published within the period of review, have been highlighted as:", "• of special interest", "•• of outstanding interest" ]	[]
Formation and homogenisation of Sn–Cu interconnects by self-propagated exothermic reactive bonding	We produced Sn–Cu interconnects by self-propagated exothermic reactions using Al–Ni NanoFoil at ambient conditions, through the instantaneous localised heat across the interfaces between Sn electroplated Cu substrates. This technique presents a great potential for electronics integration with minimal thermal effects to the components. However, the metastable phases resulted from the non-equilibrium interfacial reactions and solidification were inevitable under a highly transient regime due to a drastic heating/cooling (over 10 7 K/s). In this study, Finite Element Analysis was performed to predict the temperature profiles across bonding interfaces, which were subsequently correlated with the formation and homogenisation of the bonded structures during the bonding and post-bonding ageing process. It has been revealed that, for nano-sized metastable phases, their formation, morphologies and distribution were primarily attributed to the convective mass transportation, liquid-solid inter-diffusion, and directional non-equilibrium solidification of Sn in molten zone of the bonding interfaces. The non-equilibrium phases initially formed in the Sn–Cu interconnects can be homogenised towards the equilibrium status by accelerated ageing. This was achieved through the coalescing and subsequent growth of the original nano-sized metastable phases, as a result of the solid-diffusion of Cu and Ag atoms at intergranular boundary regions of Sn grains, Al–Ni NanoFoil/Sn. and Cu/Sn interfaces.	[ "ENGI", "MATE" ]	[ "Al-Ni NanoFoil", "Exothermic reactive bonding", "Homogenisation", "Interfacial reactions", "Non-equilibrium metastable phases", "Sn–Cu interconnects" ]	[ "2210", "2211", "2500" ]	[ "Self-propagating exothermic reaction for synthesizing composite materials and intermetallic compounds has shown its effectiveness in providing a localised heating source for bonding various metals [1–6].", "The reaction is based on an Indium NanoFoil® [7] prepared by vapour-deposition of hundreds of alternating layers at the nanoscale (e.g. AlNi, AlTi, and AlSi).", "Once initiated with a small burst of energy in the form of either heat, electrical spark, laser or other means, the reaction starts due to a large enthalpy of mixing (e.g. 1050–1250 J/g for AlNi) and proceeds self-sustainingly at fast propagation velocities (7–10 m/s) [7–9].", "The released energy is capable of melting adjacent metals followed by subsequent solidification creating a solid joint, thus known as reactive bonding.", "Solder interconnects through reactive bonding, particularly with AlNi NanoFoils, offers numerous advantages [10–13].", "The localised heat, extremely rapid heating/cooling under a steep temperature gradient towards adjacent regions can cause a small heat-affected zone (HAZ) [9,12].", "This can reduce the ambient processing temperature, and achieve fluxless joining due to the breakdown of oxidation layers on the metal surface for bonding by significant transient thermal stress [6].", "A buffer layer (normally metals) deposited on the substrate can create a transient zone to facilitate interfacial interaction to form a strong bond.", "This unique approach can enable the robust bonding of high-temperature Pb free solderable metals for heterogeneous integration of multi-functional devices.", "J. Wang et al. bonded Au-coated stainless-steel specimens under ambient condition using free-standing AlNi multilayer foils and Au80Sn20 solder layers.", "The joints formed has enabled a 30% increase of the shear strength compared to conventional reflowed solder joints [11].", "Bonding of bulk metallic glasses with an extremely high strength over 480 MPa, was also achieved by A. J. Swiston et al. using AlNi multilayer foils of 87–274 μm thick with 40–69 nm thick bilayer [3], where the structure of the glasses was not affected.", "T. Fiedler et al. successfully joined Mg60Cu40Y10 with Pd40Ni40P20 amorphous alloys using the reactive bonding technique [13] without forming a crystalline structure, and it has also been demonstrated to make strong bonds between Ti, ceramics, super-alloys and MEMS devices through reactive bonding with AlNi NanoFoils [1,4,14,15].", "The exothermic thermodynamics of AlNi systems has been investigated experimentally and numerically.", "The diffusion-induced peritectic reaction has been counted for the reactive bonding process, primarily dominated by the heat conduction and the solid-liquid reactions between liquid Al and Ni nanolayers.", "However, the instantaneous and intensive nature of localised exothermic reactions can inevitably govern the interfacial reactions; hence the mechanical integrity of obtained interconnects.", "The heating rate of over 106 K/s, followed by rapid cooling of up to 400 K/s [3], causes a drastic temperature gradient > 104 K/mm across the bonding interface [11].", "Under such conditions, the reactive bonding process is immensely non-equilibrium in the adjacent meals or alloys.", "For instance, the original nanolayers in multi-layered AlNi NanoFoils can be entirely replaced with coarsened and equiaxed NiAl grains after reaction based on an in-situ TEM observation [16].", "In the Au80Sn20 based stainless-steel bonds, a fine lamellar microstructure (~50 nm spacing) and columnar inter-mixing regions at the AlNi NanoFoil/Au-Sn alloy interface were observed [11], with significant segregation of their composition in comparison with equilibrium AuSn alloys.", "However, very little research has been performed to elaborate the interfacial interactions due to the exothermic reactive bonding, in particular, in the uses of such technique for electronics interconnects, where it is important to understand the fundamental details of such non-equilibrium process.", "In this study, bonding trails for SnCu interconnect were implemented using the exothermic AlNi NanoFoil.", "Both computational and experimental approaches were carried out to examine the non-equilibrium exothermic reactive bonding process between Sn electroplated Cu substrates.", "Finite element analysis provides complementary, simulative insights into the localised temperature profiles and temperature gradients during the exothermic reactive bonding.", "The interfacial analysis using SEM/TEM and EDX techniques were also conducted to reveal the formation mechanism of non-equilibrium metastable phases and the microstructural homogenisation in the subsequent ageing process of the SnCu interconnects.", "As shown in Fig. 1(a), a 40-μm thick AlNi multi-layered NanoFoil® (Indium Corporation) with a 1:1 atomic ratio of Al to Ni consisting of alternating nanolayers of Al (60 nm thick) and Ni (40 nm thick) was used as the exothermic material.", "In the NanoFoil, 5 at.", "% Vanadium was added to refine the reaction products and improve the mechanical performance, with a 1 μm thick Incusil layer (59 wt% Ag-27.25 wt% Cu-12.5 wt% In-1.25 wt% Ti, an alloy used for high-temperature brazing) precoated on both side of the NanoFoil to protect the Ni or Al nanolayers from oxidation and providing a better wetting and reaction interface for common solder alloys [7].", "As shown in Fig. 1(b), the NanoFoil was placed between two pieces 5 × 5 mm2 Cu plates (Copper, 99.95%), 1 mm-thick as the bonding substrate, forming a sandwich bonding structure.", "The Cu plates were coated with 20 μm-thick electroplated Sn layer composed of fine equiaxed Sn grains, η-Cu6Sn5 IMC particles, and a continuous 0.5 μm-thick interfacial η-Cu6Sn5 layer (Fig. 1(c)).", "NanoFoil inserted was slightly larger (5 × 7 mm2) than Cu plates thus accessible for ignition through electrical discharge.", "The reactive bonding was implemented under the ambient condition using 1 MPa pressure and 373 K preheating, the optimal parameters as previously determined [17].", "The exothermic reaction was initiated by an electrical spark produced through a DC power supply at 5 V and 1A.", "A FEI Nova 600 Nanolab dual-beam Focused Ion Beam/Scanning Electron Microscope (FIB/SEM) was employed to lift-out cross-sectional TEM samples across the interfaces of bonded structures.", "The thin samples of approximately 100 nm-thickness were mounted on molybdenum (Mo) supporting grids and examined using a FEI Tecnai F20 TEM equipped with Oxford Instruments X-maxN 80 TLE SDD detector for compositional mapping by energy dispersive X-ray analysis (EDX).", "The TEM was operated in scanning mode at 200 kV with a scanning probe tuned to be ~2 nm.", "High angle annular dark field (HAADF) images were recorded together with the chemical maps.", "Then, the bonded structures were subjected to an accelerated ageing test at 175 °C for the various time in order to understand the thermal effects on the microstructural evolution.", "The TEM samples were aged for 5, 15 and 30 mins, while the as-bonded joints were aged for 2, 8, 24, 72 h for various analysis and mechanical testing.", "The shear tests were also conducted on these as-bonded and aged sandwich bond structures using a DAGE 4000+ tester at a shear speed of 5 μm/min.", "The time-dependent FEA of the temperature profiles across the reactive bonding interfaces of the Cu/Sn/NanoFoil/Sn/Cu sandwich structure (Fig. 2(a) and (b)) indicates the local temperature increases abruptly at the reaction front with the propagation progresses.", "The exothermic reaction was completed at 0.014 ms, where the maximum temperature 1765 K can be attained in NanoFoil at an average heating rate of 106–7 K/s.", "A drastic temperature decrease, down to 1434 K at the NanoFoil/Sn interface and 539 K at the Sn/Cu interface, causes a steep temperature gradient, 3.06 × 107 K/m, extended into the Sn layer.", "Meanwhile, with a less steep gradient in the Cu within 60 μm, the temperature levels off to ambient temperature.", "After the temperature at the Sn/substrate interface reaches its peak point, 610 K, at 0.45 ms, the Sn layer remains molten until the temperature drops sharply to 505 K within 0.226 ms, with a cooling rate about 105–6 K/s.", "Because of the rapid heating and cooling, the interfacial reactions and crystallization of metals can be of highly non-equilibrium.", "The planar temperature distributions at both NanoFoil/Sn and Sn/Cu interfaces (Fig. 2(b)) shows the peak temperature proceeds with propagation front (Fig. 2(b)).", "However, the heat released from the exothermic reaction (~3.9 × 108 W/cm3) is far outweighed the heat transfer capability of the Sn (66.8 W/m·K), causing a severe heat accumulation in the Sn layer, at a gradient of up to 107 K/m, compared with the 105 K/m in the Cu substrate.", "TEM cross-sectional view of the bonded structure revealed the fine NiAlgrains with an in-plane size of 30–100 nm formed in the NanoFoil after reaction (Fig. 3(a)), which was in contact and bonded with the compact Sn layer (Fig. 3(b)).", "In the Sn layer, submicron long-thin columnar grains with nano-sized intergranular IMC precipitates at the grain boundary were observed.", "These grains formed as groups at different orientations, the boundary-like domains.", "The EDX results in Table 1 confirmed the columnar grains are composed of Sn with trace Ni, Cu and Ag, whilst both the intergranular IMCs and boundary-like domains consist of non-stoichiometric phased of CuSn2–4 or AgSn1–3, which cannot be found in the phase diagrams and are hence deemed to be metastable.", "Among these Sn grains and IMCs, voids with a dimension of up to tens of nanometres were observed, likely due to the rapid volume shrinkage at the cooling transient stage.", "Further details of the NanoFoil/Sn and Sn/Cu interfaces are revealed through HAADF analysis in Fig. 4.", "Clearly, at the NanoFoil/Sn interface (Fig. 4(a), (b) and (c)), a 10–20 nm-thick interfacial Cu-rich IMC layer was indicative of the annihilation of the Ag59-Cu27.25-In12.5-Ti1.25 Incusil metallization due to the exothermic reaction.", "The formation of significant Ag-enriched IMCs in the Sn layer confirms the dissolution and migration of Ag from the Incusil coating.", "However, the multi-layered structures of NiAl foils remains in the mixture with the equiaxed NiAl grains resulted from the exothermic reaction, whilst an inter-diffusion region (~100 nm) can be seen between the NanoFoil and Cu-rich IMC layer.", "The inter-diffusion region was found to contain Ag, Sn and Cu, surrounded by Al-enriched segregations, suggesting a considerable atomic scale diffusion of Sn into NanoFoil.", "At Sn/Cu interface (Fig. 4(d), (e) and (f)), discrete η-Cu6Sn5 IMC grains in the Sn layer, with metastable CuSn1–2 IMCs distinctive from the continuous IMC layer are evident.", "In addition, the grain refinements of Cu near the interface are also observed as a result of heat effect due to the exothermic reaction.", "SEM morphological evolution of the bonds due to the ageing (Fig. 5(a)) clearly shows the growth of CuSn IMCs at the Cu/Sn interface with the ageing time, but hardly observed at NanoFoil/Sn interface.", "In the Sn layer, after long time 72 h ageing, only small numbers of IMCs particles of hundreds of nanometers remain, of which the dark phases were identified to be η-Cu6Sn5 and the bright particles were the Ag3Sn.", "At the Cu/Sn interface, the thickness of the interfacial IMCs (fully η-Cu6Sn5) layer increased exponentially with ageing time, and a ε-Cu3Sn layer was gradually appeared and grow between η-Cu6Sn5 and Cu, as observed in the conventional interconnects formed by, for instance, reflow process.", "However, in-situ TEM analysis of the interconnects with ageing in Fig. 5(b)) has revealed that, even within 15 min of ageing, the nano-sized intergranular metastable IMC precipitates had been dissolved into the Sn matrix, as evidenced by the significantly decreased number of AgSn and CuSn IMCs.", "This is particularly true near the Cu/Sn interface accompanying the growth of interfacial IMCs.", "However, in the Sn layer, it appeared these metastable IMC precipitates had migrated to the boundary-like regions and merged into large sizes of IMCs with an increased Cu/Ag concentration (near-equilibrium).", "When the ageing was long enough, the concentration of Sn (Ag, Cu) at the inter-diffusion region decreased evidently, and equilibrium IMC particles up to 300 nm can be formed.", "However, no noticeable changes of IMCs layer have been found at NanoFoil/Sn interface, apart from a seeming growth of a void locally.", "It can also be expected that the dissolution of Incusil coatings and metastable IMCs at NanoFoil/Sn interface can be homogenised due to ageing.", "Further analysis of the constituents in the IMCs by EDX in TEM shows the changes in atomic ratios of Cu, Ag and Sn in the IMCs at different locations (Fig. 6(a) and (b)).", "Overall, across the entire Sn layer from AlNi NanoFoil to Cu substrate, the Cu content in CuSn IMC or the Ag in AgSn IMCs increases with ageing time, because non-equilibrium IMCs formed due to insufficient inter-diffusion during the limited time of exothermic reaction were homogenised from metastable status to become more stabilised.", "However, the solute ratio of Cu which also co-existed in the solidified columnar Sn grains slightly increased in the central Sn layer and near the NanoFoil, but drastically decreased near Cu substrate during the ageing process (Fig. 6(c)).", "This is expected as the result of the transformation from non-equilibrium to stable phases owing to the continuous supply of Cu from the substrate.", "Nevertheless, it is hard to see any major changes for the solute element Ag in the Sn grains due to ageing, primarily due to the limited amounts of Ag available in the 1 μm thick Incusil layer.", "The compositions of the interfacial IMCs at both NanoFoil/Sn and Cu/Sn interfaces were also homogenised with the ageing time (Fig. 6(d)).", "With the significant increase of Ni and reduction of Sn and Cu, the initial continuous (CuNi)6Sn5 layer with the embedded Ag3Sn particles at the NanoFoil/Sn interface evolves with ageing time till they reach the equilibrium status.", "However, at the Cu/Sn interface, as expected, the Cu content in the IMCs slightly increases, but the element Sn decreases with the ageing time.", "This is likely attributed to the formation of Cu3Sn through further diffusion and supply of Cu from the substrate, and the conversion of initial Cu6Sn5 IMCs into Cu3Sn as the IMCs layer continues to grow thicker.", "The experimental results from the shear tests of the bonded structures were collected and plotted in Fig. 7.", "Clearly, the shear strength reached the peak point after aged for 24 h, showing an approximately 20% increase from 21 MPa to 25 MPa, and this was followed by a drastic decrease thus arrived at the lowest point, 16 MPa.", "After 120 h ageing, the shear strength remained more or less unchanged.", "Such changes in the bonding strength should reflect the microstructural evolution, i.e. equilibration, morphological characteristics and growth of IMCs.", "Considering the diffusivity of Ni in liquid Al (0.386–1.8 × 10−8m2 s−1 in the range of 980–1773 K), Cu and Ag in liquid Sn (0.413–1.075 × 10−8m2 s−1 in the range of 560 K to 750 K, and 0.59–1.18 × 10−8 m2 s−1 in the range of 560 K to 1108 K, respectively) [23], the diffusions of Ni, Cu and Ag are expected to be within the range of tens of nanometres during self-propagating reaction.", "However, Table 1 clearly shows the IMCs were enriched by Ag and Ni across the entire CuSn interconnected structures, primarily in the form of intergranular IMCs precipitates along the grain boundaries (Fig. 3).", "This implies the elements such as Ni, Ag, Cu must migrate through an entirely different mechanism across the 20 μm-thick Sn layer between NanoFoil and Cu substrates, which could have ~103 higher speed than the solid-liquid diffusion process.", "Among a variety of potential routes, the model of the convective motion of liquid [16] can be confirmed to enable such fast transportation of the elements across the Sn layer; this is supported by the numerical predictions of temperature profiles (Fig. 2(a)).", "The extreme high localised temperatures can melt the entire Sn layer instantaneously.", "The re-distribution and mass transfer of individual components can occur given the micro-scale thermal gradients and the applied pressure.", "These can force the convective motion of the molten Sn, facilitating a long-range atomic migration across the Sn layer, forming non-equilibrium metastable phases through simultaneous solidification as the exothermic reaction propagates further at the front of the bonding.", "Phase transformation of a single AlNi NanoFoil in the exothermic reaction, as reported in [16], is accomplished through the peritectic reaction between Ni and Al nanolayers by the active solid-state diffusion upon the ignition with heating (i.e. 493 K) or sparks.", "This reaction spontaneously produces localised energy to rapidly heat up the NanoFoil and accelerate the AlNi inter-diffusion process [9,16].", "The heat generated is sufficient to melt the Al nanolayers initiating the convective motion of liquids in the solid-liquid mixture under the thermal transient stress developed between nanolayers.", "The transient stress and the volume reduction induced by the formation of NiAl IMCs can fragment Ni nanolayers into fine solid Ni nano-particles.", "As inter-diffusion regions between Ni and Al expand, a drastically increased temperature at the central area of the NanoFoil could exceed the melting point of Ni (1728 K), which allows the complete NiAl reaction in the liquid state, forming nano NiAl IMC grains by cooling.", "Due to the primarily homogeneous nucleation, the equiaxed IMCs nano-grains with random orientations were observed in the central part of the NanoFoil after solidification (Fig. 8(a)).", "However, since the temperature in the region near the NanoFoil/Sn interface, which is ~1434 K, may not exceed the melting point of Ni and NiAl IMCs, the incomplete NiAl reaction of nanolayers was expected.", "This can result in a mixture of liquid Al with a wide range of solid components including fragmented Ni and NiAl IMCs nano-particles, where further refinement and alignment of these mixed nano-grains due to the solidification under fast cooling and the steepest thermal gradient resulted in the lamellar textures (Figs. 4(a) and 8(b)).", "The EDX results in the table of Fig. 8 confirm that the NiAl atomic ratio of the reacted NanoFoil near the NanoFoil/Sn interface was significantly higher than that in the central part of the reacted NanoFoil, indicating the incomplete NiAl reactions in this zone.", "In addition, as can be expected, substantial other metallic elements such as Sn, Ag and Cu had penetrated into the NanoFoil through the convective motion of liquid phases as proposed above.", "Given the combined liquid phases such as Al in NanoFoil, the elements in the Incusil coatings (melting point is 988 K) and Sn with the fragmented Ni or NiAl solid nanoparticles makes a mushy status at the NanoFoil/Sn interface zone, the proposed solid-liquid diffusion through the convective mass transportation in the liquids is most likely to cause the atomic migration which can in turn govern the interfacial interactions (Fig. 9(a)).", "As stated above, the incomplete NiAl reactions near the NanoFoil/Sn interface cause a mixture of multiple phases, likely in two forms, i.e. NiAl IMC particles and NiAl-Ni core-shell particles in an orderly alignment (Figs. 4(a) and 8(b)).", "Taking the local mushy region at NanoFoil/Sn interface, one can propose the interactions of multiple components through convective motion schematically shown in Fig. 9(b).", "The convective flow in this zone can assist to release the transient thermal stress and change the density of different liquids; as such the elements from the NanoFoil, Incusil coating and Sn can lead to the formation of interfacial high-melting-point metastable IMCs, i.e. (CuNi)6Sn5 and Ag3Sn (Fig. 6(d)).", "The Cu atoms could be supplied from Incusil layer, and possibly transported directly from Cu substrate, through the ‘nearest neighbour jump’ mechanism [24–26].", "However, given the transient nature of propagating bonding process, the rapid heating and cooling do not permit sufficient time for elemental redistribution or further growth of the phase precipitates (e.g. IMCs) once formed.", "The preferential transient state aggregations of these IMCs along the columnar Sn grain boundaries are likely to occur forming the non-equilibrium metastable IMC phases under the extremely high temperature gradient [27–30].", "The solid-liquid convective inter-diffusion process also occurs through the interfacial reactions at the Sn/Cu interface.", "Firstly, the high temperature at this region can exceed the melting point of original Cu6Sn5 IMCs (Fig. 2), which is likely to dissolve the original IMCs and restart the reactions with the molten Sn.", "This can induce a solute Cu enrichment, which causes the formation of a large number of high Cu content IMC particles, particularly due to the limited time for Cu to be redistributed through the fast cooling and solidification, and formation of the IMCs particles accompanies with the nucleation and growth of Sn grains (Fig. 10(a)).", "Akin to the IMCs formed at the NanoFoil/Sn interface, these IMCs could not fully reach the equilibrium state, but a continuous quasi-equilibrium η-Cu6Sn5 IMCs layer could be formed and grow thicker at the Cu/Sn interface (Fig. 5(a)), given sufficient element source and relatively longer interaction time.", "The heat generated by exothermic reaction can also anneal the surface of the Cu substrate, i.e. causing the grain refinement, the lattice distortion of Cu grains and numerous vacancies, even dislocations [31–33].", "The formation of columnar Sn grains with the nano-sized IMCs aggregated along the grain boundaries is expected under the non-equilibrium transient state in the mushy zone (Fig. 10(b)).", "This could be initiated from the Cu substrate, given the steep temperature gradient and rapid cooling, the Cu, Ag enriched IMCs (Fig. 6(a) and (b)) which had high melting temperature can thus be formed in the mushy zone.", "These Sn grains solidify and grow rapidly but directionally forming long thin columnar structures, given the great heat dissipation along the perpendicular direction to the Cu/Sn interface [34].", "The IMC precipitates are expected to aggregate at grain boundaries to minimize the interfacial energy in the mushy zone, hence forming the intergranular IMCs after solidified (Fig. 10(b) and (c)) [35].", "Similar to the IMC phases formed at both NanoFoil/Sn and Cu/Sn interfaces, the elemental redistribution and subsequent growth of Sn grains and IMCs in the intergranular region cannot be fully complete due to the transient heating and cooling nature of reactive bonding.", "This explains why a number of components and IMCs in the as-bonded SnCu interconnects primarily consist of metastable phases at the nanometre scale.", "Accelerated ageing process allows the nano-scale non-equilibrium metastable phases to be fully homogenised towards the equilibrium status, and this has been observed by the merging and growth of the IMCs (Fig. 4(b)).", "This can be quantitatively evaluated by counting the number of IMCs and measuring their mean value of size with ageing time as statistically derived in Fig. 11(a).", "The evolution of the IMCs has been completed through the dissolution, equilibration and final growth almost occurring simultaneously, with the composition and energy states being varied at different locations.", "While the pronounced nano-scale IMCs precipitates exist prior to or after a short time (e.g. 5 mins) ageing, with the ageing time further extended, the merging and subsequent growth of these original IMCs are apparent (Fig. 11(a)).", "Driven by compositional gradients in each component IMCs or Sn grains, the solid-diffusion of Cu and Ag atoms from metastable IMCs and intergranular regions into Sn can take place [36], leading to the homogenisation of entire SnCu interconnect at elevated temperature (Figs. 5 and 6).", "The relation of mean IMC size vs ageing time embedded in Fig. 11(a) indicates the IMCs increases from the initial 50 nm to ~200 nm as the result of ageing.", "Similarly, at the NanoFoil/Sn and Cu/Sn interfaces, the IMCs evolve with ageing time as the homogenisation process.", "The “over-saturated” atoms from the inter-diffusion region and substrate, serving as elemental sources, enable the atomic replacement in metastable IMCs.", "As a consequence, the concurrent homogenisation and growth of interfacial IMCs were evident, which abides the kinetics which substantially differentiates from the one with originally equilibrium IMCs, for instance, in the case of Sn joints formed by conventional reflow process (Fig. 11(b)).", "In particular, it is interesting to observe a significant change of kinetics of the IMCs growth at Cu/Sn interfaces, which is likely due to the availability of the large amount of solute Cu to reach their equilibrium status.", "Given the thickness of IMC layers, the growth rates of interfacial IMCs with ageing time, especially in the short-term ageing, can be calculated, and the result indicates the value is much greater than what has been reported for reflow soldering [37].", "The increased growth velocity is likely attributed to the presence of the non-equilibrium metastable IMCs formed during propagating exothermic reaction.", "According to the Arrhenius equation, the growth constant of phases was dominated by the activation energy that is responsible for the interfacial reactions to form IMCs.", "In the case of metastable IMCs, their Gibbs free energy was lower than the original Sn but much higher than the relevant equilibrium IMCs, such that less activation energy is required to complete the growth [38].", "The formation and growth velocity of IMCs can, therefore, be accelerated, leading to a relatively thicker IMC layer than conventional CuSn interconnects given the same ageing conditions at the initial ageing period since the metastable IMCs are not fully equilibrated.", "The fluctuation of the measured shear strengths of the aged CuSn joints in Fig. 7 was resulted from the microstructural evolution and homogenisation.", "From the SEM images in Fig. 5(a), the initial voids formed mainly at the interfaces during the bonding process were reduced at the initial ageing stage, possibly due to the atomic migration and growth of phases occurred in the homogenisation process.", "This can be correlated with the initial shear strength increase in Fig. 7.", "However, further extended ageing time can cause significant CTE mismatch and induce the interfacial “Kirkendall voids” due to extensive diffusion process [39], thus leading to the deterioration of the mechanical strength of the SnCu interconnects (Fig. 7).", "To summarise, we have successfully developed an ambient bonding process to form the SnCu interconnects through self-propagating exothermic reactions.", "For the optimal design of the bonding structures and processing parameters, the formation and subsequently homogenisation of SnCu interconnects have been investigated to understand the interfacial interactions and phase transformations during the bonding and subsequently ageing processes.", "Both FEA simulations and SEM/TEM analysis confirmed that the metastable and very often non-stoichiometric IMCs was formed through a non-equilibrium process at an extremely short time (~0.014 ms).", "This was involved with the liquid-solid diffusion and convective mass transportation of multiple components in the mushy zone of NanoFoil/Sn and Cu/Sn interfaces at the propagating front point.", "It has been found that the incomplete NiAl reaction near the NanoFoil/Sn interfaces resulted in a mushy zone of an orderly aligned residual fragmented Ni and NiAl IMC-Ni core-shell particles.", "This has caused non-equilibrium metastable IMCs and their insufficient growth and aggregations through a transient phase transformation due to the rapid heating and cooling.", "The solid-liquid convective inter-diffusion at the Sn/Cu interface also produced a solute Cu enrichment in the IMC particles precipitated alongside the grain boundaries of columnar Sn under the high perpendicular temperature gradient.", "However, a continuous quasi-equilibrium η-Cu6Sn5 IMCs layer grow thicker at the Cu/Sn interface, given sufficient Cu supply and relatively longer interaction time.", "Accelerated ageing allowed the nano-scale non-equilibrium metastable phases to be fully homogenised towards the equilibrium status as observed by cross-sectional TEM.", "While the pronounced nano-scale IMCs precipitates exist prior to or after short time (e.g. 5 mins) ageing, with the extended ageing time, the merging and subsequent growth of these original IMCs are apparent.", "This is driven by the solid-diffusion of Cu and Ag atoms into Sn grains, causing the homogenisation of entire SnCu interconnect at elevated temperature.", "At the NanoFoil/Sn and Cu/Sn interfaces, the IMCs also evolved with ageing time through the concurrent homogenisation and growth of interfacial IMCs, but it significantly differentiates from the kinetics of equilibrium IMCs, for instance, in the case of Sn joints formed by the conventional reflow process.", "It has been reflected that the CuSn joints were consolidated as such the shear strengths increased at initial ageing.", "However, further extended ageing time can deteriorate the mechanical strength of the SnCu interconnects.", "The fundamental insight into the formation and homogenisation of SnCu interconnects by self-propagating exothermic reactive bonding in this study will effectively guide the design and manufacture of such joints by considering the materials and processing windows." ]	[ "SnCu bonds were developed by self-propagated exothermic reaction of AlNi NanoFoil.", "Nano-metastable phases were formed due to a non-equilibrium transient regime.", "The formation process is via convection diffusion and directional solidification.", "Nano-metastable phases were homogenised under an accelerated ageing." ]
An error diffusion based method to generate functionally graded cellular structures	The spatial variation of cell size in a functionally graded cellular structure is achieved using error diffusion to convert a continuous tone image into binary form. Effects of two control parameters, greyscale value and resolution on the resulting cell size measures were investigated. Variation in cell edge length was greatest for the Voronoi connection scheme, particularly at certain parameter combinations. Relationships between these parameters and cell size were identified and applied to an example, where the target was to control the minimum and maximum cell size. In both cases there was an 8% underestimation of cell area for target regions. © 2014 Elsevier B.V. All rights reserved.	[ "COMP", "ENGI", "MATE", "MATH" ]	[ "Additive manufacturing", "Cellular structure", "Delaunay", "Error diffusion", "Functional grading", "Voronoi" ]	[ "1706", "2205", "2210", "2500", "2611" ]	[ "Cellular structures, also commonly described as lattice structures, consist of a number of connected members or tessellated unit cells that form a complex structural network.", "Modern manufacturing techniques, such as additive manufacturing (AM), enable the fabrication of highly complex small to medium sized parts which can include cellular structures.", "Large scale truss structures are commonly seen in architectural applications where strength and stiffness can be combined with freedom of design.", "Smaller scale structures have other potential applications such as tailored impact absorption capacity [1–3] and heat dissipation [1,4–11].", "An example of such a structure manufactured using an AM process: selective laser melting (SLM) is shown in Fig. 1.", "This demonstrates a regular tessellation of a unit cell connected to an outer skin.", "The outer skin can be useful to provide mating faces for adjacent parts, to provide protection for the cellular structure and for maintenance reasons.", "In this case, a solid outer skin has been used; however, other forms of skin may also be used, where appropriate, such as a ‘net’ skin [2,3].", "Compared to many other manufacturing methods, AM has a comparatively inexpensive cost of complexity [12], and this can actually decrease as part complexity increases as the necessity for support structure for SLM can reduce.", "The design of these structures, however, remains a challenge.", "With increased geometric complexity comes increased design complexity, and this is exacerbated when including computational analysis methods and mathematical optimisation techniques in the design process [13–15].", "Hence, there is a general requirement for efficient methods for the design and analysis of cellular structures.", "This paper focuses on the problem of generating a cellular structure with a spatial variation in the cell size.", "Different regions of a component generally require different extents of internal structure support.", "With a fixed, uniformly tessellated cellular structure, this variation can only be achieved by varying the structural member dimensions, which leads to a large number of design variables.", "As the variation in functionality can only be achieved by varying structural member dimensions, this is restricted by the unit cell size defined prior to optimisation.", "In addition, some efforts have been made to vary the individual cell design such that the structure is constructed of differing cells [16]; however, this has many challenges and still does not address the cell size variation.", "This leads us to consider how the design space could be explored more fully by allowing variation of the cell sizes.", "Some existing cellular structure design approaches do exhibit different sized cells as a by-product of requiring the structure to conform to a shape with curved faces; instead of trimming tessellated cells to this shape, they are either swept [17] or based upon an unstructured mesh [18]; however, these variations are slight.", "A novel method is proposed that employs dithering (also known as half-toning) method to achieve spatial variation of cells and subsequent control over component behaviour.", "Learning from developments linking dithering to meshing techniques [19–22], the dithering defines spatially varying points which are then connected with structural members.", "These points determine the spatial variation of the cell sizes (area/volume).", "This paper first introduces the error diffusion methodology used for dithering, and then applies this to some examples to demonstrate the functionality using two point connection schemes.", "The relationships between the method parameters and the resulting cell size are investigated and their use for controlling cell size and distribution demonstrated.", "Dithering converts a continuous tone image into a binary (white–black) representation.", "This is useful for bi-level printers and displays.", "When viewed from a certain distance, the binary representation appears similar to the continuous representation to the human eye.", "There are several dithering methods, which can be divided into two categories: ordered dither and error diffusion [23].", "Error diffusion was used for this work as it does not exhibit undesirable grid-like patterns in the dithered result while ordered dithering does [24].", "Error diffusion uses an adaptive algorithm based on a fixed threshold to produce a binary representation of the original input.", "Following this update, the procedure is repeated for each subsequent location in turn.", "The proportions of the error diffused to adjacent pixels are determined heuristically and a typical filter for this in 2D is shown in Fig. 2a [25].", "An empty location in the filter represents zero error diffused to this pixel.", "3D error diffusion, while less common than 2D (as there are fewer potential applications), is a simple extension of the 2D process, where the error is diffused in 3D space to voxels around the current voxel.", "Again, the proportions of the original error to diffuse are determined heuristically and the filter used in this work is shown in Fig. 2b [23].", "In this case, Eq. (3) will consist of 20 locations of Pxyz corresponding to the locations and fractions shown in Fig. 2b.", "In the 1D case, the error is diffused completely to the following pixel.", "Fig. 3 presents a coarse numerical example that shows the actual value changes resulting from following this procedure for a 2D case.", "A better visual binary representation would be possible with a higher resolution array.", "The application of 1D and 2D dithering methods to enable design of a cellular structure with variably sized cells is demonstrated with a larger example problem in the following section.", "The method can be split into 3 main stages:", "Definition of functional grading.", "Error diffusion to generate dithered points of boundary and area.", "Application of connection scheme to generate structure cells.", "To generate a variable cellular structure, a continuous representation of the desired functional variation is first required.", "This could be a result of a finite element analysis (FEA), for example, providing a temperature (for thermal problems) or stress (for structural problems) distribution, or a result of a density based topology optimization [26] without penalisation of the intermediate densities.", "However the functional variation is defined, it represents the magnitude distribution of the analysis response.", "To demonstrate this method, the functional variation representation used is shown in Fig. 4a.", "This stage consists of two steps: the part boundaries are tackled first, followed by the part area (or volume for a 3D part).", "In the 2D example, the boundary is identified as a single layer of pixels, as shown in Fig. 4a.", "This layer is transformed into a single row of pixels per boundary using a boundary tracing method to preserve pixel order.", "For the part shown in Fig. 4, this gives a single one-dimensional row of variable greyscale pixels; this row is then dithered using 1D error diffusion.", "The dithered pixel row can then be transformed back to the correct shape, as shown in Fig. 4c.", "This boundary dithering stage serves two purposes.", "Firstly, it provides a more precise positional boundary than just using 2D dithering alone, as this does not necessarily place node points on the boundary.", "Secondly, it enables identification of the edges between the node points that are to be constrained for stage 3 of the method where the connection schemes are applied.", "Once the boundary points have been generated, the area can be tackled.", "Firstly, the area is reduced inwards by a pixel, as the boundary pixels have already been used.", "Secondly, by passing the filter from Fig. 2 over this representation and diffusing the error of binarisation accordingly, a point distribution is generated representing the cell spacing.", "The 1D (boundary) and 2D (area) dithered results are then combined to form a complete set (Fig. 5a).", "Due to the different filters used for the boundary and area, there is calibration required to ensure the resulting dithered points from the stages spatially correspond.", "This requires insight into how each filter affects the spacing of the dithering points which is investigated in Section 3.", "With this information the appropriate calibration parameter can be set.", "The third stage of the method is to apply a connection scheme to the dithered points to form the basis for the cellular structure.", "In this work, two example connection schemes were used: Delaunay triangulation [27] and Voronoi tessellation [28,29].", "The triangulation generates structural members that connect up the points whilst the Voronoi tessellation generates members that lie in the line/plane equidistant from the two nearest points.", "It is the edges of the polygons generated that form the member longitudinal axes onto which a geometric shape can be mapped, for instance a cylinder.", "These cellular structures are shown in Fig. 5 and the mapping of cylindrical members to these edges, along with spherical end caps, is shown in Fig. 6.", "While the triangular cellular structure is more suited to applications where high stiffness is required as these cells are stretch-dominated [30], a Voronoi cellular structure, which has cells of a similar shape to those found in natural foams that should deform readily under load [2], is more suited to impact absorption applications as the cells are bending-dominated [30].", "The minimum and maximum cell sizes shown in the previous example are determined by two parameters: the image pixel/voxel resolution and greyscale value.", "Specifying real design limits for the cellular structure ensures that it can be optimised for a given load case, but this requires determination of the effect of these parameters on the cell size range, which in turn enables local control over the generated node spacing and the overall quantity of generated nodes.", "As mentioned in the previous section, a calibration is required between the boundary and area/volume dithers owing to the different filters used.", "Without this calibration, there is a mismatch between the minimum and maximum sizes of the resulting 1D boundary and 2D area geometries.", "This is also the case when combining 2D and 3D dithering stages in a 3D example.", "It is therefore useful to be able to link 1D, 2D and 3D dithering methods so that the required sizes of the cells throughout the area or volume and the boundaries are matched.", "With this in mind, experiments were carried out to investigate the effects of image pixel/voxel resolution and greyscale value on the cell size range for both Delaunay and Voronoi connection schemes.", "A range of test 1D, 2D and 3D greyscale arrays were generated of varying image pixel/voxel resolution, L between 10 and 100 pixels in increments of 10, and greyscale level, V between 0 and 240 in increments of 10.", "The greyscale ranges from 0 to 255, but an upper limit of 240 was used because in a pure white region, no dithered points would be generated.", "This means that the samples with greyscale values above 240 there would be very few points or none at all to connect.", "On application to a real part, the boundary points would be included and allow connection but the samples did not include boundary points to reduce edge effects skewing the results.", "Each of the constant greyscale level samples was subjected to the relevant 1D, 2D or 3D error diffusion filter.", "Following dithering, in the case of the 1D samples, the average distance between the points was determined.", "In the case of the 2D and 3D samples, two connection schemes: Delaunay and Voronoi were used to generate either triangular/tetrahedral cells, or Voronoi cells respectively.", "From these cells, the average element edge length, and area or volume was calculated.", "These measures are required to control the sizes of the cells within the cellular structure.", "The results are presented in Sections 3.2 and 3.3.", "The Voronoi cells on the boundary, i.e. those that have a vertex at infinity were excluded from the calculations.", "In addition, vertices that were closer together than a specified distance were merged to avoid skewing the edge length results.", "For the Delaunay connection scheme, highly distorted (sliver) triangular/tetrahedral cells, generally located on the perimeter, were filtered out based on a radius tolerance.", "This filtering of edge effects increased the accuracy of the sample results.", "To investigate relationships between the parameters and the cell edge length for the five cases: 1D, 2D and 3D for both Delaunay and Voronoi connection schemes, a surface defined by Eq. (4) was fitted to the sample results using a nonlinear least squares method as implemented in MATLAB using variable bounds determined from parametric studies.", "The relationships between the two parameters, V and L, and the mean cell area (2D) or cell volume (3D) were found to be a close fit to Eq. (4) as demonstrated by the goodness of fit measures in Table 1.", "The corresponding coefficients for the five cases are also presented in Table 1.", "It was found that there was a power relationship between L and edge length and there was a double exponential relationship between V and edge length.", "The same samples were used to evaluate the effect on the cell size, specifically the area in 2D and volume in 3D.", "Results for the 2D cases for Delaunay and Voronoi connection schemes are shown in Fig. 8.", "Similar to the edge length results, the relationships between V and L and the mean cell area or volume were found to be a close fit to Eq. (4) as demonstrated by the goodness of fit measures in Table 2.", "In contrast to the edge length results, the Voronoi results are more closely represented by the fitted surface.", "The variability in edge length and area was investigated for both the Delaunay and Voronoi connection schemes.", "Each greyscale integer point was evaluated for a fixed sample size, the results of which are shown in Figs. 9 and 10.", "The mean is represented by the solid central line and the standard deviation is shown by the grey region either side.", "The inset squares show the sample cells for different regions over the greyscale range.", "Across both sets of samples, the variability in the member edge length was more significant than that of the cell area.", "For the area, the variability dropped to zero or close to zero at several positions along the greyscale range.", "At these regions, the cellular structure consists of identical or very similar size cells as can be seen from the samples shown.", "For the Delaunay results, as the triangular cells are not equilateral this decrease in variability is only marginally apparent in the edge length variability results.", "However, for the Voronoi results, the reductions in variability correspond to square cells which do have equal edge lengths.", "Also of interest in the Voronoi edge length results is the increase in the mean when the variability decreases.", "Very short cell edges were filtered out before analysis by merging vertices, but due to the greater variation in cell shape (as can be seen in Fig. 11) compared with Delaunay, there is still more variation in edge length for Voronoi cells.", "This means that it will be more difficult to correlate the member length at the boundary and area or volume of cells generated using the Voronoi connection scheme compared with Delaunay.", "This updated grayscale value is accommodated by modifying the input image grayscale distribution by multiplying by the difference (grayscale darkening/lightening) between the two V values (for 5 mm2 and 20 mm2).", "Because the lowest value of the range is 0, the previously defined minimum cell size is unchanged.", "The desired extreme element sizes are therefore defined by modifying the image size for the minimum size and then modifying the greyscale level for the maximum size.", "Both the Delaunay and Voronoi connection schemes were evaluated for these two size requirements and the results are shown in Fig. 13.", "It was found that in both cases, there was an underestimation of the cell area in both the minimum and maximum regions.", "For the Delaunay case these errors were 7% and 8.4%, and for Voronoi, 8.6% and 8.5%.", "This error is due to deviation of the fitted surface from the data; however, because it is reasonably consistent, it could be empirically compensated for.", "As the boundary and area or volume dithering stages are carried out independently using different error diffusion filters, the resulting spacing of the dithered points are unlikely to match.", "This mismatch can be seen in Fig. 14 which shows the fitted surfaces for the 1D case and 2D Delaunay case, with the only matching instance shown by the plotted intersection curve.", "The difference as shown in Fig. 15 can be compensated for by modifying V whilst keeping L as previously calculated.", "Depending on the application of the cellular structure, local control of member orientation can be beneficial, primarily for load bearing applications.", "However, the error diffusion filter does not inherently allow local orientation control for two reasons: firstly, the filter is passed row by row across the part and does not follow its geometric profile, and secondly, the Delaunay triangulation and Voronoi tessellation connection methods do not handle direction constraints.", "The end result of this is likely to be a sub-optimal solution and so the approach may be more suited to applications where the orientation of members has less of an effect or is not known in advance, potentially for heat dissipation and impact absorption applications.", "There are some instances of the use of a variable diffusion filter to allow local control of the dithered result, for example, to emphasise edges [32,33].", "A similar approach could potentially be adopted to improve upon the current method by taking account of principal stress tensors or force flow directions to influence the structural member orientation by making a corresponding adjustment to the error diffusion filter.", "However, it is difficult to see how this would have a significant effect on the member orientation, especially with the Voronoi connection scheme where members are generated around the dithered points.", "An alternative approach could be to identify members that exist in a particular orientation and increasing the cross sectional dimensions of these members, while also reducing the dimensions of members in differing orientations.", "In the example shown in Section 2.2, the boundary dithering is carried out using a 1D filter.", "This requires that the boundary pixels be transformed into a straight path as shown in Fig. 16a.", "The filter is then passed along this path from the start to the end diffusing the pixel error accordingly.", "However, because the start and end of the path are no longer adjacent, these adjacent pixels are not included in the error diffusion processing causing incorrect dithering to some extent.", "In the specific case presented in Section 2.2, this is not of significance as the greyscale values of the pixels at A and B are consistent, but in other cases this may not be the case.", "This issue is more complex when tackling 3D problems as the boundaries are surfaces and so require 2D error diffusion.", "For a simple shape such as the cube shown in Fig. 16b the process of transforming the boundaries to 2D is straightforward, but for a more complex shape, mapping procedures [34] could be applied.", "In the same way that for the 1D case, the adjacent pixels A and B are no longer adjacent following transformation, in the 2D case several adjacent edges are no longer adjacent following transformation.", "For instance, in Fig. 16, edges 2 and 3 are separated while edges 15 and 16 remain adjacent.", "The error diffusion filter is passed over the rectangular region surrounding the unwrapped boundary surface from A to B in a row by row fashion and so does not account for the separated adjacent edges in the process.", "This paper has presented a novel method of generating a functionally graded cellular structure based on a functional variation representation input.", "The spatial variation in the area/volume of the cells is achieved using error diffusion to convert a continuous tone image into binary form.", "It is not claimed that this is an overall optimal solution, but it is an efficient and novel way to create a functionally graded cellular structure with a small number of design variables.", "The effect of the two parameters, image pixel/voxel resolution and greyscale value on the resulting cell edge length and area/volume was investigated.", "For both the edge length and area/volume results, Eq. (4) represented the mean data well.", "In the case of the Voronoi edge length results, the variation in the cell shape causes peaks at certain parameter combinations resulting in a worse fit.", "At these combinations, the edge lengths of the cells are equal and so the variability becomes zero and the mean value increases relative to the other combinations.", "The fitted relationships were applied to an example, where the target was to control the minimum and maximum cell size for the two connection schemes.", "In both cases there was approximately an 8% underestimation of cell area for both minimum and maximum target regions.", "A comparison of the edge length results for the boundary and area was also made, and a step outlined for adjusting the boundary results to ensure a closer match between the edge lengths.", "The limitations of using this error diffusion based approach were discussed, the main ones being the difficulties of specifying structural member orientation and the complexities of error diffusion of boundaries especially when tackling 3D problems.", "The first of these issues could be addressed by using a variable diffusion filter or by applying local dimensional changes based on structural member orientation.", "The second of these issues could be addressed to some extent by using mapping techniques, but a more advanced filtering process where edges that are adjacent are considered simultaneously would be useful.", "To extend the proposed method to 3D, a volumetric functional variation representation is required as an input, which can be obtained by taking slices through a 3D FEA mesh fringe at the same resolution as the in plane dimensions.", "It is important that these resolutions are the same or the cellular structure will be distorted in a particular dimension.", "As would be expected, a 3D error diffusion filter such as that shown in Fig. 2b should be used.", "A constrained 3D Delaunay algorithm capable of handling non-convex point envelopes and connecting interior and boundary dithered points could be used to generate the 3D cellular structure." ]	[ "A novel approach to generate functionally graded cellular structures was evaluated.", "The relationships between the method parameters and cell size were investigated.", "Variation in cell edge length was greatest for the Voronoi connection scheme.", "There was approximately an 8% underestimation of cell area for both target regions.", "Practical implementation implications were discussed for tackling 3D problems." ]
Energy and water use of a small-scale raft aquaponics system in Baltimore, Maryland, United States	Aquaponics is a form of aquaculture that integrates hydroponics to raise edible plants and fish. There is growing interest in aquaponics because it can be practiced in non-traditional locations for agriculture such as inside warehouses and on marginal lands, and it can provide locally grown products without using synthetic pesticides, chemical fertilizers, or antibiotics. Yet questions remain about the ecological and economic sustainability of aquaponics. The objective of this study was to describe the operating conditions, inputs (energy, water, and fish feed) and outputs (edible crops and fish) and their relationship over two years for a small-scale raft aquaponics operation in Baltimore, Maryland, United States. The system had roughly 1% water loss per day and used an average of 35,950 L for replenishment per year. Predicted values suggest rainfall could completely replace the existing water needs. The average energy use was 19,526 kWh for propane and electricity per year at a cost of $2055 US dollars. The largest uses of electricity were in-tank water heaters. Comparing inputs to outputs, 104 L of water, 0.5 kg feed, and 56 kWh energy ($6 in energy costs) were needed to produce 1 kg of crops; and 292 L of water, 1.3 kg feed, and 159. kWh of energy ($12 in energy costs) were needed to produce a 1 kg increase in tilapia. Raising tilapia was a net loss, while raising crops was a net gain when comparing market prices to energy costs. Understanding energy, water, and feed use in aquaponic systems is essential to inform farm business plans. These data can serve as a point of comparison to other small-scale aquaponic systems, and inform future work on life cycle assessments of aquaponics.	[ "AGRI" ]	[ "Aquaculture", "Aquaponic raft culture", "Aquaponics", "Recirculating aquaculture", "Tilapia" ]	[ "1104" ]	[ "Aquaponics is a form of aquaculture that integrates soilless crop production (hydroponics) to raise edible plants and fish.", "The fish are fed and excrete waste, which is broken down by bacteria into nutrients.", "Plants utilize some of these nutrients, and in the process filter the water in the system.", "Most aquaponics systems are recirculating aquaculture systems where water is continuously recycled through an interconnected series of fish tanks and waste treatment systems (Timmons and Ebeling, 2002).", "Early attempts at recirculating aquaculture were challenged by the accumulation of ammonia, a potentially toxic by-product of fish waste (Bohl, 1977; Collins et al., 1975).", "In one approach to improve water quality, researchers incorporated plants as biofilters (Lewis et al., 1978; Naegel, 1977; Sneed et al., 1975; Sutton and Lewis, 1982), which was an early application of aquaponics.", "A common application of aquaponics today is raft (deep water culture) aquaponics, in which water from the fish tanks flows into a series of solid filtration and biofilter tanks, which respectively serve to remove large solids and use bacteria to break down ammonia into nitrate.", "From these tanks water flows through the plant beds before returning to the fish tanks.", "To create a stable ecological system and maximize crop and fish production, aquaponics practitioners now control a variety of factors such as the water temperature, pH, micro- and macronutrients, dissolved oxygen, and sunlight/photo-period.", "Several studies have attempted to optimize various factors and report the commercial production associated with these optimized states (Rakocy, 1984, 2012; Rakocy et al., 2006; Savidov, 2005; Watten and Busch, 1984), and much of this literature has been reviewed by Tyson et al. (2011).", "Aquaponics has been discussed as a part of sustainable intensive agriculture, however there are several limitations to aquaponic food production that may make aquaponics a better or worse fit at certain scales or in some climates or regions of the world.", "The weaknesses of aquaponics, as described in a United Nations Food and Agriculture report, include: it is knowledge intensive, expensive to start-up, energy/resource demanding, requires daily maintenance, has fewer management choices than agriculture or aquaculture, requires access to fish and plant seed, the fish in the system have narrow temperature ranges, and mistakes or accidents can result in catastrophic collapse of the system (Somerville et al., 2014).", "The benefits of aquaponics are the efficient use of water, limited waste, organic-like management, colocation for producing two agricultural products (i.e., edible fish and plants), increased density of crop production, and it addresses a growing interest in locally grown food (Somerville et al., 2014).", "These benefits must outweigh the limitations for aquaponics to be economically viable for the farmer, environmentally sustainable, and beneficial for the community.", "The field of aquaponics has grown dramatically in the past few years (Love et al., 2014), however, data gaps exist on the resource use, cost–benefit analysis, and life cycle assessment (LCA) of aquaponics.", "The objective of this study was to describe the operating conditions, inputs (energy, water, and fish feed) and outputs (edible crops and fish) over two years for a small-scale, raft aquaponics operation in Baltimore, Maryland, United States (U.S.), and explain the relationships between inputs and outputs.", "These data can help fill gaps on energy use in aquaponics, serve as a point of comparison to other small-scale aquaponic systems in other regions with different climates, inform farm business plans, and serve as a starting point for future work on systems-level (i.e., LCA) studies of aquaponics.", "We describe our operation as a “farm,” which fits within the U.S. Department of Agriculture (USDA) definition of a farm as a place where over $1000 in agricultural products were produced and sold during a year (USDA, 2015).", "Over the two-year study period our operation had roughly $10,000 in sales.", "Within the USDA Farm Classification system, our operation most closely fits with a “Residential/Lifestyle farm,” which is a small farm whose operators have a primary occupation that is not farming (in our case educators and researchers) and have gross sales less than $250,000 per year (USDA, 2013).", "The 10.3 m3 aquaponics system was sited in a 116 m2 hoophouse on the grounds of the Cylburn Arboretum in Baltimore, Maryland, U.S.", "The system was operated with fish and plants for six months (starting in June 2012) prior to the beginning of the study period to allow the biofilter to ripen and nutrient levels to increase sufficiently to support consistent crop growth.", "The period under study was January 1, 2013 to December 31, 2014.", "The design and specifications of the system are presented in Fig. 1.", "Four fish tanks were part of the same system and should be considered one experimental unit.", "It is typical for aquaculture systems to have more than one tank so that fish at varying stages of development can be raised and harvested in a staggered fashion.", "The mechanical systems and their energy demands are reported in Table 1.", "Mechanical components drawing electricity were a water pump, an air blower, four in-tank electrical water heaters, a 4-ft wall-mounted greenhouse fan, an inflation blower to maintain a pillow of insulation between the layers of greenhouse film, several box fans to distribute air throughout the greenhouse, and fluorescent lights.", "In cold weather, thermostat-controlled, propane-fired space heaters maintained the air temperature at no less than 4–7 °C.", "If the water temperature dropped below 22 °C, the thermostat-controlled electric heaters operated.", "The system did not have an electric water-cooling mechanism and in summer months the water temperature would increase above 22 °C.", "To mitigate excessive temperature increases, in summer months a 50% shade cloth (Aluminet, Maryland Plants and Supplies, Inc.) was installed above the hoophouse, a reflective plastic tarp was hung 1.5 m above the fish tanks, and a thermostat-controlled 4-ft greenhouse fan was used to pull air through two sets of louvered windows.", "Additional cooling was achieved by rolling up the sides of the hoophouse to 1 m in height.", "In the event of a power outage, backup power was supplied by a propane-driven generator.", "Consistent with state regulations for commercial finfish aquaculture operations, a permit was obtained from the Maryland Department of Natural Resources (DNR).", "The permit requirements included a site inspection, a map of the location, fish health certification and species origin documents, a plan for the treatment of non-native species to prevent introduction into the wild, a waste management plan, and annual reporting of activities under the permit.", "The DNR permit also allows for the commercial sale of live unprocessed fish.", "Fish tanks were stocked with 21 Nile tilapia (Oreochromis niloticus) to ripen the system, and 227 blue tilapia (Oreochromis aureus) were stocked for grow-out.", "For the first year of the study period the fish were fed two different plant based diets: for 9 months a slow sinking feed with 50% protein provided by Watson et al. (2013) and for 3-months an expensive and less palatable USDA Organic feed with 32% protein (AquaOrganic diet from The Aquaponics Source).", "For the second year of the study, a more consistent, commercially available feed was introduced, a slow sinking feed with 35% protein (Finfish Bronze, Ziegler Brothers Inc.", "Gardners, PA).", "Fish were fed by hand once or twice a day in quantities based on the number of fish in the system and their body weight and the water temperature, feeding to satiation.", "Water was continuously cycled through the system at a rate of 93 Lpm throughout the study period.", "Water additions were made from a 625 L storage tank into the aquaponic system.", "The storage tank allows for a waiting period in which chlorine can dissipate from the municipal water supply, which can then be gravity-fed into the hydroponic tanks.", "Sources of water loss were evaporation, evapotranspiration, spillage, leakage, and water exchange (38 L of 10% fish solids per day).", "Originally, fish tanks were operated without covers.", "After experiencing significant condensation during winter months on the interior of the greenhouse film, additional measures were taken to cover the fish tanks in the winter using a radiant barrier (TekFoil) to reduce the heat and water loss due to evaporation out the top of the tanks and reduce the relative humidity in the hoophouse.", "The potential for rainwater use was calculated based on the local water data for monthly inches of rainfall, the square footage of the hoophouse, and an estimated collection efficiency of 70%.", "Rainfall collection potential is reported in Eq. (1) as Lpm.", "In the equation, RW = rainwater, P = collection efficiency (70%), z = amount of rainfall per month, l = hoophouse length, h = hoophouse height, and times 2 because the hoophouse height is ½ the hoophouse width in this case.", "Water treatment was performed using four 190-L cone-bottom clarifiers (one per fish tank) followed by two 132-L biofilter tanks in series (the first one filled with orchard netting, the second filled with aerated Kaldnes K1 media).", "Data on water quality monitoring and the addition of chemical amendments are presented in Fig. 2.", "Alkalinity and pH were maintained using a combination of hydrated lime (Good Earth Organics, Cave Junction, OR) and potassium bicarbonate (Nuts.com, Cranford, NJ), typically on a daily basis in 50–150 g amounts.", "The water pH was measured daily using a colorimeter (model DR850, Hach, Loveland, CO) and a phenol red indicator (Hach).", "The alkalinity of the system water was measured weekly (Lamotte, Chestertown, MD).", "Ammonia and nitrite were measured routinely using a colorimeter (Hach) to confirm that levels were below known tolerances for tilapia.", "Powdered iron chelate (Grow More, Gardena, CA) was added in roughly 20-80 g amounts on a semi-weekly basis to the base addition tank whenever yellowing was observed in plant leaves.", "Greenhouse temperature and water temperature were measured daily using a greenhouse thermometer and an in-tank digital thermometer.", "Dissolved oxygen was not measured routinely.", "There were five main contributors to electrical energy usage in the system: water heaters, air blowers, box fans, a pump, and lights in the winter for seedling germination.", "Propane was used to power a backup generator in case of power outage and heaters to ensure that night-time temperatures in the hoophouse remained above 7 °C.", "Data for electrical and propane usage were collected from the site.", "Energy calculations for the radiant solar heat gain (RSHG) and solar heat gain factor (SHGF) (American Society of Heating, 2013) were estimated based on the orientation of the site (Google Maps, Mountain View, CA), the specific latitude, the cloudiness for the zip code, and estimates of infiltration and exfiltration of outside air due to the prior construction effectiveness of the hoophouse (Eq. (2)).", "In the equation, RSHGN = radiant solar heat gain in the X-direction (i.e., north, south, east, west), SHGFN = solar heat gain factor in the X-direction, AN = area of the hoophouse in the X-direction, Cloud% = percent of cloud cover, ɛ = material emissivity.", "Prior to the study period, sprayfoam insulation and foam-board insulation were installed to reduce thermal losses through doors, leakage through vents and along cracks, and repair holes.", "The electricity consumption during the study was recorded over time from an electric meter.", "Propane use was recorded from the propane tank dial and converted to kWh based on the energy content at sea level.", "Energy costs were estimated based on local energy rates: $0.10/kWh for electricity and $0.11/kWh for propane (or $3.00 per gallon).", "A sample of five to ten fish from each tank were weighed on a semi-monthly basis (13 times over the course of the study), and these weights were multiplied by the estimated number of fish in a tank to calculate a tank weight.", "Samples were obtained by dipping the net into each tank five times and weighing the fish caught.", "Fish were not crowded prior to sampling because the small tank size and a center stand pipe made this impractical.", "The feed conversion ratio (FCR) was calculated by dividing the total feed administered over the time between fish weightings by the change in tank weight over that time.", "A variety of crops were grown in the aquaponics system and harvested on weekly basis starting in January 2013 and sold at market rate at a local farmers market.", "Weights of the harvested portion of each crop were recorded.", "Seasonal plantings included herbs (basil, chives, dill, etc.), loose-leaf greens (salad mix, mustard mix, etc.), greens harvested by the head (lettuces, bok choi, tat soi, etc.), fruiting crops (cucumbers, peppers), and bunched greens (kale, chard, mustard).", "Initially, there was a large water demand to fill the system, and thereafter there was a daily or weekly need to “top-off” the system to replace water lost due to evaporation, evapotranspiration, spillage, leaks, and water exchanges (Fig. 3a).", "The daily water loss of about 1% was near the expected range of 0.5–10% reported previously (Rakocy et al., 2006).", "The total annual water demand to maintain the system was 36.9 m3 in 2013 and 35.0 m3 in 2014.", "Comparing between years, there were natural variations in the amount of monthly water use, for example May 2013 had less water use than May 2014.", "The need for water addition was greater in warmer months than cooler months, and relatively similar between the two years in the study period (Fig. 3a).", "At the scale of the operation, however, natural rainwater collection could supplement or replace any water losses.", "Fig. 3b provides the monthly amount of rainwater in Baltimore, MD in 2013 that could be collected and used in the aquaponics system (93.5 m3 in total), which was greater than the amount needed due to water loss.", "Agriculture is one of the major users of fresh water globally.", "While aquaponics offers a water-efficient method for both aquaculture and hydroponics, in a previous survey of 809 aquaponics operations, 90% of respondents used drinking water (community piped water or well water) as their water source for aquaponics (Love et al., 2014).", "In the survey, however, some respondents (39%) used rainwater to supplement potable water use (Love et al., 2014) indicating that they may be willing to use other biosecure water sources.", "For aquaponics to become more ecologically sustainable, aquaponic system operators and designers should be encouraged to make full utilization of available rainwater.", "To assess the net energy demand in the study system, the RSHG in the hoophouse was calculated for 2013 (Fig. 4) and monthly energy usage was monitored in 2013 and 2014 (Fig. 5).", "Hoophouses are designed for passive solar heating, and the total annual RSHG for the orientation of the hoophouse and its latitude was predicted to be 71,924 kWh, which does not account for shading from nearby trees.", "When accounting for losses due to the hoophouse building envelope, the total effective RSHG annually was determined to be 50,443 kWh, and reported by month in Fig. 4.", "During some periods in the summer, solar energy can completely replace both propane and electric water heating, barring extreme conditions.", "Excess solar energy was deflected in the summer using a 50% shadecloth and heat in the hoophouse was exhausted using fans and roll-up sides.", "The total electricity use in 2013 and 2014 was 10,903 kWh and 10,844 kWh, respectively and varied by month due to seasonal changes in temperature (Fig. 5a).", "Water heating consumed the largest amount of electricity in the aquaponic system in all months except June, July, August and September, when the water temperature was maintained almost exclusively by RSHG.", "Propane use (to maintain air temperature > 7 °C in cold months) totaled 8451 kWh and 8553 kWh in 2013 and 2014, respectively (Fig. 5b).", "These values equate to approximately 315–330 gallons of propane each year.", "The propane volume dial was less precise than the electrical digital readout, and propane readings were taken less frequently than electrical readings, which resulted in variability in reported propane use.", "Due to the use of propane to maintain hoophouse air temperatures, the energy loss from tank conduction decreases.", "Therefore, both the propane and electricity were indirectly and directly heating the water, respectively, and need to be considered as the total resources required for heating water.", "The total combined propane and electricity use was 19,354 kWh and 19,698 kWh in 2013 and 2014, respectively (Fig. 5c).", "January was the month with the highest energy demand, using roughly a third of total yearly energy use.", "The total energy costs in 2013 and 2014 were $2035 and $2074 (Table 2).", "Energy costs in January represent one third of total annual energy costs.", "Total electricity and propane costs were roughly equal, however, the electricity costs had a narrower range ($12–$203 per month) than propane costs ($0–$600 per month), which was due to high propane use in cold weather months.", "These data indicate it is more cost effective to heat water than air during cold weather months.", "The driving factors in heat losses in the study system was water loss through evaporation and evapotranspiration, reheating influent water when “topping up” the system, conduction through the tank walls, and leaks in the hoophouse.", "These sources of heat loss can be minimized through structural improvements to the hoophouse building envelope, more effective insulation of tanks, and more effective lid enclosures.", "Hoophouses have very little insulation and drop to ambient temperatures at night, therefore strategies to capture RSHG during the day using solar thermal water heaters would help reduce energy demands.", "Removing nearby coniferous trees that shaded the hoophouse in the winter and changing the hoophouse orientation so that the broad side faced south, are strategies that would likely have reduced the need for heating in our study.", "There are also opportunities to use heat recovery hot water heaters in line with the aquaponics piping, for example from ice makers and refrigeration.", "Heat recovery provides thermal heat at a cheaper rate than electric resistance heaters, in a more direct and effective delivery method than propane, and improves the cooling performance of the refrigeration equipment.", "Additionally, row covers could be use to protect plants at night from frost to reduce winter air heating needs.", "Warm air rising from the heated water would likely be trapped under the row cover, potentially reducing the need for heating of the air.", "The fish in aquaponics have much higher temperature requirements than the plants, and hardy winter greens are commonly raised in hoophouses throughout the winter in the mid-Atlantic, U.S. often without supplemental heating.", "While taking the winter off may be one possible strategy to lower energy costs, this may be impractical because of the length of time needed for biofilter ripening upon start-up and the need to maintain a fish population to support hydroponics.", "Energy demand and access to electricity are notable limitations of small-scale aquaponics (Somerville et al., 2014).", "In a survey of commercial aquaponics operators, those in temperate to warm climates were four times as likely to be profitable as those in colder climates (Love et al., 2015), suggesting that heating costs could be a constraint.", "Over 70% of commercial systems are sited in a greenhouse or use a greenhouse in combination with other growing locations such as indoors or outside (Love et al., 2015).", "In colder climates there may be a benefit to siting aquaponics operation indoors instead of greenhouse because buildings can be insulated to minimize heat loss.", "Among the drawbacks of such siting, however, is the loss of RSHG and the need to use artificial light for plant production.", "Future studies are needed to compare the benefits and drawbacks of farming in greenhouse versus inside building in cold climates, or whether it is more efficient to use other means such as shipping in food from other regions.", "Fish weight and crop harvests were tracked over the two-year study period.", "Tilapia gained a total of 129 kg in 2013 and 117 kg in 2014 including the weight of harvested and unharvested fish (Fig. 6a).", "The feed conversion ratio (FCR) was 1.29 over 2 years, which was based on a fish community of predominantly blue tilapia (94% blue tilapia, 6% Nile tilapia).", "These findings are similar to previous reported values for single populations of tilapia (Al Hafedh, 1999; Rakocy et al., 2006; Shnel et al., 2002).", "The tilapia grow-out period in this study was longer than a typical grow-out period (Rakocy, 1989), which led to growth rates suppressed relative to feeding rates in 2014.", "Additionally, the system was maintained with colder water temperatures (min.", "22 °C) than what is typical for tilapia in aquaculture.", "There was a concern that warmer water temperatures (>27 °C) could cause plants to bolt prematurely.", "Admittedly, tilapia were not the main profit center of the operation, and balancing the needs of the plants and fish requires some compromises that may affect the productivity of each crop, which is a common limitation facing aquaponic managers.", "Crops were harvested weekly for sale at a local farmers market, with 294 kg harvested in 2013 and 422 kg harvested in 2014.", "These crops were raised from the nutrients from fish with occasional supplemental nutrient additions of potassium, calcium and chelated iron (as described in Section 2).", "The 43% increase in harvests in 2014 can be attributed to improved farm management, and more gains in production efficiency could be possible over time.", "Crop plantings and productivity varied by season, and harvests in March through July were greater than other months due to favorable growing conditions (Fig. 7).", "Low production in December, January, February and the peak of the summer (August) was consistent with others’ experiences growing in greenhouses in the region (personal communication Scott Ritchie, Baltimore City Recreation and Parks, Horticulture Division).", "Winter plant harvests decreased due to ambient air temperature and fewer hours of direct sunlight reaching the hoophouse.", "Summer plant harvests decreased due to heat stress and pest outbreaks of aphids, spider mites, and harlequin beetles that suppressed plant yields.", "Integrated pest management strategies were employed, including the addition of beneficial insects and removing diseased plants, which helped control pest pressure.", "Seasonal variations in production are typical in agriculture.", "Others have also reported seasonal a drop in aquaponics production; in one case cucumber yield dropped in the winter due to root rot caused by Pythium (Savidov, 2005).", "The system promotes nutrient cycling, however not all biomass was utilized.", "A small amount of fish waste solids were discarded.", "Roughly 38 L of 10% fish waste solids were removed daily from the system daily to prevent the buildup of anoxic conditions and were used to fertilize outdoor plants near the facility.", "On a larger scale, others have used geotextile bags to collect, dewater, and compost aquaponic fish waste solids (Danaher, 2009).", "Additional unused biomass included fish mortalities, inedible plant parts (roots, stalks, etc.) and damaged or blemished crops that were not sold.", "Table 3 presents the monthly inputs of water, feed and energy required to produce 1 kg of crops in a small-scale raft aquaponics system.", "An average of 104 L of water, 0.5 kg feed, and 56 kWh energy was needed to produce 1 kg of crops.", "The most efficient season for converting feed to plant biomass was the spring where 1 kg of fish feed was converted into 5 kg of plants, which were mainly leafy green crops such as chard, lettuce, and kale.", "Others have reported as much as 9 kg of lettuce grown using 1 kg of fish feed (Love et al., 2015).", "The average monthly energy cost to grow 1 kg of crops was $6, and varied by month with a maximum of $55 in January to a minimum of $1 in May through August.", "In a study of the economics of small-scale aquaponics in Hawaii, average energy costs were $0.73/kg of lettuce, which were similar to the costs we observed during summer months (Tokunaga et al., 2015).", "At the farmers market where our crops were sold, loose-leaf salad greens sold for $26.52/kg and bunched greens (such as kale and chard) sold for $8.82/kg, which shows that for most months, sales were greater than energy costs when selling at farmers markets.", "Farmers receive higher farm gate prices when selling direct-to-consumers compared to lower farm gate prices for indirect outlets (distributors, retailers, and institutional buyers).", "Moreover, certain growing practices can allow farmers to receive a premium for their product.", "For example, hydroponic and USDA organic greens often fetch a higher price than conventional products.", "Tilapia production was compared to water, feed and energy inputs in Table 4.", "Comparing inputs to outputs, 292 L of water, 1.3 kg feed, and 159 kWh of energy were needed to produce 1 kg increase in tilapia weight.", "Relative to crops, fish require roughly 3-fold more water, feed, and energy per month to produce 1 kg of product.", "The energy costs to produce 1 kg of tilapia was on average $12.", "The market price for whole tilapia sold to restaurants in Baltimore, MD was $8.82/kg, which was less than the energy costs for raising the fish.", "A recent case study of small scale aquaponics in Hawaii had lower input costs for producing fish than in Baltimore, with a total costs $4.80/kg and $1.70/kg from energy costs (Tokunaga et al., 2015), which can be attributed to the warmer climate in Hawaii.", "The sales price for tilapia in the Hawaii case study was $11.02/kg, and assuming that all other factors were equal, the authors speculate that aquaponics would be more profitable than hydroponics given the additional profit from fish (Tokunaga et al., 2015).", "We found the opposite situation in Baltimore, Maryland where fish sales prices were lower than input costs due to higher winter energy costs.", "Approaches to minimizing heating for fish would be raising species that can survive at lower water temperatures and require less heating, implementing renewable sources of energy such as photovoltaic (although there are higher start-up costs), raising higher value species to enhance the market price, or move location to sites with warmer winters or a better insulated space.", "Many factors such as markets, production scale, and start-up and recurring input costs need to be carefully considered when planning aquaponics businesses to avoid pitfalls.", "A limitation of this study was the focus on a single operation as a case study, which can limit generalizability to other sizes and types of aquaponics systems.", "Other limitations were the lack of information on labor costs, which can contribute to half of the input costs (Tokunaga et al., 2015), and capital costs which is another hurdle for starting small-scale operations.", "The operation studied was subject to many of the same challenges and constraints typical of small-scale agriculture: labor shortages, learning curve of management, budget constraints, pest control, etc.", "As such, these data can be useful to aspiring aquaponic growers by providing a real-world example to inform business plans.", "The strength in the present study is in reporting values related to the inputs (water, energy, and feed) and outputs (fish and crops) for a facility located in the Mid-Atlantic and describing relationship between inputs and outputs.", "These data could support future work on cost benefit analyses or life cycle assessments to better flesh out the strengths and weaknesses of small-scale aquaponics.", "Additional case studies are needed to confirm our findings in small-scale raft aquaponics systems.", "One recent case study in Hawaii found small-aquaponics was economically feasible, however the authors were not as optimistic as previous reports and found economic outcomes were sensitive to market prices (Tokunaga et al., 2015).", "Further comparisons are needed across medium and large facilities in operation, in a range of climates, and under different management regimes to see if our findings are relevant for slightly larger or much larger operations." ]	[ "Inputs and outputs of a small-scale raft aquaponics system were measured.", "Edible plants require fewer inputs than fish and are the profit center.", "Small-scale aquaponics needs further study related to resource use." ]
Alkyl-imino sugars inhibit the pro-oncogenic ion channel function of human papillomavirus (HPV) E5	Despite the availability of prophylactic vaccines the burden of human papillomavirus (HPV) associated malignancy remains high and there is a need to develop additional therapeutic strategies to complement vaccination. We have previously shown that the poorly characterised E5 oncoprotein forms a virus-coded ion channel or viroporin that was sensitive to the amantadine derivative rimantadine. We now demonstrate that alkylated imino sugars, which have antiviral activity against a number of viruses, inhibit E5 channel activity in vitro. Using molecular modelling we predict that imino sugars intercalate between E5 protomers to prevent channel oligomerisation. We explored the ability of these viroporin inhibitors to block E5-mediated activation of mitogenic signalling in keratinocytes. Treatment with either rimantadine or imino sugars prevented ERK-MAPK phosphorylation and reduced cyclin B1 expression in cells expressing E5 from a number of high-risk HPV types. Moreover, viroporin inhibitors also reduced ERK-MAPK activation and cyclin B1 expression in differentiating primary human keratinocytes containing high-risk HPV18. These observations provide evidence of a key role for E5 viroporin function during the HPV life cycle. Viroporin inhibitors could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation.	[ "IMMU", "PHAR" ]	[ "EGFR signalling", "HPV", "Viroporin" ]	[ "2406", "3004" ]	[ "Human papillomaviruses (HPV) are small non-enveloped double-stranded DNA (dsDNA) viruses that infect squamous epithelial cells.", "They are sexually transmitted causing approximately 5 million new infections per year in the USA (Cates, 1999).", "Mucosal, low-risk HPV types cause benign anogenital warts, whereas a limited number of high-risk HPV types cause the majority of anogenital cancers as well as a subset of oropharyngeal malignancies; HPV16 and HPV18 are responsible for the largest proportion of carcinomas (de Sanjose et al., 2010; Prigge et al., 2017; Walboomers et al., 1999).", "Prophylactic vaccines are available, yet these protect against only a sub-set of HPV types with little impact upon pre-existing infections (Schiller and Müller, 2015).", "Moreover, HPV vaccination programmes are poorly available in most developing countries where cervical cancer is more frequent (Bruni et al., 2016).", "Effective antiviral agents represent an alternative approach to treating HPV infections, yet no such drugs are currently available.", "High-risk HPV encodes three early oncogenes (E5, E6 and E7) that influence cellular proliferation, differentiation and survival (Moody and Laimins, 2010).", "E6 and E7 functions are well characterised as the major drivers of keratinocyte proliferation, and their expression is retained within tumours.", "By contrast, E5 is less well understood.", "High-risk E5 expression induces anchorage-independent growth in murine fibroblasts and growth in low serum, as well as transformation of primary human keratinocytes (Müller et al., 2015a).", "Mice transgenic for epithelial HPV16 E5 expression display hyperplasia and spontaneous tumour formation (Maufort et al., 2007), with increased cervical dysplasia (Maufort et al., 2010a).", "E5 mRNA is highly abundant in HPV lesions (Stoler et al., 1992), and the protein is likely expressed in the early stages of malignancy, when episomal viral genomes are present (Chang et al., 2001).", "Thus, E5 represents a potential target for early stage intervention in HPV positive cancers.", "E5 potentiates epidermal growth factor receptor (EGFR) signalling, resulting in enhanced mitogen activated protein kinase (MAPK) activity (Straight et al., 1993; Wasson et al., 2017).", "E5 modulation of EGFR is implicated in pathogenesis, and expression of a dominant negative EGFR attenuates epithelial hyperplasia observed in E5-transgenic mice (Genther Williams et al., 2005).", "The generation of high-risk HPV genomes lacking E5 expression has identified a key role during the productive stages of infection (Fehrmann et al., 2003; Genther et al., 2003; Wasson et al., 2017).", "Recently, EGFR activation has been shown to be critical for E5 function during the productive stages of the HPV lifecycle (Wasson et al., 2017).", "In the absence of EGFR signalling, HPV18 containing keratinocytes fail to maintain cyclin B1 expression in the suprabasal layers of the epithelium and instead initiate default differentiation programmes.", "Precisely how EGFR signalling is augmented by E5 is unknown, but may involve E5-mediated alkalinisation of EGFR-containing endosomes, preventing degradation, and resulting in recycling of activated receptor to the plasma membrane (Straight et al., 1995).", "How E5 perturbs endosome acidification remains unknown.", "E5 belongs to an expanding group of viral membrane proteins termed viroporins (Royle et al., 2015; Scott and Griffin, 2015).", "These small proteins (50–120 residues) contain at least one pore-forming helix, which oligomerises to create a hydrophilic pore across membranes.", "Viroporins perturb host cell homeostasis at various stages during infection, often involving viral entry and/or release (Scott and Griffin, 2015).", "However, other cellular functions can also be altered by these proteins, including vesicle trafficking and apoptosis.", "Inhibition of viroporin function represents an ideal target for antiviral drug development (Scott and Griffin, 2015), with clinical precedent set by prototypic adamantane drugs targeting the influenza A virus (IAV) M2 proton channel (Pinto et al., 1992).", "We previously demonstrated that HPV16 E5 forms a hexameric viroporin complex, with activity sensitive to the adamantane rimantadine, as well as novel unrelated scaffolds (Wetherill et al., 2012).", "Here, we now identify alkyl imino sugars as inhibitors of E5 viroporin activity.", "Ensuing blockade of channel activity prevents E5-mediated activation of ERK-MAPK resulting in diminished cyclin B1 expression within suprabasal keratinocytes and concomitant block to the HPV18 life cycle.", "Thus, targeting E5 channel activity represents a viable drug target with potential for both antiviral and anti-tumour applications.", "FLAG-E5 was expressed as a glutathione S-transferase (GST) fusion in Escherichia coli cells and then cleaved and purified as described previously (Wetherill et al., 2012).", "C33A cells were transfected by using polyethyleneimine (PEI) reagent as described in (Müller et al., 2015b) with plasmids expressing GFP and GFP-18E5 in combination with a reporter plasmid in which the human cyclin B1 (CCNB1) promoter drives the expression of firefly luciferase (Wasner et al., 2003).", "A constitutively expressing Renilla luciferase plasmid was used to assess transfection efficiency.", "Transfected cells were serum starved for 12 h and then lysed and assayed for luciferase activities by using Dual-Luciferase Stop and Glo reagent (Promega) and a luminometer (EG&G Berthold) as described (Richards et al., 2015).", "Where appropriate, cells were treated with rimantadine or NN-DNJ (10 μM) for 12 h prior to analysis.", "All assays were performed in triplicate.", "Fold promoter activity was calculated by dividing the relative luciferase activity of stimulated cells by that of mock-treated cells.", "Rhodamine-labelled liposomes comprised of phosphatidic acid and phosphatidylcholine containing self-quenching concentrations of carboxyfluorescein (CF) were generated as described (Wetherill et al., 2012).", "FLAG-E5 induced CF release was assessed by real-time fluorimetry (FLUOstar Optima microplate reader, BMG Technologies).", "Excitation and emission filters set to 485 and 520 nm, respectively) in reactions typically comprising 1 μg protein and 50 μM liposomes in a total reaction volume of 100 μl with HBS, giving a final E5 concentration of ∼1 μM, as described (Wetherill et al., 2012).", "1 μM melittin (Sigma) served as a positive control.", "All samples were repeated in triplicate, and data were averaged.", "Endpoint readings were taken or initial rates were calculated from the initial linear dye release kinetics (ΔFU s−1), where FU are fluorescence units.", "Compounds used were rimantadine-HCl (Sigma), hexamethylene amiloride (HMA) (Sigma), and the imino sugar derivatives N-nonyl deoxynojirimycin (NN-DNJ) and N-nonyldeoxygalactonojirimycin (NB-DGJ) (Toronto Biochemicals).", "All inhibitors were reconstituted as 40 mM stock solutions in DMSO, then diluted as appropriate in media or buffer.", "The secondary structure of E5 was predicted by using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred) and MEMSAT3.", "Models of the E5 monomer were constructed by using Maestro (Schrodinger Inc.) as previously described (Wetherill et al., 2012).", "Monomers were built amino acid by amino acid with energy minimization (Merck molecular force field [MM/FF] in a simulated water dielectric).", "Monomers were docked into an oligomer and subjected to further energy minimization.", "Docking against E5 complexes employed the LigPrep module of Maestro in the Schrodinger docking program Glide (Friesner et al., 2004).", "The LigPrep-treated and energy-minimized ligand was docked into the prepared receptor grid, and the binding affinity was evaluated with the Glide score (GScore) parameter (Eldridge et al., 1997).", "The best-docked pose was selected as the one with the lowest GScore, the highest negative value.", "C33A cells were maintained as described previously (Müller et al., 2015b).", "Transfection of primary human foreskin keratinocytes (NHK) isolated from neonate foreskin tissues (ethical approval no. 06/Q1702/45) was performed as described previously (Delury et al., 2013; Wilson et al., 2005).", "To account for donor-specific effects, cell lines harbouring wild type or E5 knockout (E5KO) HPV18 genomes were generated in NHK isolated from two donors.", "HPV18 containing NHK were grown in complete E media until 90% confluent.", "Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.", "Cells were maintained in this media for between 48 and 72 h before lysis and analysis.", "Total protein was extracted from keratinocytes in lysis buffer (Mankouri et al., 2010) and resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for cyclin B1 (H-433, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), phospho-ERK1/2 (43705, Cell Signalling Technology), GFP (sc-9996, Santa Cruz Biotechnology) and GAPDH (G-9, Santa Cruz Biotechnology).", "Immunoblots were visualized with species-specific HRP conjugated secondary antibodies (Sigma) and ECL reagent (Thermo/Pierce).", "Cells were plated in 96-well plates 24 h prior to treatment with E5 inhibitors.", "Cell viability was determined 24 h post-treatment by MTT assay as described previously (Samson et al., 2016).", "Membrane lipid packing was studied using the fluorescent probe, MC540 (Sigma), assessing the degree of membrane insertion by fluorescence flow cytometry (Suzuki et al., 2010; Williamson et al., 1983).", "GFP or GFP18E5 transfected C33A cells were incubated for 48 h, then detached using an enzyme-free/PBS-based cell dissociation buffer.", "Cells were suspended in 10 μg/ml MC540/PBS for 10 min at 37 °C, then washed thoroughly in PBS before resuspension in 250 μl PBS.", "Cell surface fluorescence was analysed with a BD Fortessa used at 488 nm excitation and 575 nm emission wavelengths.", "Several prototypic classes of inhibitor compounds have been shown to abrogate viroporin function in vitro including rimantadine, nonylated imino sugars (e.g. NN-DNJ) and hexamethylene amiloride (HMA) (Scott and Griffin, 2015).", "These compounds have since been shown to exert antiviral effects against a number of viruses including HCV, BVDV, Dengue and SARS-CoV (Pavlović et al., 2003; Stgelais et al., 2009; Wilson et al., 2006; Wu et al., 2002; Zitzmann et al., 1999).", "As rimantadine effectively blocks HPV16 E5 activity (Wetherill et al., 2012), we examined whether other prototypic viroporin inhibitors might block 16E5 viroporin activity in an in vitro liposome dye release assay (Fig. 1A) (Wetherill et al., 2012), namely HMA, the long alkyl-chain imino sugar NN-DNJ and the short alkyl-chain imino sugar NB-DGJ (Fig. 1B).", "Interestingly, HMA (400 μM) treatment enhanced both the initial rate of CF release from liposomes and likewise increased endpoint fluorescence compared to the DMSO control (Fig. 1C and D).", "In these assays Triton X-100 treatment yielded maximal fluorescence.", "This increase was likely due to either HMA induced fusion events, non-specific disruption of liposomes, or HMA holding the E5 channel complex in a more open conformation.", "In contrast addition of both NN-DNJ and NB-DGJ led to a significant reduction in 16E5-mediated CF release from liposomes, as measured by initial rate or endpoint fluorescence (Fig. 1E and F).", "Next, FLAG-16E5 was pre-incubated with increasing concentrations of NN-DNJ (1–400 μM).", "NN-DNJ potently inhibited E5 viroporin activity in a dose-dependent fashion (Fig. 2A and B); plotting percentage initial rates were plotted against log10[NN-DNJ) (μM) and the approximate IC50 for NN-DNJ calculated to be 6.84 μM (Fig. 2C).", "To confirm that imino sugars did not non-specifically block CF release from liposomes, the bee venom pore forming peptide Melittin was incubated with a high concentration (400 μM) of each imino sugar and the impact on CF release assessed by endpoint fluorescence.", "Compared to a Melittin only control neither imino sugar exerted any inhibitory effect on CF release (Fig. 2D).", "Finally, we confirmed that neither imino sugar could quench CF fluorescence intensities by incubating 400 μM compound with liposomes prior to disruption using Triton X-100 (0.5% w/v).", "As expected, none of the compounds displayed any ability to quench fluorescence (Fig. 2E), confirming that inhibition of FLAG-16E5 was protein-specific.", "To interrogate the potential mode of E5 inhibition by imino sugars we revisited our previous docking studies (Wetherill et al., 2012) and determined the probable binding modes of NN-DNJ and NB-DGJ using the Glide program (Schrodinger).", "Inhibitor compounds were docked into hexameric 16E5 models with the Glide grid extended to allow free docking of the each compound to the entire channel surface.", "Unlike previous studies where rimantadine occupied a lipophilic pocket in the lining of the channel lumen (Wetherill et al., 2012), NN-DNJ instead intercalated between E5 monomers at the protomer interface with a GlideScore of −5.1 kcal/mol (Fig. 3A).", "This was predicted to disrupt the pi stack generated by TM1 Phe15 and TM3’ Phe60 (Fig. 3B), potentially disrupting oligomerisation of E5 protomers.", "NB-DGJ adopted a similar binding pose although was located closer to the pore, and also likely impairs the monomer interaction by disrupting the Thr38 – Thr40 hydrogen bond (Supplementary Fig. 1A).", "NB-DGJ had a similar estimated Glide docking score to NN-DNJ of −5.4 kcal/mol.", "We next wished to assess whether viroporin activity is a conserved feature of E5 proteins from other HPV types and ultimately to determine whether it contributes to the biological function of E5 in cells.", "Our laboratory has begun to study the functions of the E5 protein coded by HPV18 (Wasson et al., 2017).", "As such, we asked the question does 18E5, like 16E5, also possess viroporin activity?", "Despite relatively poor amino acid conservation, the two proteins share similar hydrophobicity profiles indicating the presence of three TMD (Fig. 4A), and in silico modelling predicts the formation of an analogous 18E5 hexameric channel complex (Fig. 4B).", "We were not able to purify recombinant 18E5 using our standard viroporin purification methods (Wetherill et al., 2012), and so were unable to undertake in vitro liposome assay studies with this protein.", "As an alternative strategy, we set out to examine potential 18E5 viroporin activity in cells.", "For this, we assessed the effect of 18E5 on membrane integrity using the lipophilic fluorescent dye Merocyanine 540 (MC540), which provides an indirect measure of lipid packing (Lelkes et al., 1980; Williamson et al., 1983) and has previously been used to investigate viroporin function (Suzuki et al., 2010).", "GFP-18E5/GFP transfected C33A cells were assessed for MC540 labelling using flow cytometry, which showed that the MC540 intensity in 18E5 cells was significantly higher than in GFP expressing cells (3 fold increase; p = 0.01) (Fig. 4C), suggesting that 18E5 disrupts lipid packing and modifies the structure of cellular membranes.", "Consistent with previous observations, levels of ERK-MAPK phosphorylation, but not total protein, were increased in GFP-18E5 expressing cells compared to GFP alone and this correlated with an increase in cyclin B1 expression (Fig. 4D).", "This was reversed by addition of either rimantadine, NN-DNJ or NB-DGJ (Supplementary Fig. 1B) at 100 μM.", "This was not due to cytotoxic effects of these inhibitors (Supplementary Fig. 2).", "A similar reduction in ERK phosphorylation and cyclin B1 levels was observed in C33A cells expressing E5 proteins from high-risk HPV16 (Fig. 4E) and HPV31 (Fig. 4F) types treated with rimantadine or NN-DNJ yet compounds alone had no effect upon levels of basal ERK phosphorylation and cyclin B1 expression in GFP expressing cells (Fig. 4D–F and Supplementary Fig. 1B).", "Moreover, ERK phosphorylation and cyclin B1 expression were also unaltered in HeLa cells, which are HPV positive but lack E5, treated with Rimantadine or NN-DNJ (Fig. 4G).", "Next, we assayed human cyclin B1 promoter driven transcription of firefly luciferase (CCNB1-luc) (Wasner et al., 2003) in cells co-transfected with this reporter and either GFP-18E5 or GFP.", "Luciferase activity was significantly higher in GFP-18E5 expressing C33A cells compared with GFP alone, and this was specifically reversed following treatment with either Rimantadine or NN-DNJ (Fig. 4H).", "Accordingly, neither rimantadine nor NN-DNJ affected luciferase activity in GFP-expressing C33A cells stimulated with EGF.", "Thus, E5 specific up-regulation of cyclin B1 expression following EGFR activation is sensitive to structurally unrelated prototypic viroporin inhibitors, which in turn display no off-target effects upon relevant pathways.", "Hence, we conclude that E5 viroporin activity is directly responsible for these effects.", "Finally, we assessed the biological function of E5 viroporin activity in the context of the productive HPV life cycle.", "Primary human keratinocytes harbouring the wild-type (WT) or an E5KO HPV18 genome were differentiated in high calcium media in the presence/absence of 50 μM Rimantadine (Fig. 5A), NN-DNJ (Fig. 5B) or NB-DGJ (Supplementary Fig. 1C).", "Again, inhibitors were able to reverse the retention of ERK phosphorylation and cyclin B1 expression in the presence of differentiation stimuli, reducing them to similar levels observed in cells harbouring the E5KO genome (compare lanes 2 and 3 plus 3 and 5).", "As a consequence of the loss of mitogenic signalling we noted that wild type cells treated with viroporin inhibitors exhibited an increase in differentiation marker expression.", "Thus, taken together our data demonstrate that E5 viroporin activity is critical for maintenance of mitogenic signalling and the delay in differentiation marker expression observed during the productive stages of the HPV18 life cycle.", "This work provides evidence linking the viroporin activity of high risk HPV E5 proteins (types 16, 18 and 31) with a critical aspect of their function within cells.", "We also show that alkylated imino-sugars represent a second class of prototypic viroporin inhibitor compounds which, in addition to the previously identified E5 blocker, rimantadine, prevent E5-mediated upregulation of cyclin B1 expression via its effects upon EGFR activation.", "Hence, E5 represents the first example of a potentially oncogenic viroporin, making it an excellent prospective target for both antiviral and stratified anti-tumour strategies.", "Treatment with both long and short chain alkyl-chain imino sugar derivatives reduced HPV16 E5 viroporin activity in vitro.", "In particular, NN-DNJ exhibited considerable in vitro potency ([IC50] ∼ 6.84 μM), representing an improvement compared with previous studies of rimantadine or bespoke scaffolds including MV006 (Wetherill et al., 2012).", "However, the potency of both compounds appeared similar in cell-based assays in the low-mid micromolar range.", "This is reminiscent of activity vs. other viroporins for which such prototypic molecules are shown to have activity, including e.g. hepatitis C virus p7 and IAV M2.", "However, whilst lacking true drug-like potency, prototypic viroporin inhibitors are useful for identifying both potential binding sites and inhibitory modes of action that can subsequently be targeted via rational design or compound screening approaches.", "Unlike rimantadine, NN-DNJ is predicted to intercalate between protomers within validated molecular models of the HPV16 E5 channel complex, suggesting that interrupting oligomerisation represents a potential inhibitory mechanism for these compounds.", "Specifically, NN-DNJ binding to the 16E5 channel is predicted to disrupt the pi stack generated by TM1 Phe15 and TM3’ Phe60, reducing the stability of protomer-protomer binding.", "Interestingly, alignment of E5 amino acid sequence identifies equivalent Phe residues in the mucosal HPV types, suggesting that if these residues do mediate oligomerisation then NN-DNJ may have the potential for cross-activity against several HPV types.", "Similarly, NN-DNJ was shown to prevent HCV p7 oligomerisation, with a similar inter-protomer binding site predicted by docking studies (Foster et al., 2011).", "This binding site was validated by the co-location of a resistance polymorphism, F25A, within the site, identified through the innate NN-DNJ resistance of genotype 3a p7.", "As we have employed a similar modelling/docking strategy herein, this highlights a potential mechanistic similarity between 16E5 and p7 oligomerisation, which should be further investigated.", "Lastly, it is notable that shorter chain imino sugars (e.g. NB-DGJ) could inhibit 16E5 viroporin activity in vitro.", "These compounds are ineffective against other viroporins, including p7 (Steinmann et al., 2007).", "Analysis of the NB-DGJ binding mode suggested that it also interacts with E5 at the protomer interface which would likely interfere with channel oligomerisation.", "Satisfied that both rimantadine and the imino sugars represented inhibitors of E5 channel function, we employed them as tools with which to dissect the potential link between E5 viroporin activity and key aspects of E5 function within cells.", "The role of E5 in HPV-associated cancers remains poorly defined.", "As HPV genomes often become integrated during malignant progression, disrupting the E5 ORF, it has been assumed that E5 might play a cancer-promoting role rather than driving progression or persistence post-transformation.", "However, a significant number (∼15%) of cervical cancers contain unintegrated HPV genomes and a much lower frequency of viral DNA integration occurs in HPV positive HNSCC (Morgan et al., 2017; Olthof et al., 2014).", "Moreover, E5 protein can be detected by mass spectrometry in cervical cancer cell lines containing an integrated HPV genome (Sahab et al., 2012).", "Therefore, E5 expression likely persists within a subset of advanced HPV positive tumours.", "This could expedite stratification of therapy for such patients, consistent with the growing recognition that E5 constitutes a potential therapeutic target (Kim and Yang, 2006; Maufort et al., 2010b).", "A major aspect of E5 oncogenic function is the activation of EGFR signalling to drive cyclin B1 expression via ERK in the suprabasal layers of the epithelium (Wasson et al., 2017).", "Interestingly, this likely involves alkalinisation of EGFR-containing endosomes; this was originally ascribed to interactions between E5 and cellular vATPase (Conrad et al., 1993), yet this is now thought not to be the case (Suprynowicz et al., 2010).", "Our previous observation that acidic pH activates E5 channel activity in vitro is reminiscent of other viroporins such as p7 and M2, both of which serve to promote vesicle alkalinisation.", "Consistent with a similar role for high risk E5 in cells, treatment of E5 expressing keratinocytes, or cells harbouring full-length HPV genomes, with rimantadine or imino sugars prevented increased cyclin B expression and concomitant ERK phosphorylation.", "Inhibitors had no obvious off-target effects, with neither cells expressing GFP alone, nor HPV-transformed cell lines with integrated genomes lacking E5, showing any modulation of the pathway upon treatment.", "This observation was consistent between E5 from three high-risk HPV types, suggesting that the link between E5 viroporin activity and at least one major aspect of its pro-oncogenic function are directly linked.", "Taken together, our data supports that E5 is unique amongst viroporins as the only example known to date where channel activity has a direct role regulating cellular proliferation and potentially also malignant transformation.", "Identification of prototypic inhibitors suggests that druggable sites exist within the E5 channel complex that could be exploited by dedicated small molecule drug discovery programmes.", "This could lead to new therapeutic options stratified for patients with tumours that maintain E5 expression, or indeed as an antiviral strategy for HPV positive individuals prior to the onset of malignancy.", "Prototypic viroporin inhibitors from distinct chemotypes are effective inhibitors of E5 viroporin activity in vitro and in primary cell culture models, abolishing ERK-MAPK activation and reducing cyclin B1 expression.", "The distinct mode of action of adamantanes versus imino sugars provides scope for the development of parallel yet complementary E5 inhibitor series that could comprise combination therapies.", "These could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation.", "The authors declare that there is no conflict of interest.", "The work was funded by grants to AM from Cancer Research UK (C43832/A14246 and C37059/A11941), the Medical Research Council (MRC) (MR/K012665/1), a Biotechnology and Biological Sciences Research Council (BBSRC) studentship (DK BB/M011151/1) and a Wellcome Trust studentship (GS 109157/Z/15/Z).", "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." ]	[ "Imino sugars inhibit the viroporin activity of the E5 oncoprotein.", "Imino sugars likely interact at E5 protomer interfaces within a channel to prevent oligomerisation.", "Imino sugars and adamantanes block mitogenic signalling mediated by E5 from a range of high-risk HPV types.", "Viroporin inhibitors reduce mitogenic signalling in differentiating primary keratinocytes containing high-risk HPV18." ]
Tourist-wild boar (Sus scrofa)interactions in urban wildlife management	Wild animals that reach the urban and sub-urban areas have caused various problems in the world. Human-wild boar conflicts have been increasing in these environments since the mid-1990s throughout the world. To manage human-wild boar conflicts in urban environments adequately, it is essential for a culling program to consider the distribution of wild boars and implement an awareness campaign to prevent encounters between human and wild boars. We aimed to visualize the management priority for urban wild boar population from camera trap and a questionnaire survey during August 2017 and November 2018 at Mt. Kinka, Gifu Prefecture, Japan. We investigated distribution and diel activity patterns of wild boar, and the influence of human disturbance on wild boar distribution using camera trap survey. A questionnaire survey clarified distribution of tourists. Considering the RAI for each camera, the number of tourists, areas of residential and agricultural lands for each mesh, and photographic rates in day-time for each camera, we described management priority. Wild boars showed mostly nocturnal activity and avoided the human disturbance in urban environment. In addition, we visualized management priority, suggesting that it is necessary for wildlife managers to apply various counterplans according to information on management priority. Consequently, wildlife managers would be able to select high priority management area using various parameters.	[ "AGRI", "ENVI" ]	[ "Camera trap survey", "Diel activity pattern", "Human-wildlife conflict", "Management priority", "Urban environments" ]	[ "1105", "2303", "2309" ]	[ "Wild animals that reach urban and sub-urban areas have caused various problems in the world (Soulsbury and White, 2015).", "Particularly, urbanized large mammals pose risks such as human injury and traffic accidents (Bissonette et al., 2008; Ng et al., 2008).", "In addition, the risk of bringing infectious diseases into urban areas requires attention (Raizman et al., 2013; Kilpatrick et al., 2014; Werden et al., 2014).", "On the other hand, urban wildlife provided several benefits (e.g. ecosystem engineers, important predators of pest species, and recreational value) to human, and so it is important for researcher to consider the human-wildlife relationship in a more holistic way (Soulsbury and White, 2015; Lischka et al., 2018).", "Thus, wildlife managers need to select high priority management area in urban environments, considering advantages and disadvantages between human and wildlife.", "Wild boar and feral pig (Sus scrofa) commonly come into conflict with human societies throughout the world (Chauhan et al., 2009; Licoppe et al., 2013).", "In rural areas, crop damage and digging for invertebrate prey by wild boar has often frustrated people, especially land managers (Amici et al., 2012; Lombardini et al., 2017).", "Although wild boars were considered an omnivore and consumption of agricultural crops by wild boars varied between study areas, they recognized agricultural crops as important food resources and significantly damaged to a variety of agricultural crops in Western Europe (Schley and Roper, 2003).", "Similarly, wild boars recognized agricultural crops as potentially important food resources in agricultural land and riparian habitats in Spain (Herrero et al., 2006).", "From this kind of circumstance, the number of attacks by wild boar have been increasing in urban and sub-urban environments since the mid-1990s (Mayer, 2013).", "A study in Korea showed that a total of 596 wild boars appeared in downtown areas and injured 24 people in 2012 (Lee, 2016).", "Furthermore, this study reported that 294 of 596 appearances occurred in downtown Seoul.", "In addition to human injuries, a risk of transferring leptospirosis and Streptococcus suis from wild boars to humans in urban environments has been found in Berlin, Germany (Jansen et al., 2007), in Barcelona, Spain (Fernández-Aguilar et al., 2018), respectively.", "It is known that behaviors of wild boar could be influenced by human disturbance in various environments.", "For example, activity of wild boars in rural area were changed to be nocturnal pattern by hunting activity (Ohashi et al., 2013; van Doormaal et al., 2015).", "On the other hand, several studies have compared behaviors of wild boar in urban environments with those in rural environments (Cahill et al., 2012; Podgórski et al., 2013).", "However, few studies have focused on urban environments only, and there is no information on the direct influence of human activity on behaviors of wild boars.", "In Japan, previous studies suggested a risk of human injuries and zoonoses from interactions between humans and mammals in urban environments (e.g., Asiatic black bear Ursus thibetanus and Japanese macaque Macaca fuscata, Honda, 2009; red fox Vulpes vulpes, Tsukada et al., 2000; raccoon Procyon lotor, Ikeda et al., 2004).", "There has also been a review of urban wildlife management (brown bear Ursus arctos, Sato, 2017), and an investigation of urban sika deer (Cervus nippon yesoensis) populations (Matsuura, 2018).", "Particularly, human-wild boar conflicts have increased in recent years.", "Wild boar cause not only agriculture damage (Ministry of Agriculture, Forestry and Fisheries, 2017), but also human injuries.", "For example, wild boars in downtown of Kobe city, Hyogo, caused 45 and 33 human injuries in 2015 and 2016, respectively (Hyogo Prefecture, 2017, 2018).", "To manage human-wild boar conflicts adequately, it is essential for a culling program to consider the distribution of wild boars and implement an awareness campaign to prevent encounters with them.", "Recently, camera trap survey has increased using to clarify diel activity pattern (Chen et al., 2009; Ohashi et al., 2013; Ikeda et al., 2016; Torretta et al., 2016), density (Sarmento et al., 2010; Anile et al., 2014; Soofi et al., 2017), and habitat selection (Chen et al., 2009; Sarmento et al., 2010; Curveira-Santos et al., 2017) of wildlife.", "This survey would be adequately able to evaluate biological information on wild boars.", "On the other hand, in order to make effective counterplans for management, we need to pay attentions not only to animals but also to humans (Dickman, 2010; Lischka et al., 2018).", "Therefore, we aimed to visualize the management priority for urban wild boar population from camera trap and a questionnaire survey.", "We evaluated the distribution and diel activity patterns of wild boars from camera trap survey.", "Through the questionnaire survey, we investigated the spatial patterns of tourists.", "Furthermore, we assessed the influence of human disturbance on wild boar behaviors and discussed how wildlife managers would manage wild boar population in urban environments using adequate counterplan.", "We conducted the study at Mt. Kinka, Gifu Prefecture, Japan (35°26′N, 136°45′E, Fig. 1).", "Mt. Kinka is located in the center of Gifu city, where has a population of 400,000 people, and a large-scale road network and several kindergartens, schools, and city parks are present around our study area (Fig. 1C).", "The total area of the mountain was 597 ha, and elevation was 328.8 m.", "There was Gifu castle at the top of Mt. Kinka, which is a famous tourist spot in Gifu city.", "Approximately 200,000 tourists visit the castle throughout the year via two ways.", "For one way, there were 45 mountain trails that tourists could use to visit the top of the mountain on foot.", "For another way, there was a ropeway leading to the top of the mountain from residential land northwest of this mountain that tourists could also use.", "Wild boars were found in this study area in 1997, and they have mainly caused damages to home gardens and hiking courses.", "In addition, traffic accident by wild boars were also recorded in 2011.", "Gifu city has carried on the culling program against wild boars since 2009 to reduce these damages and prevent human injuries.", "The program has culled 483 wild boars up to August 2018, and about 67–79 wild boars were culled during 2015 and 2017 (Fig. 2).", "Environmental structures within 20 survey meshes (Fig. 1C), including Mt. Kinka, mainly consisted of residential lands (31.5%), deciduous broad-leaved secondary forests (21.7%), evergreen conifer secondary forests (20.3%), and evergreen broad-leaved forests (11.1%).", "Agricultural lands accounted for only 9.5 ha (1.1%).", "The major canopy community was oak (Quercus serrata) community in deciduous broad-leaved secondary forests, Japanese red pine (Pinus densiflora) community in evergreen conifer secondary forests, and Japanese Chinquapin (Castanopsis cuspidata) community in evergreen broad-leaved forests.", "We used 20 infrared-triggered cameras (Ltl Acorn 6210; LTL Acorn Outdoors, Green Bay, Wisconsin, USA) from August 2017 to November 2018.", "To investigate the distribution of wild boar in our study area equally, we firstly divided the study area into 28 meshes, 42.5 ha (650 m × 650 m) in size.", "Second, we generated 100 m buffer from center of each mesh.", "Because this buffer in 8 meshes did not include our study area, we could not place cameras in these meshes.", "Thus, 20 cameras set within 100 m buffer from center of each mesh (Fig. 1C).", "We placed cameras near animal signs which were considered as origin to wild boars (e.g., trails and digging) of wild boars.", "The cameras were strapped to trees at approximately 1 m above the ground, and their photographic programs were 3 photos per event with 5-min delay between consecutive events.", "We checked SD cards and batteries once every 2 months.", "All photos taken by the cameras recorded date, time, and species, and we recorded the number of wild boars in 3 categories (adult, piglet, and unknown).", "Following Ikeda et al. (2013), we excluded all photographs of wild boars within <1-h period to avoid overestimation of the relative abundance index (RAI; O'Brien et al., 2003) from double counts.", "However, we used all photographs to evaluate diel activity pattern of wild boars following Ikeda et al. (2016).", "The survey was conducted from September 12 to October 21 (10 human-days) at the top of Mt. Kinka and the ropeway landing.", "We surveyed 205 tourists.", "A questionnaire was answered on site and collected.", "When respondents had questions about questionnaire, they were able to ask researchers.", "Although we gathered various information such as consciousness on wild boars and opinions on counterplans to wild boars in Gifu city from tourists, these information were excluded from our analysis because we focused on tourists’ routes.", "To compare the results with those of the camera trap survey, tourist routes were converted to the number of tourists on each mesh.", "We associated tourist routes with 20 meshes in advance.", "For example, when one person used one tourist route including M1, M2, M7, and M8, we recorded the number of tourist at M1, M2, M7, and M8.", "Then, we divided the number of tourists for each mesh into 5 levels using the natural breaks (Jenks) classification methods in QGIS (Quantum GIS Development Team, 2014).", "We calculated RAI (the number of wild boars photographed per 100 trap-days) from the number of wild boars photographed and trap-days for each camera throughout the study period.", "Thereafter, to investigate the influence of human disturbance on wild boar distribution, we calculated RAI in the day-time and night-time.", "Following Ohashi et al. (2013), we defined day-time and night-time periods according to times of sunrise and sunset each day in Gifu [35°25′N, 136°46′E] (National Astronomical Observatory of Japan, 2018) and classified all photographs into one of the two time periods.", "We used a generalized linear mixed model with a Poisson distribution in the lme4 package in R (Bates et al., 2015) to analyze the data.", "We set the total number of wild boars photographed by each camera in the day-time and night-time as the response variables and the number of tourists and areas of residential and agricultural lands as explanatory variables.", "We set each camera site as a random factor and trap-days for each camera as an offset term.", "Then, we selected the minimum adequate model (⊿AIC < 2) from all possible models using Akaike's information criterion (AIC) (Burnham and Anderson, 2002).", "We used kernel density analysis to investigate diel activity patterns of wild boars (Ridout and Linkie, 2009; Ikeda et al., 2016).", "We compared photographic frequencies (the number of wild boars photographed per hour) between day-time and night-time throughout the study area.", "To investigate relationship between diel activity patterns of wild boars and the number of tourists, in addition, we provided both photographic frequencies for each mesh.", "We used a Wilcoxon signed-rank test for comparison of both photographic frequencies.", "We photographed a total of 2868 wild boars in 9001 trap-days and investigated diel activity patterns of wild boars using these photos.", "We calculated RAI using 2260 wild boars to avoid double counts.", "The highest RAI of wild boars was at M10 (118.62), followed by M4 (47.67), M11 (46.43), M9 (38.56), and M16 (35.71) (Fig. 3A).", "Meanwhile, the lowest RAIs of wild boars were at M19 (2.69), M12 (4.27), M6 (4.64), and M15 (6.76) (Fig. 3A).", "The highest value of spatial usage of tourists was at M2 (173), followed by M7 (152), M8 (136), and M1 (122) (Fig. 3B).", "However, there were no tourists use at 9 meshes (M4, M5, M10, M15, M16, M17, M19, and M20) (Fig. 3B).", "For day-time RAI, the best model was based on the number of tourists and area of residential land (Table 1), and both variables had a negative influence on the distribution of wild boars.", "Conversely, the best model of night-time RAI was based on areas of residential and agricultural lands (Table 1).", "Residential land had a negative influence on the distribution of wild boars, while agricultural land had the opposite influence.", "Photographs of wild boars concentrated on night-time, photographic frequency in night-time (0.45 ± 0.02 wild boar/hour) was higher than that in day-time (0.07 ± 0.01 wild boar/hour) (V = 3478, P < 0.01).", "Similarly, wild boars in 17 meshes photographed in night-time frequently, while we found no significant differences in both photographic frequencies in 3 meshes (M8, M11, and M15) (Fig. 3B).", "In addition, the density of activity was low from sunrise to afternoon, increased from before sunset, and peaked at 2000 (Fig. 4).", "We clarified the spatial usage and diel activity patterns of wild boars and visualize the management priority in tourist spot in urban environments using camera trap and questionnaire survey data.", "We considered the high priority area for wild boar management to be where there was a high photographic frequency of wild boars in day-time, higher number of tourists, and greater area of residential and agricultural lands.", "It is known that the behaviors of wild boar are influenced by direct human presence and indirect human-induced habitat, and they show nocturnal activity in urban environments and forest environments (Podgórski et al., 2013; Brivio et al., 2017).", "Similarly, wild boars in this study area at Mt. Kinka avoided human disturbance and showed variation of their behaviors in urban environments.", "RAIs of wild boars were low both in day-time and night-time at several meshes, including residential lands.", "Previous studies reported that wild boars avoided habitats close to settlements to decrease the risk of encountering humans (Ohashi et al., 2013; van Doormaal et al., 2015).", "Wild boars in this study area were frequently photographed in night-time at several meshes, including more agricultural lands.", "Similarly, wild boars in Yamanashi, Japan, used orchards and abandoned orchards in night-time to avoid human contact (Ueda and Jiang, 2004).", "The results of the present study are consistent with this previous study and suggest that wild boars access food resources in areas of low human activities.", "In this study, we found a negative relationship between tourists' activity and wild boars’ activity in day-time, and total RAI was low in several meshes with a large number of tourists (excluding M11).", "Therefore, we suggest that wild boars might avoid human disturbance not to enhance encounter risk in Mt. Kinka.", "However, spatial patterns of wild boar were influenced by the availability of food and shelter and deviated from an optimal foraging strategy (Keuling et al. 2008a).", "Thus, wildlife managers are required to implement awareness campaign at several meshes (M10, M4, M11, M9, and M16), where recorded higher RAI, towards local residents.", "Further study is required to clarify the relationship between spatial patterns of wild boar and other factors, excluding human disturbance.", "As suggested from previous study (Brivio et al., 2017), wild boars in this study also showed clear nocturnal activity.", "Diel activity patterns of wildlife are also influenced by human disturbance (Gaynor et al., 2018).", "In Japan, previous studies indicated that activity of wild boars changed to nocturnal pattern in response to human hunting activity (Ohashi et al., 2013; van Doormaal et al., 2015).", "In this study area, wild boars have been culled since 2009, which might have influenced their nocturnal activity.", "However, we found no significant differences in photographic frequencies between day-time and night-time in 3 meshes.", "Surprisingly, wild boars were photographed during day-time in M8 and M11, where the number of tourists was large.", "To prevent encounters between tourists and wild boars, wildlife managers are required to cull wild boars and implement awareness campaign towards tourists.", "A previous study suggested that activity patterns of wild boars were influenced by seasonal factors, rather than human hunting activity (Keuling et al. 2008b).", "For example, activities of feral pigs in Australia were highest during April (early dry season) and lowest in October (late dry season) (Caley, 1997).", "In addition, the duration of activity of wild boars in Sweden was influenced by wind speed, relative air humidity, and snow cover (Lemel et al., 2003).", "Moreover, activity of wild boars in Italy increased when food supply was low (Massei et al., 1997).", "Although diel activity patterns of wild boars in this study area was probably influenced by the culling program, we need to investigate the influence of several factors (e.g., foods, habitats, and weather conditions) on diel activity in the future.", "Furthermore, a previous study reported the importance of anthropogenic food sources in peri-urban areas (Cahill et al., 2012).", "This previous study suggested that wild boars indirectly fed anthropogenic food sources (e.g. remnants of pet foods and discarded rubbish), not only direct feeding, and this feeding opportunities accelerate an increase in day-time activity and the loss of a fear response to humans (Cahill et al., 2012).", "In fact, the habituation of wildlife contributes to human induced human-wildlife conflicts (e.g., white-tailed deer Odocoileus virginianus, Storm et al., 2007; Coyotes Canis latrans, White and Gehrt, 2009), and these species attacked humans in urban environments.", "In this study area, there were no reports of direct feeding to wild boars, while wild boars were attracted to indirect anthropogenic food sources, such as agricultural land.", "If there are no human disturbances (e.g., culling and tourism) to wild boars, they could become habituated to humans.", "In this study, we questioned 121 of 205 tourists who knew that wild boars inhabited the study area whether they had encountered wild boars.", "In reality, 34 of 121 tourists (28.9%) had encountered wild boars, and 23 tourists (67.6%) during 0800–1700.", "Thus, it is possible that day-time encounters would increase even more in the future if human disturbance on wild boars became lower.", "In Gifu city, wildlife managers have culled wild boars since 2009, and they are required to continue culling wild boars to reduce various damages, prevent human injuries, and avoid habituation of wild boars for hereafter.", "In addition, a previous study in Barcelona recommended vegetation clearing and an awareness campaign to manage urban wild boar populations (Castillo-Contreras et al., 2018).", "To manage human-wildlife conflicts, both social and ecological information should be handled (Dickman, 2010; Lischka et al., 2018).", "In this study, we visualized management priority, considering the biological information on wild boars, the presence of tourists, and artificial environments.", "Previous study reported that home range size without feeding covered 81.4–132.4ha in Shimane, Japan (Kodera et al., 2010).", "Considering their home range size, therefore, wildlife managers in this city should implement these methods (e.g., culling programs, vegetation clearing, and awareness campaign) at M11, where encounter risks are high, and several surrounding meshes.", "Recently, human-wildlife conflicts (e.g. human injuries, traffic accidents, and infectious diseases) in urban environments are increasing throughout the world (Soulsbury and White, 2015).", "Some effective counterplans toward these conflicts have been developed, while it is unclear for wildlife managers where to carry out them.", "Thus, wildlife managers in urban environments would be able to select high priority management area, which was visualized by information on human and wild boars in this study.", "Because of the lack of data in this study, it was difficult to conduct complementary analysis, which have used to conduct spatial conservation prioritization.", "Further study is required to quantify management priority using this analysis.", "Furthermore, wildlife managers need to recognize benefits of wildlife to humans, not only considering human-wildlife conflicts.", "Although we focused on human-wildlife conflicts, wildlife managers should apply benefits of wildlife to management priority.", "In the future, wildlife managers should consider management priority for various wildlife (e.g. carnivores and medium- and large ungulates) not only in popular tourist spots and parks under urban environments, but also national parks under sub-urban and rural environments." ]	[]
Intentionality and temporal binding: Do causality beliefs increase the perceived temporal attraction between events?	Intentional motor actions and their effects are bound together in temporal perception, resulting in the so-called intentional binding effect. In the current study, we address an alternative explanatory mechanism for the emergence of temporal binding by excluding the role of motor action. Employing a sensory-based Libet clock paradigm, we examined temporal perception of two different auditory stimuli, and tested the influence of beliefs about the causal relationship between the two auditory stimuli, thus simulating a crucial feature of intentional action. In two experiments, we found a robust temporal repulsion effect, indicating that instead of being attracted to each other, the auditory stimuli were shifted away from each other in temporal perception. Interestingly, repulsion was attenuated by causal beliefs, but this effect was fragile. Furthermore, temporal repulsion was unaffected by the intensity of prior learning. Findings are discussed in the context of intentional action awareness research and multisensory integration.	[ "PSYC" ]	[ "Intentional action", "Perceived causality", "Temporal binding", "Temporal repulsion" ]	[ "3204", "3205" ]	[ "The ability to integrate sensory information into a coherent picture of the world is an important human trait.", "It enables people to build a conscious representation of complex visual scenes and auditory events that are otherwise disconnected in time or space (Serences & Yantis, 2006; Shamma, Elhilali, & Micheyl, 2011).", "Apart from integrating perceptual input, humans also form coherent representations of their own behavior (Aarts & Custers, 2009; Vallacher & Wegner, 1987).", "Self-produced actions that predict and result in sensory feedback, such as pressing keys that result in a sound or light, are perceived as coherent events of meaningful behavior (e.g., playing a tone on the piano or illuminating a room), even though action and effect are spatially and temporally separated and do not form a single entity.", "This ability to construct a coherent conscious experience of one’s own behavior has important implications for the understanding of the self and the sense of agency, i.e. the feeling of causing one’s own action and effects in the external world.", "Specifically, people perceive their intentional actions and resulting outcomes as if they are temporally bound together in conscious awareness; a phenomenon that is referred to as intentional binding (Haggard, Clark, & Kalogeras, 2002b).", "It is assumed that this temporal binding effect is rooted in internal prediction processes resulting from intentionally selected motor actions, causing the compression of the time interval between action and effect and the formation of a coherent representation of the action-effect event (Frith, Blakemore, & Wolpert, 2000).", "Recent research, however, suggests that this temporal binding effect might not be the result of intentionality per se (e.g., Desantis, Hughes, & Waszak, 2012; Dogge, Schaap, Custers, Wegner, & Aarts, 2012; Hughes, Desantis, & Waszak, 2013), but rather a product of other factors that are associated with intentional action.", "In the present study, we examine this subject in more detail and raise the question whether the temporal binding effect is similarly sensitive to events that do not require any form of action.", "Building on the notion that intentional actions are associated with beliefs in causality that might bias the perceptual relation between two events (Buehner & Humphreys, 2010; Desantis, Roussel, & Waszak, 2011; Eagleman & Holcombe, 2002), we tested whether causality beliefs cause two successive auditory stimuli to be perceptually bound together in the Libet clock paradigm.", "The emergence of such a binding effect as a function of causality beliefs would undermine the validity of the temporal binding task as a read-out of intentionality and a measure of sense of agency.", "One central argument that has been put forward in favor of the central role of intentionality in action concerns the impact of externally induced action on temporal binding.", "Some studies employed transcranial magnetic stimulation (TMS) to induce an involuntary action followed by a tone (Haggard & Clark, 2003; Haggard et al., 2002b).", "Subjects judged the time of movement onset and tone onset by using the Libet clock.", "The time judgments did not reveal a temporal binding effect but a clear and strong repulsion effect instead, indicating that subjects perceived the motor movement and subsequent tone as two separate events.", "While these TMS experiments suggest that the temporal binding task can distinguish involuntary from voluntary action in producing a coherent representation of one’s own action, it is important to note that the magnetic stimulation of motor areas results in an abrupt, reflexive and oddly felt movement of a limb (here a finger).", "Clearly, human agents have the capacity to evoke actions by direct brain stimulation, but such action onset does not fit with everyday externally induced motor movement where intentionality is ruled out, for example when someone else pushes or pulls one’s hand into a specific direction.", "Building on this notion, recent studies show that temporal binding might be reduced but does not vanish for actions that are not self-chosen or driven by internal motivation (Antusch, Aarts, & Custers, 2019; Caspar, Christensen, Cleeremans, & Haggard, 2016) or passively induced in the agent by another experimenter or a mechanical lever press (Borhani, Beck, & Haggard, 2017; Dogge et al., 2012; Engbert & Wohlschläger, 2007; Moore, Wegner, & Haggard, 2009).", "These findings suggest that it is not intentionality per se that drives the temporal binding effect, as externally induced movement does not cancel out temporal binding in settings that include clear causal relations between action and outcome.", "One explanation for the observed binding between externally induced actions and a tone might be that participants were able to predict the outcome and believed to have a causal role in producing it.", "Causal beliefs impact cognitive processes at several stages, e.g., learning, judgment and decision-making (Harris, Fiedler, Marien, & Custers, 2019; Lucas & Griffiths, 2010).", "Moreover, higher-order causal beliefs have been shown to affect low-level processes of perception (Buehner & Humphreys, 2009, 2010; Cravo, Claessens, & Baldo, 2009).", "A study by Buehner and Humphreys (2009), for example, suggests that mentally linking one’s key press to a subsequent tone following a presumed causal association enhances the anticipation of the tone.", "However, not only effects stemming from motor actions are subject to temporal binding, also external (visual) events can be spatially bound together when they share a common causal association (Buehner & Humphreys, 2010).", "Accordingly, holding a higher-order belief about the causal relationship between two events increases binding between them, even when action is not actively involved.", "In a further exploration of the role of causality beliefs in temporal binding of involuntary movement and effect, Dogge et al. (2012) subjected their participants to a temporal binding task with the Libet clock.", "Participants either executed a voluntary key press or an involuntary key press that was induced by a lever pulling down their index finger.", "In the involuntary conditions, some participants were asked to consider themselves as the agent, construing the resulting effect (tone) as being caused by their key press.", "The magnitude of the binding effect increased between the simple involuntary and involuntary self-causation condition, with mean judgment errors (indicating a stronger binding effect) being larger in the involuntary self-causation condition (Dogge et al., 2012).", "Similarly, it has been shown that explicit beliefs of being the causal agent resulted in stronger intentional binding in participants in a joint action task (Desantis et al., 2011).", "Whereas informative, these studies do not entirely rule out the role of motor prediction and intentionality in temporal binding between action and effect.", "It is for example conceivable that participants started up their motor system once they noticed a slight movement of the mechanical lever, causing them to experience a motor prediction signal and/or intentionality over the key press.", "Because of the tight link between perception and action (e.g., Decety & Grèzes, 2006; James, 1890; Prinz, 1997), a similar reasoning might account for findings that show binding effects as a result of observing others’ action (e.g., Desantis et al., 2012; Kirsch, Kunde, & Herbort, 2018).", "A more stringent test of the role of causality beliefs in temporal binding is provided by sensory paradigms that do not employ motor action.", "In one such study by Haggard and colleagues (2002a), participants judged the temporal onset of two identical successive auditory stimuli that were separated by a 250 ms time interval.", "Results revealed that especially the second stimulus was temporally repulsed from the first stimulus (Haggard, Aschersleben, Gehrke, & Prinz, 2002a).", "Similar results were obtained by Desantis et al. (2012) who compared the temporal perception of two non-motor stimuli separated by a 400 ms time interval against an operant action condition in a Libet clock paradigm.", "The findings were such that both, congruent and incongruent (with precedingly learned associations), tone pairs resulted in temporal repulsion instead of temporal binding, suggesting that temporal contiguity and identity prediction were not sufficient for binding to emerge (Desantis et al., 2012).", "The first tone was perceived as occurring earlier than in actuality, and the second stimuli was perceived as occurring later than in actuality, leading to a repulsion instead of a binding effect.", "Thus, instead of integrating the two stimuli such as in intentional binding, participants seemed to detach the two from each other by experiencing them as two separate entities and shifting them away from each other in temporal perception.", "It is important to note that these studies did not investigate the role of causal beliefs and might have suffered from flaws that reduced the probability of observing a temporal binding effect.", "For example, Haggard et al. (2002a) used successive tones of the same frequency and length.", "Hence, there might have been little inclination for participants to integrate the tones into a coherent event and to perceive them as causally related.", "Thus, participants perceptually shifted the tones away from each other.", "Using tones of different frequencies, Desantis et al. (2012) paired the tones in congruent and incongruent within-subject blocks and employed an unconventional 400 ms time interval.", "Because the tones were sometimes congruent and sometimes incongruent, a stable predictive link could not be formed.", "Furthermore, it is unclear how the extended time interval affected the results.", "Finally, while suggesting temporal repulsion, comparisons with the baseline tone condition in the Haggard et al. (2002a) study only suggest temporal repulsion of the second tone, while the first tone seems to even be bound to the subsequent tone.", "Desantis et al. (2012) only tested the temporal perception of the second tone while a comparison baseline condition was lacking completely.", "Hence, the interpretation of the findings as demonstrating temporal repulsion was neither statistically tested nor can it be claimed in the absence of a comparison baseline condition.", "Thus, it remains subject to empirical scrutiny whether external stimuli in successive contexts are repulsed or bound to each other in temporal perception.", "The present study therefore aimed to replicate the temporal binding (repulsion) effect with two different auditory stimuli by employing a unimodal sensory paradigm based on the Libet-clock.", "In our task, participants acquired the associations between two auditory stimuli pairs, such that the first stimulus predicted the onset of the second one.", "Each pair consisted of a noise sound (such as a scratch sound) followed by a sinusoidal tone 250 ms later.", "The pairings remained constant for each participant, allowing for participants to perceive the stimuli as being linked and represented as one event.", "Furthermore, as an important addition to earlier work, we examined the role of causality belief in the perceived temporal attraction between the two auditory stimuli.", "To that end, we manipulated explicit beliefs about the causal relationship between the two successively presented auditory stimuli to more stringently test their influence on temporal binding of the stimuli.", "The first experiment addresses the basic role of perceived causality in modulating the temporal binding between two different auditory stimuli.", "Experiment 2 was designed to further explore the role of perceived causality in a context where the association between the first and second auditory stimulus was strengthened by practice.", "In total, 641 individuals (49 females), with a mean age of 23 years (M = 23.02, SD = 6.15) participated in the experiment in exchange for course credit or monetary reimbursement.", "Participants were randomly assigned to one of two between-subject conditions, resulting in 33 participants in the control condition and 31 participants in the causal belief condition.", "The study was carried out in line with the guidelines of the Declaration of Helsinki and was approved by the Ethics committee of the Faculty of Social and Behavioral Sciences at Utrecht University as part of a project line (ethics approval code: FETC17-124).", "All participants gave written informed consent.", "A 2 (target: noise vs. tone) × 2 (type of trial: baseline vs. succession trials) × 2 (condition, between: control vs. causal belief) mixed design was employed.", "The experiment consisted of an altered Libet clock paradigm as used by Haggard et al. (2002) and was programmed using Eprime Software version 2.0.", "Four different auditory stimuli of 100 ms length were used: two noise sounds and two sinusoidal tones.", "The noise sounds resembled scratch sounds while the sinusoidal tones were of either 300 Hz or 1000 Hz frequency.2", "All stimuli were played via a Sennheiser headphone.", "The experiment began with an acquisition phase in which participants were familiarized with the auditory stimuli and their combinations.", "At the start, participants were told that four different auditory stimuli would be used in the experiment and that they would begin with experiencing each stimulus once.", "While participants experienced a stimulus, an on-screen message stated which stimulus they were experiencing.", "By pressing F1, they could advance to hearing the next stimulus.", "Subsequently, to facilitate the distinguishability of the stimuli, participants experienced each stimulus four additional times.", "These trials advanced automatically and like in the preceding trials, it was stated on screen which stimulus participants were experiencing.", "After participants were familiarized with the different auditory stimuli, they learned that each noise sound would be paired with one of the sinusoidal tones during the experiment.", "In the control condition, participants were told that the computer would determine the combinations of the noise and tone, and that they would therefore be independent of each other.", "Put differently, the pairs of auditory stimuli would not be causally related.", "They then heard the independent noise – tone combinations.", "In the causal belief condition, participants learned that the computer would choose the noise that would cause the second tone to occur.", "The second tone would thus be dependent on the selection of the noise.", "Put differently, the pairs of auditory stimuli would be causally related.", "They then heard the dependent noise – tone combinations.", "The noise – tone combinations were counterbalanced across participants but remained constant for a given participant.", "In both conditions, the noise – tone combinations were presented five times each (i.e., ten trials in total).", "Participants were asked to pay close attention.", "They then completed eight trials in which they heard noise – tone combinations that were either congruent or incongruent with the previously learned pairs.", "In the control condition, they were asked to indicate which combinations of tones were presented before, whereas in the causal belief condition, they were asked to indicate which combinations of tones were causally related.", "This was done to further emphasize and boost the independent (control condition) and the dependent nature (causal belief condition) of the stimuli pairs in the respective conditions before the start of the actual experiment.", "Next, participants commenced with the experimental trials.", "In both conditions, participants completed four blocks in randomized order.", "The blocks consisted of two baseline blocks (baseline noise, baseline tone) and two succession blocks (succession noise, succession tone).", "Each block comprised 24 trials, including four practice trials and 20 experimental trials.", "The practice trials preceding the experimental trials of each block were identical to and indistinguishable from the experimental trials but were not analyzed.", "Each trial began with the presentation of a dotted clock face consisting of forty grey dots arranged in a circle around the center of the screen, with a diameter of six centimeters.", "A black dot served as a clock hand and was moving alongside the grey dots with a speed of 2560 ms per rotation.", "The starting position of the clock hand varied between trials and was random.", "Participants were asked to attend to the clock face and the rotating clock hand.", "Depending on the trial, they then either perceived a single auditory stimulus or a stimuli pair in succession.", "The onset of the stimuli was programmed to always occur during the second rotation of the clock hand.", "In baseline blocks, participants either perceived one of the two noise sounds (baseline noise) or either of the two sinusoidal tones (baseline tone).", "In the succession blocks, participants always perceived an initial noise sound followed by the paired sinusoidal tone at 250 ms. After the presentation of the last stimulus, the clock hand continued moving for a duration of 1000 ms before it disappeared.", "Subsequently, the mouse cursor as well as a prompt to make a time judgment appeared in the center of the clock face.", "Participants were required to judge either the temporal onset of the noise sound (succession noise trials) or the temporal onset of the sinusoidal tone (succession tone trials) by clicking on one of the dots to indicate the position of the clock hand at the time of the event.", "In baseline trials, participants always judged the onset of the single presented stimulus (noise in baseline noise and sinusoidal tone in baseline tone trials).", "While blocks and trials were identical in both between-subject conditions, instructions differed.", "In the causal belief condition, instructions for the individual blocks emphasized the causal and dependent relationship of the two stimuli on each other.", "For example, participants in the causal belief condition read that they would hear two stimuli in succession, meaning that the tones were dependent on each other and that the first noise would cause the following sinusoidal tone.", "In the control condition, in contrast, the independence of the stimuli was emphasized.", "At the end of the experimental trials, participants filled in their age and gender.", "Moreover, participants also answered a question concerning their belief in the causal dependence of the second stimulus on the first stimulus (as indicated on a 9-point Likert scale ranging from 1 - absolutely not to 9 - very much).", "This question served as a manipulation check.", "Finally, participants were debriefed and thanked for their participation as well as reimbursed.", "Prior to the main statistical analyses, extreme temporal judgments were excluded on a trial basis.", "Following Aarts et al. (2012), temporal judgments, which were more than ±640 ms away from the actual temporal onset, were excluded as it can be assumed that participants either rated the onset of the wrong stimulus or were inattentive in general on those trials.", "Overall, the number of judgments excluded based on this criterion was very small (0.14% of all judgments).", "Based on earlier research suggesting a temporal repulsion effect between auditory stimuli (Desantis et al., 2012; Haggard et al., 2002a), we first aimed to test whether there was a significant overall temporal repulsion effect independent of condition.", "First, for each trial a judgment error (in milliseconds) was calculated as the difference between the judged time of an event and its actual time of occurrence.", "A positive judgment error corresponds to delayed awareness of the event, and a negative judgment error corresponds to anticipatory awareness.", "Next, for each participant separate shifts for the first (noise) and second (sinusoidal tone) stimulus were computed by subtracting the mean baseline judgment errors from the mean judgment errors in succession trials (noise shift = mean judgment error in succession noise trials – mean judgment error in baseline noise trials; tone shift = mean judgment error in succession tone trials – mean judgment error in baseline tone trials).", "Finally, overall binding scores were computed by subtracting the shift of the second stimulus from the shift of the first stimulus (i.e., overall score = noise shift – tone shift).", "Positive overall binding scores thus indicated temporal compression of the interval between the stimuli (temporal binding) while negative scores were indicative of temporal repulsion.", "See Fig. 1 for the distribution of the raw data in the full sample.", "First, we examined the direction of the overall temporal shift.", "To that end, we conducted a one-sample t-test of the overall binding score against zero.", "As we had no hypothesis pertaining to either a temporal binding or a temporal repulsion effect, we conducted a two-tailed test.", "Next, we aimed to test whether temporal perception of the stimuli in the two conditions was statistically different.", "Specifically, we assumed that stimuli in the causal belief condition would be bound more strongly than in the control condition.", "In line with that, we conducted a one-tailed independent samples t-test on the overall binding scores in both conditions.", "Finally, we tested the direction and significance of the temporal shift in the two conditions.", "That is, we tested whether the perceptual shift was equally present in both conditions.", "For that purpose, two one-tailed one-sample t-tests of the overall binding scores in each condition against zero were conducted.", "Additionally, to illustrate the strength of the evidence and confirm the Frequentist analyses, equivalent Bayesian analyses were performed.", "For the convenience of the reader, complete Bayesian analyses are reported.", "Prior to the main analysis, we conducted an independent samples t-test, testing the belief in causality rating between control and causal belief condition.", "Confirming our manipulations, the results revealed that participants in the causal belief condition (M = 6.68, SD = 2.15) believed more strongly in the causal dependency of the second stimulus on the first stimulus than participants in the control belief condition (M = 5.24, SD = 2.48), t(62) = −2.4688, p = .016, two-tailed, d = 0.61, 95% CI [−1.12, −0.11].", "Next, we proceeded with the main analysis as outlined in the data analysis plan.", "To examine the direction of the perceptual shift and its statistical significance, a one-sample t-test of the overall binding scores against zero was conducted.", "Results revealed a highly significant repulsion effect equaling 77 ms (M = −77.20, SD = 65.94), t(63) = −9.366, p < .001, two-tailed, d = −1.17, 95% CI [−1.49, −0.85].", "This temporal repulsion effect occurred bi-directionally.", "That is, noises were significantly more anticipated on succession trials (M = 13.12, SD = 46.97) as compared to baseline trials (M = 50.97, SD = 47.94), t(63) = 5.949, p < 001, two-tailed, dz = 0.74, 95% CI [0.36, 0.80].", "Similarly, tones were perceived significantly later on succession trials (M = 90.17, SD = 49.34) than on baseline trials (M = 40.83, SD = 50.82), t(63) = −6.932, p < .001, two-tailed, dz = −0.87, 95% CI [−1.31, −0.66].", "In addition, to test whether temporal perception differed for participants in the control and the causal belief condition, overall binding scores were subjected to an independent samples t-test based on condition.", "The results revealed that participants in the causal belief condition (M = -52.50, SD = 67.24, N = 31) perceived the stimuli as significantly closer to each other in time than participants in the non-causal control condition (M = −100.40, SD = 56.35, N = 33), t(62) = −3.096, p = .0015, one-tailed.", "The difference of −47.9 ms was of moderate effect size, d = −0.77, 95% CI [−1.28, −0.26].3", "See Fig. 2 for a visualization of the overall shifts for the separate between-subject conditions.", "Following, two one-sample t-tests were conducted to test whether the judgments were significantly different from zero.", "In the control condition (M = −100.40, SD = 56.35), judgments showed a statistically significant temporal repulsion effect, t(32) = −10.24, p < .001, one-tailed, d = −1.78, 95% CI [−2.33, −1.22].", "Similarly, also in the causal belief condition (M = −52.50, SD = 67.24), individuals showed a significant temporal repulsion effect, t(30) = −4.35, p < .001, one-tailed, d = −0.78, 95% CI [−1.18, −0.37].", "The separate shifts for the noise and the tone in the between-subject conditions are visualized in Fig. 3.", "To assess the strength of the evidence, we performed Bayesian analyses in JASP (JASP Team, 2018, Version 0.9).", "In the absence of sufficient information regarding a reasonable effect size and given the novelty of the paradigm, the default Cauchy prior of 0.707 instead of an informed prior was used for the Bayesian t-tests (Bartlett, 2018; Quintana & Williams, 2018).", "To assure the robustness of Bayes Factors, robustness checks were conducted.", "The results indicated that the chosen prior did not inflate the Bayes Factors.", "Hence, the results of all tests can be assumed to be robust.", "The results of all robustness checks are reported in the Supplementary Materials.", "To test the strength of the temporal repulsion effect, we conducted a Bayesian two-tailed one-sample t-test on the overall temporal repulsion score.", "The alternative hypothesis stated that the temporal repulsion effect differed from zero (zero meaning no perceptual shift).", "The results revealed a BF10 = 5.199e + 10 (=51,990,000,000), 95% CrI [−1.46, −0.84], indicating extreme evidence for the alternative hypothesis.", "Next, to test the strength of the difference between the two conditions, a one-tailed Bayesian independent-samples t-test was conducted.", "The alternative hypothesis assumed that the temporal judgments in the causal belief condition would be smaller than in the control condition, indicating less repulsion.", "The BF+0 was 25.01, 95% CrI [0.19, 1.19], suggesting that the data was 25 times more likely to occur under the alternative than under the null hypothesis.", "Thus, there was strong evidence for the idea that temporal repulsion was smaller in the causal belief condition than in the control condition.", "Consecutively, two Bayesian one-sample t-tests were conducted.", "Both overall binding scores were tested against a population mean of zero.", "For the causal belief condition, a BF10 = 183.4, 95% CrI [−1.13, −0.34], was found, indicating very strong evidence for the alternative hypothesis.", "For the control condition, a BF10 = 7.136e + 8 (=713,600,000), 95% CrI [−2.36, −1.13], was found, indicating even extreme evidence for the alternative hypothesis.", "In this first experiment, we aimed to replicate the temporal repulsion effect between two auditory stimuli suggested by existing literature (Desantis et al., 2012; Haggard et al., 2002a).", "Employing a sensory based Libet clock paradigm, strictly excluding the possibility for motor action, for the first time, we empirically tested and statistically demonstrated this temporal repulsion effect (i.e., corroborated by Frequentist and Bayesian analyses).", "Additionally, we found that participants who held an explicit belief about the causal dependency of the stimuli on each other, perceived the stimuli as being less strongly repulsed than participants who did not hold such belief.", "This finding suggests an increased perceived coherence between the stimuli in the causal belief condition.", "However, temporal binding was still absent, indicating that the temporal integration of the two auditory stimuli was weak and thus did not produce a full coherence experience.", "To test the reliability of the temporal repulsion effect in the perception of successive auditory stimuli and the attenuation thereof in the causal belief condition, a second experiment was set up.", "It is conceivable that temporal repulsion persisted in the light of causal belief because of the arbitrary nature of the stimuli and their associations.", "For example, recent research using a different paradigm suggests that stimuli that have a naturalistic link (such as the picture of a hand and a clapping sound) are more easily bound in temporal perception (Thanopoulos, Psarou, & Vatakis, 2018).", "Research suggests that these associative links between stimuli are acquired through learning and repeated exposure (Turk-Browne, Scholl, & Chun, 2008).", "Especially in the absence of motor action such learning might be pivotal for forming a stable predictive link.", "Hence, in an attempt to strengthen these associations, we increased the amount of learning trials from ten to sixty trials in the additional learning conditions.", "The sample of the second experiment consisted of 1004 (66 females) volunteers with a mean age of 22 years (M = 22.22, SD = 2.03) who participated in the experiment in exchange for course credit or a monetary reimbursement.", "The study was carried out in line with the guidelines of the Declaration of Helsinki and was approved by the Ethics committee of the Faculty of Social and Behavioral Sciences at Utrecht University (ethics approval code: FETC17-124).", "All participants gave written informed consent.", "The design of the second experiment resembled the design of the first experiment with the addition of another between-subject factor.", "The complete design was a 2 (target: first vs. second tone) × 2 (type of trial: baseline vs. succession) × 2 (condition, between: control vs. causal belief) × 2 (additional learning, between: no vs. yes) mixed design.", "The addition of the learning factor resulted in two additional between-subject conditions.", "That is, participants could be randomly assigned to either one of the following conditions: control condition or causal belief condition (replication of the conditions of Experiment 1), control learning condition or causal belief learning condition (resulting from the addition of the learning factor to the design).", "The general procedure of the experiment resembled that of the first experiment.", "Also the stimuli used were identical to the stimuli in the first experiment.", "The experiment was programmed using Eprime 2.0.", "The procedural differences between the first and the second experiment are outlined below.", "The experiment began with an acquisition phase.", "In the control and causal belief condition, the same acquisition phases as in the first experiment were employed (for details see Experiment 1).", "In the additional learning condition, after having acquired the stimuli, participants completed extensive learning phases of the stimuli pairs.", "In total, participants completed 60 learning trials (2 × 30 trials, separated by a 30 s break).", "The repeated exposure to the stimuli pairs thus would strengthen the association between the stimuli.", "Like in the first experiment, in the causal belief condition, the causal relationship between the two stimuli of a pair was emphasized whereas in the control condition their independence was emphasized.", "The control and causal belief condition were identical to the first experiment.", "The instructions for experimental blocks in the learning conditions were borrowed from the respective control conditions.", "As in Experiment 1, we excluded temporal judgments that were more than ±640 ms away from the actual temporal onset of the stimulus on a trial basis (Aarts et al., 2012).", "Overall, the amount of excluded trials was very small (0.15% of the total amount of judgments).", "Furthermore, we calculated separate shift scores as well as overall binding scores per participant using the same formula as in Experiment 1.", "Upon inspection of the overall binding scores, one participant seemed to be an extreme outlier with an average mean judgment score of −532.38 ms. As this score was more than six standard deviations away from the average overall binding score of the remaining participants (M = −67.28, SD = 74.34), we decided to remove this person from further analysis.", "A visualization of the raw data including the outlier is provided in Fig. 4.", "We proceeded with testing the significance of the overall temporal perceptual shift.", "Since we had no directional hypothesis regarding the perceptual shift in the overall sample (either temporal binding or temporal repulsion), we conducted a two-tailed one-sample t-test on the overall temporal binding scores.", "After establishing the significance of the temporal repulsion effect in the sample, we subjected the overall binding scores to an ANOVA with causal belief and learning as between-subject factors to assess their influence on temporal perception.", "Following, four separate one-tailed one-sample t-tests were conducted to test the significance of the temporal repulsion shift in the separate between-subject conditions.", "Finally, equivalent Bayesian analyses are reported to give an impression of the strength of the evidence.", "To check that the belief in the causal relationship between paired stimuli was indeed stronger in the causal conditions than in the control conditions, we conducted an ANOVA with causal belief condition and learning as between-subject factors on causal belief.", "The results revealed that, on average, participants in the causal belief conditions (M = 5.6, SD = 2.24 , assessed on a 9-point Likert scale) believed more strongly in the causal dependency of the second stimulus on the first stimulus than participants in the control belief conditions (M = 3.67, SD = 2.17), F(1, 95) = 19.615, p < .001, η2 = 0.17, 95% CI [0.05, 0.30].", "Learning was not found to have an influence on the causal dependency belief, F(1, 95) = 0.101, p = .751, η2 = 0.00, 95% CI [0.00, 0.04].", "Similarly, also the interaction between the causal belief manipulation and learning was not significant, F(1,95) = 1.631, p = .205, η2 = 0.01, 95% CI [0.00, 0.10].", "A one-sample t-test on the overall binding score across conditions was conducted to assess the direction of the perceptual shift and to test its statistical significance.", "The results revealed a significant repulsion effect of around 67 ms (M = −67.28, SD = 74.34), t(98) = −9.005, p < .001, two-tailed, d = −0.91, 95% CI [−1.14, −0.67].", "Simple main effects confirmed that temporal repulsion occurred on both sides – the noise and the tone side, meaning that they were both perceptually repulsed from each other.", "While noises were perceived as occurring earlier on succession trials (M = 3.49, SD = 39.06) than on baseline trials (M = 35.44, SD = 41.05), t(98) = 7.805, p < .001, two-tailed, dz = 0.78, 95% CI [0.31, 0.73], tones were perceived significantly later on succession trials (M = 64.58, SD = 60.34) than on baseline trials (M = 29.25, SD = 44.54), t(98) = −6.018, p < .001, two-tailed, dz = -0.60, 95% CI [-0.90, -0.43].", "The overall binding scores were then subjected to an ANOVA with condition and learning as between-subject factors.", "Neither the main effect for condition, F(1, 95) = 0.203, p = .653, η2 = 0.00, 95% CI [0.00, 0.05], nor the main effect for learning, F(1, 95) = .061 p = .806, η2 = 0.00, 95% CI [0.00, 0.03], was significant.", "Moreover, also the interaction between condition and learning was not significant, F(1, 95) = 0.117, p = .733, η2 = 0.00, 95% CI [0.00, 0.05].", "Also the planned comparison between the control and the causal belief condition did not yield a significant effect, F(1, 95) = 0.331, p = .566, η2 = 0.00, 95% CI [0.00, 0.06].", "The causal belief effect that we found in the first experiment was thus not replicated.", "See Fig. 5 for a visualization of the overall binding scores in the four between-subject conditions.", "The scores of all four between-subject conditions were negative, indicating temporal repulsion of the two stimuli from each other: control condition (M = −74.94, SD = 57.46, N = 27), causal condition (M = −62.90, SD = 68.02, N = 25), control learning condition (M = −66.01, SD = 98.73, N = 24) and causal learning condition (M = −64.37, SD = 73.30, N = 23).", "Separate one-sample t-tests were conducted to test if temporal repulsion in the conditions was significantly different from zero.", "The results were as follows: t(26) = −6.777, p < .001, one-tailed, d = −1.30, 95% CI [−1.81, −0.78] (control), t(24) = −4.624, p < .001, one-tailed, d = -0.92, 95% CI [−1.39, −0.45] (causal), t(23) = −3.275, p = .0015, one-tailed, d = −0.67, 95% CI [−1.11, −0.22] (control, learning), t(22) = −4.211, p < .001, one-tailed, d = −0.88, 95% CI [−1.35, −0.39] (causal, learning).", "Fig. 6 provides an overview of the separate temporal shifts for the perception of the noise and the tone as compared to baseline.", "Furthermore, Bayesian analyses were conducted using JASP software (JASP Team, 2018, Version 0.9).", "Like in Experiment 1, the default Cauchy prior of 0.707 was used for the Bayesian t-tests as a lack of existing information regarding a realistic effect size did not allow for the determination of an informed prior (Bartlett, 2018; Quintana & Williams, 2018).", "To assure the robustness of the Bayes Factors, robustness checks were conducted.", "The results indicated that the chosen prior did not inflate the Bayes Factors and that all test can be assumed to be robust.", "The results of all robustness checks are reported in the Supplementary Materials.", "Like in the first experiment, to test the strength of the temporal repulsion effect, we conducted a Bayesian two-tailed one-sample t-test on the overall temporal repulsion score.", "The alternative hypothesis stated that the temporal repulsion effect differed from a population mean equal to zero.", "The BF10 = 3.979e + 11(=397,900,000,000), 95% CrI [−1.46, −0.84] indicated extreme evidence for the alternative hypothesis.", "Subsequently, we conducted a Bayesian ANOVA on the overall binding scores with causal belief and learning as between-subject factors.", "The results showed that, given the data, there was no evidence that the separate effects of causal belief (BF10 = 0.234) and learning (BF10 = 0.219) were a good addition to the null model explaining the extent of temporal binding or repulsion.", "Similarly, neither a model including both factors (BF10 = 0.051) nor a model including both factors and the interaction term (BF10 = 0.015) was favorable given the data.", "Next, to test the likelihood of decreased temporal repulsion in the normal causal belief condition as compared to in the normal control condition (replication of Experiment 1) given the data, a one-tailed Bayesian independent-samples t-test was carried out.", "The null hypothesis that there was no difference between the two conditions was tested against the alternative hypothesis that the temporal repulsion in the causal condition was smaller than in the control condition.", "The BF+0 = 0.497, 95% CrI [0.01, 0.7], suggested no evidence for the alternative hypothesis.", "Finally, we conducted separate Bayesian one-sample t-tests to test each group judgment against zero.", "The results were as follows: control condition BF10 = 47998.34, 95% CrI [−1.75, −0.75], causal condition BF10 = 250.6, 95% CrI [−1.34, −0.39], control learning condition BF10 = 12.36, 95% CrI [−1.05, −0.19], and causal learning condition BF10 = 87.08, 95% CrI [−1.23, −0.30].", "That is, while there was extreme evidence in favor of the alternative hypothesis in the control and causal condition, the evidence for the alternative hypothesis was (very) strong in the two learning conditions.", "This suggests that the evidence for temporal repulsion was strong in all conditions but slightly less strong in the learning conditions.", "In short, we replicated the temporal repulsion effect of the first experiment, but we could not replicate the moderating influence of causal belief, even when participants received more extensive practice in causally linking the two auditory stimuli.", "Research on action awareness consistently finds voluntary actions and their effects to be attracted to each other in temporal perception.", "This temporal binding effect is said to be rooted in factors inherent to intentional action – hence the name, intentional binding.", "Here we explored a potential confound of intentional action in producing temporal binding.", "Specifically, we explored the role of beliefs of causality, and cancelled out any role of action by examining temporal perception of auditory stimuli.", "Employing an adapted sensory-based version of the Libet clock paradigm, in two experiments we found a temporal repulsion effect for the perception of auditory stimuli in the absence of motor action.", "This finding supports past research that observed a similar effect (Desantis et al., 2012; Haggard et al., 2002a).", "However, we were able to statistically test it with a sufficiently large sample, and unambiguously demonstrated the existence and robustness of such an effect.", "That is, both - the noise and the sinusoidal tone - were significantly shifted away in comparison to baseline.", "We also found evidence for a moderating effect of causal belief on temporal binding of auditory stimuli.", "Specifically, in Experiment 1 we showed that making participants believe that the first auditory stimulus is causally related to the second auditory stimulus reduced the repulsion effect, suggesting an increased form of temporal integration and perceived coherence.", "The repulsion effect was not fully reduced, and we could not replicate the moderating influence of causal belief in a second experiment, even though we exposed participants to more training in applying causal beliefs to the auditory stimulus combinations.", "Hence, while we demonstrated a robust and clear temporal repulsion effect for sensory stimuli in the absence of motor action, the influence of higher order beliefs was less robust and not sufficient to cause actual temporal binding, as would be the case in intentional action.", "One possible explanation for the absence of a causal belief effect on temporal binding of auditory stimuli might lie in the naturalistic character of the stimuli combinations.", "Recent research on voluntary action, for example, suggests that naturalistic as compared to non-naturalistic links between action-events carry an implicit (learned) causality experience which makes them likely to bind to an effect (Dogge et al., 2012; Dogge, Hofman, Custers, & Aarts, 2019; Thanopoulos et al., 2018).", "Following this line of reasoning, the absence of temporal binding may be due to the lack of a natural association between the two auditory stimuli.", "Although previous research showed that spatial binding of visual stimuli could emerge rather rapidly (Buehner & Humphreys, 2010), auditory sequences might require practice for temporal binding and perception of coherence to occur (Hsu, Le Bars, Hämäläinen, & Waszak, 2015; Lange, 2009; Turk-Browne et al., 2008).", "We found that the number of trials that we used was not enough and that it is unclear how much prior experience is necessary to induce temporal binding between non-naturalistic stimuli links.", "While there is research suggesting that a few trials might suffice (Shimi & Logie, 2018), other research suggests that more elaborate prior learning is necessary (Ernst, 2007).", "The failure to replicate the causal belief effect on temporal binding thus indicates the fragility of building a coherent representation of a non-naturalistic combination of sounds (Grotheer & Kovács, 2014; Jacobsen & Schröger, 2001), which seems less problematic for intentional action where one produces sounds by key-presses that might be more naturalistic and more easily to acquire.", "All in all, the present finding might give reason to assume that intentionality has a special status in binding action and effect.", "As far as the evidence in the current literature can tell, temporal binding mainly occurs when people perform, observe and simulate actions (such as key-presses) to produce effects.", "Despite potential flaws in experimental designs and testing (Hughes et al., 2013), these overall findings strongly suggest that sensorimotor processes are vital to conscious experiences of action coherence and agency.", "It is unclear, though, whether the predictions involved in this sensorimotor mechanism stem from intentional action, actual body movement or action simulation, or whether temporal binding arises from multisensory causal integration that links physical sensations to predicted sensory effects (Ehrsson, Spence, & Passingham, 2004; Prikken et al., 2019).", "Future research might therefore design temporal binding studies that more indirectly operate on the motor-sensory system, possibly ruling out action simulation and using conditions that resemble sensory experiences (e.g., tactile stimulation of the finger) that often accompany intentional motor action during interaction with the world.", "Finally, the present experiments employed the Libet clock paradigm to examine how two successive auditory stimuli are temporally bound together in conscious awareness.", "A key feature of the Libet clock in a full temporal binding within-subject design is that one can specifically examine whether repulsion (or attraction) is caused by a shift away from the first stimulus or from the second stimuli, or both.", "Our data suggest that the temporal repulsion effect occurred bi-directionally, such that subjects perceived the first stimulus earlier and the second stimulus later in time than actually was the case.", "Repulsion in the temporal binding task thus represents an instance in which two successive stimuli are disconnected and separated in conscious experience.", "It is important to note that recent research uses alternatives for the Libet clock, in particular time interval estimations.", "Whereas these time estimations seem to yield findings that correspond with a temporal binding effect, the measure does not offer a clear inspection of the effect: time interval estimations are compared with the actual time intervals, leading to under- or overestimations of time intervals.", "Notably, in some studies underestimations are considered to represent temporal binding, whereas in other studies overestimations are treated as temporal binding when comparing conditions that are hypothesized to modulate binding effects (Damen, Van Baaren, Brass, Aarts, & Dijksterhuis, 2015; Kühn, Brass, & Haggard, 2013; Wen, Yamashita, & Asama, 2015).", "Thus, although the time interval estimation measure is practical and easy to administer, it is ambiguous in showing whether attraction or repulsion effects are at stake (for studies on time interval estimations with respect to auditory and visual stimuli links, see e.g., Humphreys & Buehner, 2009; Imaizumi & Tanno, 2019).", "Therefore, whereas the Libet clock paradigm has downsides on its own (Pockett & Miller, 2007), it allows for reliable assessments of the relative temporal shifts compared to baseline time perception, which may be preferred over time interval estimation to examine whether, how and when a positive temporal binding (attraction) binding or negative temporal binding (repulsion) effect will occur.", "To conclude, the present research clearly shows that two auditory stimuli occurring in the absence of motor actions are not subject to temporal binding but temporal repulsion, even when people believe that the stimuli are causally related.", "Understanding the role of causal beliefs in binding action and effect has received some attention lately, but most studies still involve motor movement, and hence, it remains unclear whether and how intentional action shapes coherent representations and a sense of agency of our own behavior.", "We believe that ruling out any role of motor movement (albeit overtly or covertly) might be crucial in examining alternative mechanisms for temporal binding between action and effect, such as multisensory causal binding that integrates associated information from different sensory modalities.", "The current research might serve as a first step in this enterprise.", "S. A., R. C. and H. A. conceived the idea and planned the experiment.", "S. A. programmed the experiment, supervised data collection and analyzed the data.", "S. A. and H. M. interpreted the data.", "S. A., H. A. and R. C. wrote the manuscript.", "H. M. provided useful comments." ]	[ "Successive auditory stimuli are repulsed from each other in temporal perception.", "Belief about causality attenuates the temporal repulsion of auditory stimuli.", "Prior learning does not facilitate temporal binding of auditory stimuli." ]
Rock-slope failure following Late Pleistocene deglaciation on tectonically stable mountainous terrain	The ages of 31 postglacial rock-slope failures (RSFs) in Scotland and NW Ireland, derived from 89 cosmogenic isotope exposure ages, are employed to analyse the temporal pattern of failure and its relationship to the timing of deglaciation, rates of glacio-isostatic crustal uplift and periods of rapid climate change. RSF ages span almost the whole period since ice-sheet retreat, from 18.2±1.2ka to 1.7±0.2ka, or from 17.1±1.0ka to 1.5±0.1ka, depending on the production rate used in 10Be age calculation, but catastrophic failure of rock slopes was ~4.6 times more frequent prior to ~11.7ka than during the Holocene. 95% of dated RSFs at sites deglaciated during retreat of the last ice sheet occurred within ~5400 years after deglaciation, with peak RSF activity 1600-1700 years after deglaciation. This time lag is inferred to represent (1) stress release initiated by deglacial unloading, leading to (2) time-dependent rock mass strength degradation through progressive failure plane development, and ultimately (3) to either spontaneous kinematic release or failure triggered by some extrinsic mechanism. By contrast, 11 dated RSFs at sites reoccupied by glacier ice during the Younger Dryas Stade (YDS) of ~12.9-11.7ka exhibit no clear temporal pattern, suggesting that glacial reoccupance during the YDS was ineffective in preconditioning a renewed cycle of enhanced RSF activity. Comparison of timing of individual RSFs with that of deglaciation and rapid warming events at ~14.7ka and ~11.7ka suggests that glacial debuttressing, enhanced joint water pressures during deglaciation and thaw of permafrost ice in rock joints could have triggered failure in only a small number of cases. Conversely, the timing of maximum RSF activity following ice-sheet deglaciation corresponds broadly with maximum rates of glacio-isostatic crustal uplift, suggesting that the two are linked by enhanced seismic activity. A seismic failure trigger is consistent with full-slope failure at all sites where failure planes are clearly defined. Our results indicate that numerous RSFs must have occurred in areas that were reoccupied by glacier ice during the YDS, but have not been identified because runout debris was removed by YDS glaciers. More generally, they provide the first reliably-dated body of evidence to support the view that retreat of the last ice sheets in tectonically-stable mountainous terrain initiated a period of enhanced rock slope failure due to deglacial unloading and probably Lateglacial seismic activity, implying that most undated RSFs in such areas are probably of Lateglacial or very early Holocene age. They also demonstrate, however, that a low frequency of RSF activity extended throughout the Holocene. © 2014 Elsevier Ltd.	[ "AGRI", "ARTS", "EART", "ENVI", "SOCI" ]	[ "Cosmogenic isotopes", "Palaeoseismicity", "Paraglacial", "Rock-slope failure", "Stress-release" ]	[ "1105", "1204", "1907", "2306", "3302" ]	[ "Many formerly-glaciated mountain environments are characterized by a high spatial density of large-scale postglacial rock-slope failures (RSFs) in the form of major rockfalls, rockslides, rock avalanches or deep-seated gravitational slope deformations.", "Such RSFs are often described as paraglacial, implying that failure was conditioned by the preceding episode of glaciation and deglaciation (Ballantyne, 2002; Wilson, 2009; Kellerer-Pirklbauer et al., 2010; McColl, 2012).", "Several authors have inferred a causal connection between deglaciation of steep rockwalls and subsequent rock-slope failure, with regard to both recent retreat of mountain glaciers (e.g. Evans and Clague, 1994; Holm et al., 2004; Arsenault and Meigs, 2005; Allen et al., 2010) and shrinkage of Late Pleistocene ice sheets (e.g. Abele, 1997; Soldati et al., 2004; Blikra et al., 2006; Longva et al., 2009; Mercier et al., 2013; Cossart et al., 2013a).", "Proposed explanations for this association include stress-release fracturing, uplift and dilation of rock masses, debuttressing (removal of supporting glacier ice from unstable rockwalls), freeze-thaw activity, thaw of permafrost ice in ice-bonded joint networks, enhanced joint-water pressures during deglaciation and seismic activity (Prager et al., 2008; McColl, 2012).", "In the case of RSFs following Late Pleistocene or early Holocene deglaciation, assessment of the manner in which deglaciation has affected rock-mass stability has been impeded by limited data on the timing of postglacial failures.", "Over the past two decades, however, surface exposure dating using cosmogenic isotopes (3He, 10Be, 21Ne and 36Cl) has been employed to establish the age of numerous individual postglacial RSFs, particularly in tectonically-active mountain belts such as the Alps (Bigot-Cormier et al., 2005; Van Husen et al., 2007; Hormes et al., 2008; El Bedoui et al., 2009; Hippolyte et al., 2009; Ivy-Ochs et al., 2009; Prager et al., 2009; Sanchez et al., 2010), Himalaya (Mitchell et al., 2007; Dortch et al., 2009), Andes (Hermanns et al., 2001, 2004; Fauqué et al., 2009; Welkner et al., 2010) and Karakoram (Seong et al., 2009; Shroder et al., 2011).", "Most of these studies, however, involve the dating of a single RSF or small number of RSFs, so the regional temporal pattern of RSF activity since deglaciation cannot be assessed.", "Moreover, almost all exposure-dated RSFs are located in tectonically-active mountain belts.", "In such areas earthquakes caused by postglacial fault movements due to tectonically-driven uplift or regional crustal stresses may trigger major RSFs, so that RSF ages may be largely independent of the timing of Late Pleistocene deglaciation (e.g. Hewitt et al., 2008, 2011; Antinao and Gosse, 2009; Sanchez et al., 2010; Stock and Uhrhammer, 2010; Hermanns and Niedermann, 2011; Penna et al., 2011).", "Numerous postglacial RSFs are also present in mountainous parts of seismically-quiescent intraplate terrains, such as Scandinavia and much of the British Isles, that were completely buried under Late Pleistocene ice sheets.", "The Scottish Highlands, for example, are estimated to contain over 550 postglacial RSFs, including 140 with areas exceeding 0.25 km2 (Jarman, 2006, 2007).", "For such areas, several authors have championed the idea that active faulting driven by glacio-isostatic rebound produced large magnitude (M > 6.0) earthquakes following ice-sheet shrinkage, with progressive reduction in seismic activity during the Holocene as the rate of uplift slowed (Davenport et al., 1989; Gregerson and Basham, 1989; Ringrose, 1989a; Dehls et al., 2000; Morner, 2004, 2005; Lagerbäck and Sundh, 2008).", "One implication of this view is that many RSFs in mountain areas presently characterized by low-level seismic activity may have been released by earthquakes in the aftermath of ice sheet retreat, with diminishing RSF activity thereafter.", "Some support for this view is provided by research on the stratigraphy of inferred rock avalanche deposits in Norwegian fjords: Blikra et al. (2006) detected a peak of RSF activity during deglaciation and Longva et al. (2009) inferred that ∼89% of the volume of postglacial rock avalanche deposits in Storfjorden (western Norway) represents deposition during ice-sheet deglaciation (∼14.7–11.7 ka), though the frequency of rock avalanches apparently peaked in the early Holocene (∼11.7–10.0 ka).", "Similarly, Strasser et al. (2013) concluded that a high frequency of Lateglacial (∼16.7–11.6 ka) landslide events represented stratigraphically in lacustrine sediment sequences in Central Switzerland represents enhanced seismic activity attributable to glacio-isostatic crustal movements.", "Over the past 15 years we have amassed a database of 31 dated rock-slope failures in the Scottish Highlands, Inner Hebrides and NW Ireland (Fig. 1) based on a total of 89 10Be or 36Cl exposure ages obtained on runout debris (Ballantyne et al., 1998, 2009a, 2013a, 2013b; Ballantyne and Stone, 2004, 2009, 2013).", "The aim of this paper is to synthesise all the dating evidence from this research to characterize the timing of postglacial RSFs in this tectonically-stable region and to examine the implications in terms of the long-term causes and short-term triggers of failure.", "More specifically, we employ this unique dataset to test various hypotheses concerning the relationship between the timing of deglaciation and RSF response, namely: (1) that failure represents a rapid, immediate response to deglaciation; (2) that RSF activity was focused within a few millennia during the Lateglacial and Early Holocene, as suggested by several previous studies (Blikra et al., 2006; Prager et al., 2008; Fauqué et al., 2009; Longva et al., 2009); (3) that the timing of failure involves elements of both rapid and delayed response to deglaciation, as proposed by Ballantyne and Stone (2013); and (4) that the timing of failure extended throughout the postglacial period, with limited temporal clustering, as suggested by an inventory of dated RSFs compiled by Prager et al. (2008) for the Austrian Tyrol.", "Our sample of RSF ages confirms that retreat of Late Pleistocene ice sheets in tectonically-stable, mountainous intraplate terrains initiated a period of enhanced rock-slope failure over a period of roughly 5000 years spanning the Lateglacial period and very early Holocene.", "It follows that the great majority of undated RSFs in such areas are also of Lateglacial or early Holocene age.", "Equally, however, the late Holocene ages of a few dated RSFs imply that future catastrophic RSFs are possible, especially in areas occupied by glacier ice during the YDS.", "Future research priorities include dating of postglacial fault scarps to investigate more directly the temporal connection between palaeoseismicity and Lateglacial RSFs (cf. Sanchez et al., 2010), and establishing the ages of deep-seated gravitational deformations, which constitute many of the largest RSFs in the British Isles but have not hitherto been dated.", "The mountains of the Scottish Highlands, the Inner Hebrides and NW Ireland are largely underlain by metasedimentary rocks or sandstones of Neoproterozoic age, or by granitic rocks of Devonian age.", "Of the 31 dated RSFs, all those in NW Ireland and on the Isle of Jura (1–14 in Table 1 and Fig. 1) are seated on massive, well-bedded quartzites of Dalradian age, though most exhibit no relationship between the locus of failure and local dip of the bedding planes (Ballantyne et al., 2013b).", "Of the dated RSFs in the Scottish Highlands, four are located on granite (15, 16, 18, 19), two on Torridon sandstone (17, 28), and most of the remainder on metamorphic rocks (pelitic, semi-pelitic or psammitic schists, quartzite or granulite), though one (31) occurred within rhyolitic ignimbrite.", "The single dated RSF on the Isle of Skye (20) represents failure of stacked basalt lavas seated on underlying Jurassic shale.", "Summit altitudes at the dated RSF sites range from ∼550 m in NW Ireland to ∼1150 m in the Cairngorm Mountains in the north-eastern Highlands.", "Vertical travel distances of individual dated RSFs generally range from ∼100 m to ∼400 m, except in the case of the Beinn Alligin rock avalanche, where runout debris from a 500 m high failure plane extends a further 1.25 km downvalley, giving a total vertical descent of ∼650 m (Ballantyne and Stone, 2004).", "During the last glacial maximum (∼26–21 ka) the last British-Irish ice sheet covered all mountain summits in Scotland and NW Ireland, extended to the Atlantic shelf edge in the west and was confluent with the Fennoscandian Ice sheet in the North Sea Basin (Bradwell et al., 2008; Hubbard et al., 2009; Clark et al., 2012; Fabel et al., 2012; Ó Cofaigh et al., 2012).", "By ∼19 ka the ice sheet margin had retreated to the present coast in NW Ireland (Clark et al., 2009), and by ∼15 ka the ice margin was located along the western seaboard of Scotland, extensive areas of the eastern Highlands were ice free, and the summits of mountains in the Scottish Highlands had emerged from the ice sheet as nunataks (Phillips et al., 2008; Ballantyne, 2010; Ballantyne and Stone, 2012; Fabel et al., 2012).", "Following rapid warming at the onset of the Lateglacial Interstade at ∼14.7 ka, when mean July temperatures in Scotland rose by ∼6 °C (Brooks and Birks, 2000; Brooks et al., 2012), glacier ice completely disappeared from all mountain area of the British Isles, or survived only in favourable locations such as cirques or high plateaus in the northern and western Highlands (Finlayson et al., 2011; Ballantyne and Stone, 2012).", "Rapid temperature decline at the onset of the ensuing Younger Dryas Stade (YDS) of ∼12.9–11.7 ka resulted in renewed expansion of glacier ice, which formed a major ice cap or icefield in the western Highlands, several smaller peripheral icefields and numerous independent cirque and valley glaciers (Golledge, 2010, Fig. 1).", "Postglacial RSFs in NW Ireland and in Scotland outside the limits of YDS glaciation may therefore have occurred at any time since local deglaciation as the British-Irish Ice Sheet retreated (19–14 ka), whereas postglacial RSFs inside the YDS glacier limits must have occurred since YDS glacier retreat, which occurred within the period 12.5–11.5 ka (Golledge et al., 2008; Golledge, 2010; Ballantyne, 2012).", "Glacier ice has been absent from the British Isles throughout the Holocene.", "Permafrost is known to have developed down to sea level in the wake of the retreating ice sheet prior to warming at ∼14.7 ka, and again during the YDS (Ballantyne and Harris, 1994).", "It is possible that residual permafrost survived under high ground during the intervening Lateglacial Interstade (∼14.7–12.9 ka), but evidence for this is lacking.", "NW Ireland and Scotland occupy intraplate terrain of relative tectonic stability.", "The region is presently characterized by low magnitude (ML < 4) seismic activity focused mainly in the western Highlands (Musson, 2007), possibly reflecting interaction of NW–NNW directed tectonic stress and residual glacio-isostatic rebound (Firth and Stewart, 2000; Muir-Wood, 2000).", "The characteristics of the 31 dated RSFs and details of sampling are summarised in Ballantyne and Stone (2013), and Ballantyne et al. (2013a, 2013b).", "Almost all sampled RSFs represent catastrophic translational rockslides or rock avalanches involving kinematic release of 0.2–10.0 Mt of rock and runout of coarse bouldery debris (Fig. 2), though failure at The Storr on Skye (20) took the form of foundering of basalt lavas on underlying shale in the form of a lateral block glide.", "Although deep-seated gravitational slope deformations are common on metamorphic rocks in the Scottish Highlands (Jarman, 2006, 2007), these lack runout debris and hence were not included in our dating campaign because of the difficulty of finding outcrops suitable for exposure dating.", "In all cases but one, samples for exposure dating were chiseled from near-horizontal surfaces of large boulders in the RSF runout zones; the exception was the RSF at The Storr (20), where we sampled the tops of shattered bedrock pinnacles.", "Sampling was focused wherever possible on the distal part of the runout zone to reduce the possibility of sampling boulders emplaced by rockfall events subsequent to the main failure event.", "In two cases (22 and 23) we sampled boulders from RSF runout debris that had been deposited on, and transported downslope by, residual YDS glaciers (Fig. 2f), implying that these RSFs occurred towards the end of the YDS (Ballantyne and Stone, 2013).", "At all other sites there is no evidence for displacement of RSF debris by glacier ice, indicating that failure occurred after final deglaciation of these sites.", "For four sites (20, 21, 24 and 25 in Fig. 1 and Table 1), two samples were submitted for analysis; for all other sites three samples were submitted.", "At all but two sites, quartz-rich lithologies were submitted for analysis of cosmogenic 10Be in quartz, the exceptions being the two RSFs in igneous rocks (20 and 31) which were analysed for whole-rock cosmogenic 36Cl.", "Full details of the analytical data and procedures are given in Ballantyne and Stone (2013) and Ballantyne et al. (2013a, 2013b).", "Calculation of the 36Cl exposure ages obtained for RSFs 20 and 31 is described in Ballantyne and Stone (2013).", "10Be exposure ages for all other sites were calculated using the CRONUS-Earth online calculator (Balco et al., 2008) and calibrated (or recalibrated) using two local 10Be production rates (LPRs) to minimise scaling uncertainty (Balco, 2011).", "The first, the Loch Lomond LPR (LL LPR) is based on 10Be concentration in samples from boulders on an independently-dated YDS terminal moraine (Fabel et al., 2012), and implies a reference production rate (Lm scaling) of 3.92 ± 0.18 atoms g−1 a−1, similar to the Arctic 10Be production rate (3.96 ± 0.15 atoms g−1 a−1) of Young et al. (2013).", "The second is the NWH11.6 LPR, which is based on 10Be concentration on samples from bedrock surfaces and glacially-deposited boulders inside the limits of small YDS glaciers in the NW Highlands (Ballantyne and Stone, 2012).", "An assigned deglacial age of 11.6 ± 0.3 ka, based on the timing of rapid summer warming at the YDS-Holocene climatic transition (Brooks and Birks, 2000; Brooks et al., 2012), yields a reference 10Be production rate (Lm scaling) of 4.20 ± 0.14 atoms g−1 a−1 for an assumed surface erosion rate of 1 mm ka−1, similar to that of 4.26 atoms g−1 a−1 calculated by Balco et al. (2009) for Lateglacial sites in northeastern North America.", "As the small glaciers at the sites from which the NWH11.6 production-rate calibration samples were obtained may have retreated before 11.6 ka (Golledge et al., 2008; Ballantyne, 2012), this production rate should be regarded as maximal, producing the youngest possible ages for our RSF samples.", "The two LPRs thus effectively bracket the range of possible exposure ages (Fabel et al., 2012).", "The NHW11.6 LPR produces exposure ages 6.8–7.0% lower than the LL LPR, or roughly 1000 years younger for LL LPR ages of 14–15 ka.", "We report ages using the time-dependent Lm scaling of the CRONUS-Earth calculator (Lal, 1991; Stone, 2000), which is widely employed in studies of deglaciation chronology within the British Isles.", "Other scaling schemes available using the CRONUS-Earth calculator produce ages up to 1.5% older or 0.5% younger.", "We assume a surface erosion rate (ɛ) of 1 mm ka−1 for all samples.", "Although this is a reasonable assumption for crystalline rocks (Ballantyne, 2010), it probably overestimates erosion rates for some lithologies, such as quartzite, and may underestimate it for others, such as sandstone.", "However, assumption of ɛ = 0 reduces our reported ages by only ∼1% and assumption of ɛ = 2 mm ka−1 increases reported ages by a similar margin, with negligible effect on the temporal pattern of RSF ages reported here.", "Moreover, use of a particular LPR, scaling scheme or erosion rate has negligible effect on analysis of RSF ages relative to local deglaciation ages (which were also derived from cosmogenic isotope exposure ages), as all ages are affected proportionately.", "Below we cite ages calibrated using LL LPR first, followed by ages calibrated using NWH11.6 LPR in brackets.", "Uncertainties are cited as external (total) dating uncertainties at ± 1σ.", "To derive a best-estimate age for each of the 31 dated RSFs we first excluded ‘outlier’ ages that differ from others obtained from the same site at p < 0.05, to produce pairs or triplets of statistically-indistinguishable individual ages.", "We then calculated the uncertainty-weighted mean age for each RSF from the remaining ages.", "Ten of our weighted mean ages (Table 1) are based on three consistent individual ages and 19 on two consistent individual ages.", "The age of two sites (10 and 13) are based on the single oldest postglacial age obtained at these sites, where significantly younger ages probably reflect sampling from boulders emplaced by later rockfall or debris-flow events (Ballantyne et al., 2013a).", "The timing of deglaciation at RSF sites outside the limit of YDS glaciation in the Scottish Highlands and Skye was determined from independent local deglaciation chronologies based on 10Be or (in one case) 36Cl exposure age dating of bedrock surfaces or boulders on moraines (Stone et al., 1998; Everest and Kubik, 2006; Ballantyne et al., 2009b; Ballantyne, 2010; Ballantyne and Stone, 2012), recalculated where appropriate using LL LPR and NWH11.6 LPR.", "For sites in the Scottish Highlands that were reoccupied by glacier ice during the YDS (∼12.9–11.7 ka) we assume a deglaciation age of 11.6 ± 0.3 ka, consistent with very rapid warming after 11.7 ka (Brooks and Birks, 2000; Hubbard et al., 2009; Brooks et al., 2012) and 14C-based evidence that some YDS were still advancing to their maximum extent as late as 12.0–11.7 ka (Macleod et al., 2011).", "It is probable, however, that some YDS glaciers reached their maximum extent and began to retreat a few centuries prior to 11.6 ka (Golledge et al., 2008; Ballantyne, 2012), implying that the time elapsed between deglaciation and RSF occurrence should be regarded as minimal for RSFs sampled inside the limits of YDS glaciation.", "For eight RSFs in NW Ireland (1,2 and 4–9) we derive deglaciation age from the weighted mean (17.4 ± 0.9 ka (16.3 ± 0.7 ka)) of three 10Be exposure ages for bedrock samples from an adjacent col (Ballantyne et al., 2013b), and deglaciation age for the Slieve League RSF (3) is approximated by a 10Be exposure age of 19.3 ± 1.0 ka (18.1 ± 0.8 ka) reported by Ballantyne et al. (2007) for a nearby site.", "The timing of deglaciation for the Jura RSFs is represented by the mean 10Be exposure age of 16.8 ± 0.9 ka (15.8 ± 0.5 ka) obtained for two samples from a nearby moraine (Ballantyne et al., 2013a).", "The pattern of exposure ages alone, however, does not take into account differences in the timing of deglaciation at individual sites.", "Fig. 4 depicts probability density distributions (PDDs) for all RSFs (n = 31), for all RSF sites deglaciated during retreat of the last ice sheet and not subsequently reoccupied by ice during the YDS (n = 20) and for all RSFs that were sampled inside the limits of YDS glaciation (n = 11), in all cases calculated as time elapsed since deglaciation (t) for each RSF site.", "Because the PDDs take into account both mean age and the associated ± 1σ uncertainties, the PDD curves extend to the left of t = 0, even though postglacial landslides cannot have occurred prior to deglaciation, except in the case of the two RSFs (22, 23) that occurred on to residual glacier ice at the end of the YDS.", "The weighted mean age obtained for four other RSFs (1, 15, 16 21) slightly exceeds the assumed deglaciation age for these sites, and for these sites t = 0 was assumed in generating the PDDs.", "The plots for all RSFs (Fig. 4a and d) highlight a tendency for RSFs to be clustered within a few millennia following deglaciation, but with two modal values.", "The earlier mode peaks 100–300 years following deglaciation and represents a subset of nine (29%) ‘rapid response’ RSFs (1, 2, 15, 16, 17, 21, 22, 23, 24) whose weighted mean age is statistically indistinguishable from the timing of deglaciation; the age of all other RSFs is significantly younger than the timing of local deglaciation at p < 0.05.", "The second mode peaks ∼1700 years after deglaciation.", "By calculating areas under the curves for the region t > 0, it is possible to retrodict probability of failure within given time periods: Fig. 4a (based on LL LPR ages) implies 70% probability of failure within 3000 years following deglaciation and 88% within 5000 years; the equivalent figures for Fig. 4d (based on NWH11.6 LPR ages) are 73% and 92%.", "The most striking feature of Fig. 4 is the different pattern of response for sites deglaciated during retreat of the last ice sheet (Fig. 4b and e) and those deglaciated during retreat of YDS glaciers (Fig. 4c and f), particularly if the ages of the RSFs that occurred on to residual YDS glaciers (dashed lines) are excluded.", "The PDDs for the former peak 1600–1700 years after deglaciation then decline rapidly, and imply a 74% (78%) probability of failure within 3000 years following deglaciation and 93% (94%) within 5000 years after deglaciation.", "Of the 20 RSF sites deglaciated during retreat of the last ice sheet, only that at The Storr on Skye (20) failed more than ∼5400 years after deglaciation.", "By contrast, RSFs sites deglaciated during retreat of YDS glaciers exhibit no clear temporal pattern, particularly if the sample is randomized by excluding failures on to the surfaces of residual YDS glaciers (dashed lines in Fig. 4c and f).", "In terms of the four hypotheses outlined in the introduction there is only limited evidence for failure as a rapid, immediate response to deglaciation (less than 29% of sampled RSFs), but strong evidence for concentration of RSF activity within a few millennia following ice-sheet shrinkage (Fig. 4b and e) as suggested by previous studies in Scandinavia (Morner, 2004; Blikra et al., 2006; Longva et al., 2009) and Central Switzerland (Strasser et al., 2013).", "In general, the pattern of a combination of both rapid and delayed response identified by Ballantyne and Stone (2013) fits the combined data (Fig. 4a and d), though the initial ‘rapid’ response is more muted than their analysis suggested.", "The proposition that the timing of failure extends throughout the postglacial period without significant clustering is refuted by the full data set (Fig. 4a and d) and for sites deglaciated during ice-sheet shrinkage and not reoccupied by glacier ice during the YDs (Fig. 4b and d), but not for sites reoccupied by glacier ice during the YDS (Fig. 4c and f).", "Retrodiction of the factors responsible for preconditioning or triggering RSFs in formerly-glaciated mountains has focused on (1) paraglacial stress release and consequent fracture propagation associated with deglacial unloading of rock slopes and (2) the role of particular extrinsic mechanisms (‘debuttressing’, earthquakes, thaw of permafrost ice in joints, frost wedging and high joint-water pressures) in triggering failure of fractured rock masses (Braathen et al., 2004; Prager et al., 2008; McColl, 2012).", "The possible contribution of each of these mechanisms is considered below.", "Paraglacial stress release is the development of tensile stresses within a rock mass as a result of deglacial unloading, and has been widely cited as a key factor in explaining postglacial RSFs (e.g. Soldati et al., 2004; Bigot-Cormier et al., 2005; Agliardi et al., 2009; Gugliemi and Cappa, 2010; Crosta et al., 2013) but the process is incompletely understood.", "Some authors have emphasized loading by glacier ice and subsequent unloading during deglaciation in altering the state of stress within valley-side slopes (e.g. Wyrwoll, 1977; Augustinus, 1995; Cossart et al., 2008, 2013a; Mercier et al., 2013).", "This model, however, disregards both the effects of topographically-induced stresses (Miller and Dunne, 1996; Molnar, 2004) and in situ stresses resulting from the tectonic and erosional history of the bedrock (Amadei and Stephansson, 1997).", "Leith et al. (2010, 2011) suggested that differential stresses in inherently highly-stressed bedrock are responsible for at least some of the extensive fracture systems commonly observed in deglaciated terrains.", "According to this view, in situ rock stresses provide the maximum principal stress to near-surface bedrock, and the development of pronounced differential stresses during deglaciation reflects reduction in confining stress as ice loading is reduced during deglaciation.", "For tectonically stable terrains, McColl (2012) conceptualized the issue in terms of high surface-parallel stresses induced in two possible ways: (1) release of stress perpendicular to the slope by glacial erosion and/or glacial unloading, whilst slope-parallel confining stresses are maintained by the surrounding rock mass; and (2) increase in topographic stress concentration due to self-weight loading when confining stresses are removed by glacial erosion and/or unloading by ice.", "Irrespective of the cause of deglacial stress release, it is widely accepted that it is responsible for fracture propagation within deglacially-unloaded rock slopes, and in particular for the development of slope-parallel sheeting joints (Hencher et al., 2011; McColl, 2012) that form potential failure planes.", "Such joints are widespread on crystalline rocks in our study areas (Glasser, 1997) and planar or stepped failure surfaces at some RSF sites demonstrate their importance in RSF release (e.g. Ballantyne and Stone, 2004; Ballantyne, 2013).", "Some researchers, however, have rejected deglacial stress release as a cause of RSFs where a millennial-scale delay separates the timing of deglaciation from that of failure (Mitchell et al., 2007; Dortch et al., 2009; Hippolyte et al., 2009; Prager et al., 2009; Stock and Uhrhammer, 2010).", "This interpretation is convincingly challenged by Eberhardt et al. (2004) and Gugliemi and Cappa (2010), who demonstrated that stress changes accompanying Late Pleistocene deglaciation may initiate time-dependent rock mass strength degradation over millennial timescales through progressive shear plane development within fractured rock masses (cf. Kemeny, 2003; Prager et al., 2008; Brideau et al., 2009).", "This view is supported by the work of Cossart et al. (2008), who showed that, irrespective of timing, RSFs in the western Alps are located in areas where reduction of glacially-induced confining stresses was greatest.", "An implication of this interpretation is that stress release initiated by Late Pleistocene deglacial unloading may result in spontaneous kinematic release of rock long after deglaciation, without involvement of any extrinsic trigger mechanism.", "Thus the pattern of timing of postglacial RSFs in Fig. 4a and d may simply reflect the duration of progressive rock-mass weakening to the point of catastrophic failure, with ∼90% of dated RSFs occurring within 5000 years after deglaciation and the remainder over the following five millennia.", "An alternative interpretation is that although stress-release may have preconditioned failure through time-dependent rock mass weakening, the timing of failure, at least in some cases, represents extrinsic triggering.", "Possible triggering mechanisms are considered below.", "The term ‘debuttressing’ is used the describe the removal of supporting glacier ice from potentially unstable rockwalls, and has been cited by several authors as a cause of failure during or immediately after Late Pleistocene deglaciation (e.g. Agliardi et al., 2009; Ivy-Ochs et al., 2009; Hippolyte et al., 2012).", "Both large-scale rock-slope deformation and catastrophic failure of rock slopes has occurred during recent downwastage of valley glaciers (e.g. Bovis, 1990; Sigurdsson and Williamson, 1991; McSaveney, 1993; Evans and Clague, 1994; Holm et al., 2004) lending support to this view, though McColl and Davies (2013) have argued that because glacier ice is liable to deform under stresses imposed by an adjacent unstable rock mass, complete debuttressing is not a prerequisite for movement of ice-contact rock slopes.", "Debuttressing implies that slope failure occurs during or immediately after deglaciation in response to the loss of support of glacier ice, and hence that RSF age should be statistically indistinguishable from deglaciation age.", "This is true for only nine (29%) of our 31 dated RSFs, including the two that failed on to YDS glaciers; all other are significantly younger (p < 0.05) than the inferred timing of deglaciation, demonstrating that debuttressing was not instrumental in triggering failure in the great majority of cases.", "Moreover, the large uncertainties associated with both inferred deglaciation ages and RSF ages (Table 1) mean that some of the nine ‘rapid response’ RSFs could have occurred several centuries after complete deglaciation of the RSF site.", "Debuttressing therefore appears to offer a possible explanation for only a small minority of the RSFs in our sample.", "It is also notable that of >500 postglacial RSFs documented in the Scottish Highlands (Ballantyne, 1986; Jarman, 2006), only three or four cases of RSF runout on to former glacier surfaces have been reported (Ballantyne, 2013), including sites 22 and 23, again suggesting that debuttressing has been of minor significance.", "It has long been recognized that many major RSFs are triggered by earthquakes, particularly in tectonically active mountain belts (Keefer, 1984, 1994; Meunier et al., 2007), and numerous postglacial RSFs have been attributed to seismic activity (e.g. Hormes et al., 2008; Antinao and Gosse, 2009; Sanchez et al., 2010; Stock and Uhrhammer, 2010).", "Simulations by McColl et al. (2012) suggest that thick ice cover substantially reduces ground motion during earthquakes but that this mitigating effect diminishes markedly as ice downwastes during deglaciation.", "According to Eisbacher and Clague (1984), large rock avalanches are typically precipitated by seismic events of magnitude M ≥ 6.0, though smaller RSFS may occur at lower magnitudes and much depends on the pre-seismic state of stress and fracture continuity within a rock mass.", "Moreover, even where earthquake intensities are insufficient to trigger immediate kinematic release, dynamic loading during earthquakes may accelerate fracture propagation, thus promoting subsequent rock-mass failure (Prager et al., 2008).", "Our study area presently experiences relative tectonic stability.", "Recent seismic events with ML > 4.0 are rare and focused near the western seaboard of Scotland (Musson, 2007).", "Several authors, however, have proposed that tectonically-stable intraplate terrains, including the Scottish Highlands, experienced high-magnitude earthquake activity during and after shrinkage of Late Pleistocene glaciers (Sissons and Cornish, 1982; Dehls et al., 2000; Morner, 2004, 2005; Lagerbäck and Sundh, 2008; Strasser et al., 2013).", "Such enhanced seismicity has been attributed to fault (re)activation due to differential glacio-isostatic crustal rebound acting in conjunction with elastically stored tectonic stresses, though because of the prolonged response time of the resulting changes in crustal stress, fault activation and associated seismic activity may lag deglaciation by centuries or millennia (Muir-Wood, 2000; Stewart et al., 2000).", "There is evidence in northern Ireland for metre-scale normal faulting triggered by ice unloading during the final stages of glaciation (Knight, 1999).", "In the Scottish Highlands, postglacial crustal rebound and associated faulting produced metre-high scarps (Firth and Stewart, 2000; Stewart et al., 2001), and soft-sediment deformation structures of Lateglacial or early Holocene age have been interpreted as the products of M ≈ 4.6–6.4 earthquakes (Davenport and Ringrose, 1987; Ringrose, 1989a, 1989b; Stewart et al., 2001).", "The available evidence is therefore consistent with enhanced seismicity during the Lateglacial period, though the magnitude and timing of such activity is poorly constrained (Firth and Stewart, 2000).", "If earthquakes played a major role in triggering postglacial RSFs in Scotland and NW Ireland, a correspondence between RSF ages and the timing of rapid crustal rebound is to be expected.", "The available data on crustal rebound rates are limited, but suggest that at least a broad correspondence exists (Fig. 5).", "For a site at Arisaig on the west coast of Scotland (Fig. 1), for example, maximum rates of crustal uplift (14.3–26.7 mm a−1) occurred during the period ∼15.7–12.7 ka (Firth and Stewart, 2000), coinciding with the timing of all dated RSFs on Jura (10–14), 110 km to the south.", "After ∼12.7 ka average rates of uplift diminished markedly, as does the frequency of dated RSFs (Fig. 5), suggesting that the two may be linked through seismic triggering of RSFs by uplift-driven earthquake activity.", "This link may also explain the abrupt change in the frequency of dated RSFs at ∼11.7 ka (Fig. 3): prior to ∼12.7 ka, average rates of rebound at all sites for which data are available averaged 14.3–31.5 mm a−1; after ∼10.7 ka, average uplift rates at all sites fall below 7.3 mm a−1 (Firth and Stewart, 2000).", "Low-resolution temporal coincidence between uplift rates and RSF frequency (Fig. 5) is not proof of seismic causation, but the morphology of most dated RSFs is also consistent with seismic triggering.", "In every case where there is a clear failure plane it extends to, or near to, the crest of the slope (Fig. 2).", "Densmore and Hovius (2000) have shown that such full-slope failures are characteristic of coseismic RSFs, as topographic amplification of ground acceleration during earthquakes tends to trigger failure at or near ridge crests (Geli et al., 1988; Murphy, 2006).", "Both temporal association and failure plane morphology therefore suggest that the role of uplift-driven seismicity in triggering failure of fractured bedrock has been much more important in glaciated mountains of the British Isles than some studies suggest (e.g. Jarman, 2006).", "An additional possible explanation of the temporal association between Lateglacial RSFs and rapid isostatic uplift is that the latter may have been responsible for large-scale lateral dilation (spreading) of mountain rock masses.", "Cossart et al. (2013b) have suggested that such dilation can generate slope-parallel fractures, normal (extensional) faulting and deep-seated gravitational slope deformation.", "Examples of gravitational rock-slope deformations are common on mountains in the British Isles, particularly on metasedimentary rocks (Jarman, 2006, 2007), and some of the dated RSFs in this study may represent catastrophic failure of slopes weakened by progressive deformation.", "The possible role of uplift-induced lateral dilation of mountain ridges in causing fracture propagation and pre-failure creep cannot, however, be assessed without greater understanding of the age, structure and evolution of extant ‘unfailed’ gravitational slope deformations, informed by theoretical assessment of the magnitude of dilation and dilation-induced stresses likely to be induced by non-uniform isostatic rebound.", "Three climatically-driven factors have been suggested by McColl (2012) as possible triggers of paraglacial RSFs: high joint water pressures, frost wedging and permafrost degradation.", "The millennial-scale delay between deglaciation and failure at over 70% of our RSFs implies that high joint-water pressures associated with deglaciation played no part in triggering failure at these sites.", "We cannot exclude the possibility that failure was in some cases triggered by build-up of joint-water pressures during exceptional rainstorms, but as Densmore and Hovius (2000) have shown, rainstorm-triggered RSFs tend to occur on lower slopes, and in all or nearly all cases our sampled RSFs extend to the slope crest.", "Frost wedging due to recurrent (seasonal) freezing of water and thawing of ice in joint appears unlikely to trigger deep-seated RSFs, as even under propitious conditions annual freeze-thaw cycles affect at most the outermost few metres of rock slopes (Matsuoka, 1994; Matsuoka et al., 1998), and near-saturation of the joint network is required for frost wedging to be effective.", "It is notable, too that the timing of dated RSFs (Table 1; Figs. 3 and 6) spans both stadial conditions (prior to ∼14.7 ka and during the YDS of ∼12.9–11.7 ka), when annual freeze thaw cycles must have been limited to the former active layer, and cool temperate conditions (during the Lateglacial Interstade of ∼14.7–12.9 ka and the Holocene) when annual freeze-thaw depths were limited by depth of winter freezing.", "Thermistor measurements made of depth of freezing within joints on a sandstone cliff at 600 m altitude in NW Scotland (Ballantyne, unpublished data) suggest that present frost penetration is limited to the outermost 0.6–1.1 m of the joint network.", "Permafrost degradation leading to warming and thaw of ice-bonded rock represents an important cause of rock-mass weakening and failure in areas of alpine permafrost under warming temperatures (e.g. Davies et al., 2001; Fischer et al., 2006; Gruber and Haeberli, 2007; Allen et al., 2010; Krautblatter et al., 2012, 2013).", "Permafrost is known to have developed in the British Isles both in the aftermath of ice-sheet deglaciation and during the YDS (Ballantyne and Harris, 1994), so it is possible that permafrost degradation triggered RSFs during periods of climatic warming.", "During the postglacial period there have been two periods of very rapid warming that terminated stadial conditions: at the Dimlington Stade-Lateglacial Interstade transition at ∼14.7 ka, and the YDS-Holocene transition at ∼11.7 ka (Brooks and Birks, 2000; Brooks et al., 2012).", "Nine RSF ages (LL LPR) or 11 RSF ages (NWH11.6 LPR) overlap with one or other of these periods of rapid warming at ± 1σ (Fig. 6), though only four RSF ages (LL LPR) or six RSF ages (NWH11.6 LPR) either coincide with or occur within 500 years after these warming periods.", "We therefore cannot exclude the possibility that warming and thaw of permafrost ice in joints triggered kinematic release in a few cases, but the timing of the great majority of dated RSFS appears to be independent of periods of rapid warming.", "Several interesting features emerge from the temporal patterns of RSF response to deglaciation summarised in Fig. 4.", "Although almost all sampled RSFs at sites deglaciated by ice-sheet retreat occurred within 5000 years following deglaciation (Fig. 4b and e), RSF activity did not peak immediately following deglaciation as suggested by Cruden and Hu (1993), Soldati et al. (2004) and others, but roughly 1000–3000 years after ice-sheet deglaciation.", "As argued above, this long delay probably reflects progressive rock-mass weakening initiated by deglacial stress release and associated tensile rock mass damage.", "According to Eberhardt et al. (2004) and Gugliemi and Cappa (2010), deglaciated rock masses may experience millennial-scale stability without major changes in kinematic state.", "During this pre-failure period, redistributed stress concentrations intensify fracture propagation and coalescence within surface-parallel damage zones, leading to progressive loss of stability through shearing of intact rock bridges and possible ductile deformation until catastrophic failure occurs, either spontaneously or in response to an extrinsic trigger.", "The broad temporal coincidence between maximum rates of glacio-isostatic uplift and maximum RSF activity (Fig. 5) suggests that seismic activity may have been important in triggering failure of some fractured rock masses, particularly prior to ∼12 ka.", "The difference in timing of response between sites deglaciated by ice sheet retreat (Fig. 4b and e) and those deglaciated during retreat of YDS glaciers (Fig. 4c and f) may also, as suggested above, reflect reduced seismic activity during the Holocene due to diminishing rates of crustal uplift.", "An alternative explanation lies in contrasts in the duration and dimensions of ice-sheet glaciation and those of glaciation during the YDS.", "An extensive ice sheet was present over the northern parts of the British Isles for roughly 15,000 years from ∼30 ka to ∼15 ka (Bradwell et al., 2008; Ballantyne, 2010; Clark et al., 2012) during which time its volume and configuration fluctuated markedly (Hubbard et al., 2009).", "For much of this period, ice extended far out on to adjacent shelves and ice buried all summits in NW Ireland and Scotland, implying ice thicknesses at least 1000 m above valley floors, and probably much more, in the Scottish Highlands (Fabel et al., 2012).", "Conversely, most YDS glaciers in the Scottish Highlands existed for less than ∼1200 years, were confined to glacial troughs at the sites of sampled RSFs and accomplished very limited modification of the pre-YDS landscape (Golledge, 2007; Golledge et al., 2008; Golledge, 2010).", "The absence of a pronounced peak in RSF activity following YDS deglaciation may therefore reflect comparatively limited (and comparatively brief) glacial re-loading and unloading of rock slopes at this time.", "Jarman (2006) has suggested that even though most RSFs in the Scottish Highlands occur within YDS glacier limits, failure at such sites primarily reflected ‘slope stresses’ engendered by ice-sheet loading and unloading rather than the effects of glacial reoccupation during the YDS.", "The absence of a post-deglaciation peak in RSF activity in Fig. 4c and f is consistent with this view.", "We note, however, that we cannot exclude the possibility that the contrast in response represents differences in lithology (Sellier, 2008).", "The post-deglaciation peak in RSF activity evident in Fig. 4b and e was generated by dates obtained on quartzite, granite and sandstone RSFs (Table 1), whereas most dated RSFs inside the YDS glacier limits are seated on schist.", "It is possible that the pre-release lag in response to deglaciation differs systematically amongst lithologies.", "An implication of the temporal pattern of RSFs at sites not reoccupied by glacier ice during the YDS (Fig. 4b and e) is that numerous RSFs must also have occurred inside the limits of YDS glaciation in valleys that were completely deglaciated during the Lateglacial Interstade (∼14.7–12.9 ka), but these have not been recorded in RSF inventories (Ballantyne, 1986; Jarman, 2006) because YDS glacier ice subsequently removed RSF runout debris.", "Such RSF sites can be identified by ‘empty’ failure scars comprising a steep headwall and subjacent planar or stepped failure surface.", "Although no systematic survey of such sites has been carried out, it is possible that Lateglacial RSFs that occurred before or during YDS glaciation made a substantial contribution to the sediment load of YDS glaciers in Scotland and elsewhere (Ballantyne, 2002, 2013).", "More generally, our results support the results of previous research in Scandinavia (Morner, 2004; Blikra et al., 2006; Longva et al., 2009) and central Switzerland (Prager et al., 2008) indicating that the shrinkage of Late Pleistocene ice sheets on tectonically-stable intraplate steepland terrains was followed by a period of greatly enhanced RSF activity extending over a few millennia.", "It seems likely that deglacial stress release and progressive pre-failure rock mass weakening through fracture propagation, rock-mass deformation and possibly large-scale uplift-induced mountain dilation played a key role in preconditioning and triggering failure, though seismic activity driven by rapid rates of glacio-isostatic recovery may have precipitated kinematic release of fractured rock masses, particularly during the Lateglacial period of maximum uplift rates.", "Glacial debuttressing, thaw of ice-bonded rock, frost wedging and high joint-water pressures are inferred to have been of less importance in triggering postglacial RSFs in such areas." ]	[ "Ages of 31 dated postglacial rock-slope failures span the Lateglacial and Holocene.", "95% of RSFs outside Younger Dryas ice limits occurred within ∼5.4 ka of deglaciation.", "Stress release and progressive failure plane development preconditioned failure.", "Glacio-isostatic uplift-driven seismic events probably triggered many RSFs.", "Debuttressing, permafrost thaw and deglacial water pressures were less important." ]
STAT3beta, a distinct isoform from STAT3	STAT3β is an isoform of STAT3 (signal transducer and activator of transcription 3) that differs from the STAT3α isoform by the replacement of the C-terminal 55 amino acid residues with 7 specific residues. The constitutive activation of STAT3α plays a pivotal role in the activation of oncogenic pathways, such as cell proliferation, maturation and survival, while STAT3β is often referred to as a dominant-negative regulator of cancer. STAT3β reveals a “spongy cushion” effect through its cooperation with STAT3α or forms a ternary complex with other co-activators. Especially in tumour cells, relatively high levels of STAT3β lead to some favourable changes. However, there are still many mechanisms that have not been clearly explained in contrast to STAT3α, such as STAT3β nuclear retention, more stable heterodimers and the prolonged Y705 phosphorylation. In addition to its transcriptional activities, STAT3β may also function in the cytosol with respect to the mitochondria, cytoskeleton rearrangements and metastasis of cancer cells. In this review, we summarize the mechanisms that underlie the unique roles of STAT3β combined with total STAT3 to enlighten and draw the attention of researchers studying STAT3 and discuss some interesting questions that warrant answers.	[ "BIOC" ]	[ "Cancer suppressor", "JAK/STAT3 pathway", "Prognostic factor", "STAT3", "STAT3β" ]	[ "1303", "1307" ]	[ "STAT3 (signal transducer and activator of transcription 3) is one of seven STAT proteins (STATs 1, 2, 3, 4, 5A, 5B and 6) and is a highly pleiotropic protein that is activated downstream of multiple cytokine and growth factor receptors by tyrosine 705 phosphorylation (Akira et al., 1994; Lutticken et al., 1994; Zhong et al., 1994; Copeland et al., 1995) (Fig. 1).", "STAT1, STAT3 and STAT5 encode multiple forms and are structurally similar.", "However, these proteins have divergent and opposing effects on gene expression and cellular phenotypes (STAT1 is generally considered as a tumor suppressor (Zhang and Liu, 2017), and STAT3 and STAT5 are generally oncogenes (Desrivieres et al., 2006; Groner and von Manstein, 2017)) (Gouilleux-Gruart et al., 1996; Lai and Johnson, 2010; Quesnelle et al., 2007).", "STAT3 was initially identified as APFR (Acute Phase Response Factor), which is responsible for activating the promoters of acute phase genes in response to IL-6 (Wegenka et al., 1993).", "The canonical mechanism of STAT3 signalling is that it exists as a latent, un-phosphorylated monomer in the cytosol until cytokines or growth factors [e.g., IL-6, IL-10, EGF, PDGF, and TNF] engage their cognate receptors.", "The ligand/receptor interaction causes a change in the confirmation and concurrent activation of the receptor associated JAK (Janus Kinase) proteins.", "Activated JAKs trans-phosphorylate each other and the cytoplasmic tail of the receptor on the tyrosine residues, which provides docking sites for STAT3 to be recruited via its SH2 domain.", "Once recruited, STAT3 is phosphorylated on a single C-terminal tyrosine residue (Y705) by JAKs.", "This Y705 phosphorylation provides the possibility for STAT3 dimerization, which translocates into the nucleus, which is assisted by importin-α3 (Liu et al., 2005), and bind to consensus DNA sequences (γ-activated sequence (GAS) TTCNNNGAA (Becker et al., 1998)) and initiates transcription (Fig. 1).", "This pathway is tightly regulated in normal cells and transient (Heim et al., 1995; Stahl et al., 1995; Garama et al., 2016).", "However, a plenty of studies have provided evidence of nearly 70% tumor cell lines and patient samples for the incidence of constitutive STAT3 activity, such as breast (Bharadwaj et al., 2015), pancreas (Desrivieres et al., 2006; Gouilleux-Gruart et al., 1996), head and neck squamous cell carcinoma (HNSCC) (Lai and Johnson, 2010) and leukaemia and lymphoma (Quesnelle et al., 2007; Wegenka et al., 1993).", "Recently, a growing number of studies have indicated that STAT3β, a splice variant of STAT3, may play a suppressive effect, since it lacks the TAD (transactivation domain), which is in contrast to STAT3α (Schaefer et al., 1997; Maritano et al., 2004; Dewilde et al., 2008) (Fig. 2).", "Experimental data dating back to 1996 support this concept.", "Caldenhoven E. et al. found that the co-expression of STAT3β inhibited the transactivation potential of STAT3α and suggested that STAT3β functioned as a negative regulator of transcription (Caldenhoven et al., 1996).", "Conversely, STAT3β appears to regulate inflammatory factors, affect the tumour microenvironment and attract immune cells to play a role in tumour inhibition (Zammarchi et al., 2011; Wang et al., 2004; Dang et al., 2015).", "Importantly, increasing numbers of studies demonstrate that a relatively high-STAT3β (compared with STAT3α) level exerts a tumour suppressive effect in several tumour cell lines, acting as a dominant negative regulator (Yu et al., 2009; Couto et al., 2012; Musteanu et al., 2010).", "Our lab demonstrated that high STAT3β expression converts the prognostic value of pSTAT3αY705 from unfavourable to favourable in patients with ESCC (oesophageal squamous-cell carcinoma) (Zhang and Lai, 2014).", "Moreover, the induction of a splicing switch towards the beta isoform leads to apoptosis and cell-cycle arrest in STAT3-dependent cell lines via the activation of a unique gene expression signature (Musteanu et al., 2010).", "Overall, the balance between the two isoforms of STAT3 is apparently crucial to determining the occurrence and development of cancers, which can be depicted as a “spongy cushion” effect (Fig. 3).", "In the 1980s, the Darnell laboratory was investigating interferon-induced gene expression and found the existence of some transducers (Larner et al., 1984; Decker et al., 1989).", "The STAT proteins were eventually biochemically identified as the key signalling molecules in the interferon pathway in the early 1990s (Schindler et al., 1992).", "STAT3 was initially identified as APRF, a DNA-binding activity appearing in IL-6-treated hepatocytes and interacting with a cis-acting element on the promoter of acute-phase genes (Wegenka et al., 1993).", "STAT3 is a multifunctional factor protein that is involved in a striking number of functions and activates distinct repertoires of genes in different contexts via the stimulation of many factors (e.g., IL-6 family members, leptin, IL-12, IL-2, IFNs, IL-10, G-CSF, growth hormone, EGF, HGF, LIF, and v-Src (Zhong et al., 1994; Ruff-Jamison et al., 1994; Tian et al., 1994; Ihle and Kerr, 1995; Seto et al., 2015; Ram and Iyengar, 2001).", "Taking IL-6 for example (Fig. 1), receptor-associated JAKs are phosphorylated through gp130 when IL-6 combines with the IL-6 receptor.", "Subsequently, phosphorylated JAKs, in turn, cause multiple phosphorylation events at tyrosine residues within the cytoplasmic domain of the cytokine receptor, thereby providing a docking site for the SH2 domain (Src homology 2) of STAT3.", "By combining with the docking site, STAT3 becomes phosphorylated at Y705, a critical tyrosine on the C-terminal domain of STAT3, and it gains the ability to dimerize with another monomer through the reciprocal interaction of the SH2 domain (Akira et al., 1994; Yu et al., 1995; Sasse et al., 1997; Shuai et al., 1994).", "Dimeric STAT3 complexes translocate to the nucleus, where they bind to response elements in the promoters of target genes to stimulate transcription.", "In addition to the phosphorylation of Y705, which is seen as a key activating mechanism of STAT3, S727 (serine 727) phosphorylation, in the C-terminal domain, promotes the association of STAT3 with transcription co-activators (including p68, p300/CBP), providing the maximal activation of particular target genes (Schuringa et al., 2001; Heinrich et al., 2003; Frank, 2007).", "For instance, in prostate cancer and chronic lymphocytic leukaemia, the phosphorylation of S727, rather than Y705, was found to be crucial for the nuclear translocation, DNA binding, and tumour-promoting function of STAT3 (Hazan-Halevy et al., 2010).", "The JAK-STAT3 signalling pathway is engaged by many cytokines and growth factor stimuli to control diverse biological processes in a both cell- and tissue-specific manner.", "Remarkably, STAT3 is the only family member that is early embryonic lethal on inactivation, which indicates the biological importance of STAT3 (Takeda et al., 1997).", "Many STAT3 targets, such as Survivin, Cyclins and the Bcl-2 family proteins, promote cell proliferation and survival (Yu et al., 2007; Yue and Turkson, 2009).", "Studies using conditional STAT3 knockout mice provide evidence that STAT3 is required for the development and differentiation of various tissue types, such as the skin, immune system, liver, mammary gland, thymus and nervous system (Levy and Lee, 2002).", "For example, the deletion of STAT3 in the mammary glands suppresses apoptosis in glandular epithelial cells and leads to a delayed glandular involution (Wegenka et al., 1993).", "In another study, the ablation of STAT3 in keratinocytes was found to impair the migration of keratinocytes and skin remodelling (Sano et al., 1999).", "In addition, STAT3 is critical to the development and biology of immune cells.", "In one study in which STAT3 was conditionally ablated in all stratified epithelia, including the thymic epithelia, there was a dramatic increase in apoptosis in thymocytes.", "In addition, the STAT3-depleted thymocytes were more susceptible to apoptosis induced by dexamethasone and γ-irradiation (Sano et al., 2001).", "In another study, CD4+ T cell differentiation in inflammatory responses was shown to be regulated by STAT3 through Loxl3's deacetylation (Ma et al., 2017), and even in tumour cell lines, its inactivation triggers growth arrest and cell death (Bowman et al., 2000).", "These proliferative gene targets include Cyclin D1, c-Myc, PLK-1 and Pim1/2 (Avalle et al., 2012).", "Accumulating evidence suggests that STAT3 plays a critical role in promoting the self-renewal of cancer stem cells (Sherry et al., 2009; Guryanova et al., 2011; Marotta et al., 2011; Kim et al., 2013).", "In addition to STAT3’s proliferative and survival features, this protein also functions in resistance to apoptosis and the induction of angiogenesis.", "In one study, STAT3 was shown to be important in mediating the anti-apoptotic effect of IL-6 in the presence of a low-serum culture environment (Takeda et al., 1998).", "Among many cancer cell types, STAT3 transcriptionally increases the expression of various anti-apoptotic proteins, such as survivin and the Bcl-2 family members (e.g., Bcl-XL, Bcl-2 and Mcl-1) (Yu et al., 2007; Regis et al., 2008).", "Moreover, STAT3 negatively controls the manifestation of p53, which helps in the induction of apoptosis, as well as inhibiting cellular proliferation (Niu et al., 2005).", "STAT3 is shown to cooperate with c-Jun to suppress the expression of FAS, a crucial mediator of the extrinsic apoptotic pathway.", "Multiple studies demonstrate that activated STAT3 protects cancer cells from FAS ligand-induced apoptosis and p53-dependent apoptosis (Niu et al., 2005; Ivanov et al., 2001, 2002; Kunigal et al., 2009).", "It must be noted, however, that under physiological conditions, STAT3 may act as an inducer of cell death.", "In particular, STAT3 is required for lysosome-mediated epithelial cell death during mammary gland involution, where LIF functions as an activator of STAT3 (Kreuzaler et al., 2011).", "Increased VEGF expression in cultured cell lines, animal models and patient cancer specimens, as well as tumour angiogenesis in vivo, is also induced via the STAT3 pathway in diverse human cancers, such as head and neck squamous cell carcinoma, melanoma, pancreatic cancer, cervical cancer, colorectal cancer, and renal carcinoma (Wei et al., 2003a, b; Niu et al., 2002; Xu et al., 2005; Kujawski et al., 2008; Jung et al., 2007).", "STAT3 directly mediates the pro-angiogenic activity of VEGF in microvascular endothelial cells (Bartoli et al., 2003).", "Researchers reveal that STAT3 directly binds to the promoter region of the VEGF gene (sometimes combined with HIF1α (Oh et al., 2011)) and promotes its transcription, and using an inducible STAT3 knockout mouse model, it is revealed that STAT3 promotes the production of angiogenic factors (including VEGF and bFGF) in myeloid-derived suppressor cells and macrophages present in the tumour microenvironment, thereby stimulating endothelial cell migration and tumour angiogenesis (Wei et al., 2003a, b; Niu et al., 2002; Kujawski et al., 2008).", "Cellular movement and migration have long been an area of interest and importance to scientists.", "Especially for tumour cells, cellular invasion and metastasis are critical steps for the tumour prognosis.", "Many studies show that STAT3 promotes invasiveness and the metastatic potential of cancer cells by triggering EMT (epithelial to mesenchymal transition) via the upregulation of several key EMT regulators, such as Twist-1, Snail and ZEB-1 (Yadav et al., 2011; Lo et al., 2007; Guo et al., 2013; Xiong et al., 2012).", "In one study in SKOV3 cells, STAT3 signalling was revealed to be important for cell motility, and the exhaustion of STAT3 using siRNA reversed the situation; thus, the cellular migration rate was reduced (Silver et al., 2004).", "In another study, the loss of Stat3 expression in mouse embryonic fibroblasts led to an elevation in Rac1 activity, which promoted a random mode of migration by reducing the directional persistence and formation of actin stress fibres (Teng et al., 2009).", "STAT3 increases the expression of various MMPs (matrix metalloproteinases), which facilitate cancer cell invasiveness by degrading various extracellular matrix proteins.", "In mice, STAT3 reduces pancreatic cancer cell invasiveness and MMP-7 using an shRNA (Li et al., 2011).", "STAT3 protein expression is upregulated by activated STAT3, which directly binds to the promoter of the MMP-2 gene in melanoma (Li et al., 2011).", "Likewise, MMP-1 and MMP-9 is also regulated by STAT3 (Dechow et al., 2004; Itoh et al., 2006).", "As depicted in some studies, inflammation also plays a role in immune evasion.", "In 1999, Takeda et al. shows that STAT3 inhibited TH1-type inflammation after LPS stimulation by suppressing the production of specific cytokines and nitric oxide (Takeda et al., 1999).", "In another study, the activities of STAT3 in tumour cells enhanced the expression of several immune-suppressing soluble factors, such as IL-6, IL-10 and VEGF, all of which are known to prevent the maturation of dendritic cells (Sun et al., 2006).", "As indicated above, the canonical STAT3 pathway regulates many genes, the expression of which is required for cancer initiation, development and progression, including uncontrollable proliferation, anti-apoptosis, invasion, angiogenesis and immune evasion.", "Once the negative regulatory loop of STAT3 is lost (mainly SOCS, PTPs and STAT1) (Zhang and Lai, 2014; Avalle et al., 2012; Wang et al., 2012; Croker et al., 2003), STAT3 becomes constitutive activated and provides the possibility of oncogenesis.", "In addition, some pre-clinical studies demonstrate that constitutively phosphorylated-STAT3 (pSTAT3) is a common characteristic of many cancers (Yang et al., 2013; Macha et al., 2011; Huang et al., 2012; Li et al., 2015a; Shi et al., 2015).", "However, to date, no STAT3 gene mutation has been detected in any cancer.", "In addition to the canonical activities regulated by the phosphorylation of Y705 and the transcriptional function of STAT3, unph-STAT3 (un-phosphorylated STAT3) is recognized as an important transcriptional regulator (Yang et al., 2005, 2007; Yang and Stark, 2008).", "For example, Yu et al. (2002) found that unph-STAT3, through a direct physical interaction with p65, served as a dominant-negative inhibitor that suppressed the ability of ph-NFκB to induce the cytokine-dependent activation of the iNOS promoter in mesangial cells.", "In contrast, some studies show that unph-STAT3 interacts with NFκB in the nucleus to drive the expression of multiple cancer-related genes, such as RANTES, IL-6, IL-8, MET and MRAS (Yang et al., 2005, 2007).", "Another research group found that unph-STAT3 binds to similar DNA sites as the Y705 phosphorylated and dimerized STAT3 (e.g., GAS elements), but unph-STAT3 works in collaboration with transcriptional regulators, such as NFκB, to control a series of genes not normally affected by tyrosine-phosphorylated STAT3 (Timofeeva et al., 2012).", "With respect to modification, one group discovered that STAT3 interacted with DNMT1 (DNA methyltransferase1) and HDAC1 (histone deacetylase 1), by which STAT3 facilitated the gene methylation and silencing of SHP-1 in malignant T lymphocytes (Zhang et al., 2005).", "In another group, it was revealed that K685-acetylated STAT3 cooperated with DNMT1 to silence several tumour suppressor genes, including TP53, SHP-1, SOCS3 and CDKN2A, in melanomas (Lee et al., 2012).", "Intriguingly, in 2009, STAT3 was also identified in the mitochondria (Gough et al., 2009), and its import was dependent on the phosphorylation at S727 (Tammineni et al., 2013).", "This mitochondrial STAT3 supports the activity of the Electron Transport Chain (ETC), which is required for ATP production and the opening of the mitochondrial permeability transition pore (mPTP).", "The loss of STAT3 reduces the activity of the ETC, especially complex I, II and V (Gough et al., 2009; Wegrzyn et al., 2009), which is restored by reconstituting these cells with a mitochondrially restricted form of STAT3.", "These mitochondrial activities of STAT3 have consequences both in normal tissue homeostasis (e.g., neurite outgrowth and cardiac function) and in pathological conditions (e.g., tumour growth and tissue damage in response to ischaemia/reperfusion injury) (Wegrzyn et al., 2009).", "However, the deletion of STAT3 from keratinocytes results in the increased expression of mtDNA encoded genes, implying that STAT3 represses the transcription of the mitochondrial genome.", "Through an unknown mechanism STAT3 traverses two mitochondrial membranes to reside in the mitochondrial inner membrane or the matrix where it augments the activity of the electron transport chain and impedes the opening of the mPTP.", "This has consequences on cellular ATP production, ROS concentration, calcium homeostasis, and cell survival (Garama et al., 2016; Yang and Rincon, 2016; Meier et al., 2017).", "The mitochondrial pool of STAT3 is an emerging and exciting area of STAT biology.", "However, the mechanisms, especially in tumours, by which STAT3 is imported into the mitochondria and its activities within the mitochondrion still need to be illustrated.", "It is obvious that STAT3 exists in the cytosol, since mitochondrial STAT3 is imported from the cytosol.", "Importantly, some research groups also found that STAT3 functions in regulating the cytoskeleton.", "In one study, tumour-derived cell lines displayed higher migration, invasion, and metastatic abilities and showed a disrupted distribution of cell-cell junction markers, which was mediated by the STAT3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein and was also significantly upregulated in STAT3C (a continuously activated STAT3 mutant construct with two cysteine substitutions at the residues A661 and N663) mammary tumours (Barbieri et al., 2010).", "Consistent with this concept, another study indicates that STAT3 modulates the microtubule network by binding to the COOH-terminal tubulin-interacting domain of stathmin and antagonizing its microtubule destabilization activity (Ng et al., 2006).", "Moreover, Debra L.S. et al. (Silver et al., 2004) found that activated STAT3 coimmunoprecipitated with phosphorylated paxillin and focal adhesion kinase (FAK) and required paxillin and Src for its localization to focal adhesions in ovarian cancer.", "Recently, one study also showed that the depletion of STAT3 in gastric cancer cells impaired microtubule polymerization, due to the disruption of the interaction between STAT3 and Stathmin, and as a result, cell migration and invasion were decreased (Wei et al., 2013).", "Protein structure is characterized by a hydrophobic/hydrophilic equilibrium, and structural stability depends largely on the hydrophobic nature of the molecule.", "The terminal of STAT3β is approximated as a truncated form of STAT3α, but the hydrophobicity of STAT3β is better than STAT3α.", "Moreover, in a physical chemistry study, Asn466 is conserved in STAT1 to STAT4 and is critical for the sequence-specific recognition in STAT3 (Fig. 2), and the classic SH2 domain interactions are strongly conserved in both STAT3 isoforms (Becker et al., 1998).", "Because of the complexity of the STAT3 structure, there are many differences between STAT3α and STAT3β in biochemistry, and the related issues are discussed below.", "After identifying that STAT3β cooperated with c-Jun, Schaefer et al. employed COS-7 cells transfected with STAT3 expression plasmids to exploit the functional differences between STAT3α and STAT3β.", "These researchers found that activated STAT3β, in transfected COS cells, was more stable and had a greater DNA-binding activity than activated STAT3α.", "However, STAT3α exhibited a stronger transcriptional activity than STAT3β (Schaefer et al., 1997).", "Considering that STAT3αΔ48 (a mutant of STAT3α lacking its highly acidic C-terminal 48 amino acids) had properties similar to STAT3β, they concluded that this was due to the presence or absence of the acidic C-terminal tail of STAT3α rather than the STAT3β’s 7 specific terminal sequence, and the acidic tail of STAT3α may destabilize the active dimeric form of STAT3α, resulting in a lower DNA-binding activity and a more rapid dephosphorylation (Schaefer et al., 1997).", "Subsequent reports also confirm these phenomena.", "Another group measured the DNA binding strength and dimer stability in COS-7 cells and revealed that the C-terminal deletions of STAT3α increased both the DNA binding activity and dimer stability of STAT3α, suggesting that STAT3α and STAT3β have similar binding strengths via an EMSA assay (Park et al., 2000).", "Interestingly, STAT3β tends to be constitutively phosphorylated at tyrosine 705 and binds to DNA and promotes transcription in the absence of cytokine treatment, whereas STAT3α does not, indicating the increased half-life of the tyrosine phosphorylated STAT3β (Caldenhoven et al., 1996; Schaefer et al., 1995).", "Related studies were reported by two other groups.", "Firstly, U. Bharadwaj et al. revealed STAT3β's contribution to constitutive STAT3 phosphorylation in breast cancer (Bharadwaj et al., 2014).", "Secondly, Ivan H.W.NG et al. showed the sustained nuclear translocation and phosphorylation of STAT3β following cytokine exposure, which was in contrast with the transient nuclear translocation and phosphorylation of STAT3α in AD293 cells (a variant of HEK-293 cell), and they also revealed that STAT3β enhanced and prolonged the phosphorylation and nuclear retention of STAT3α.", "However, a STAT3β R609L mutant (with a disrupted SH2 domain) did not show similar phenomena (Ng et al., 2012), indicating that STAT3β’s effects need Y705 phosphorylation and dimerization.", "Our lab’s findings, in ESCC cell lines (EC109 and KYSE150), also revealed the same phenomena (Zhang et al., 2016).", "In addition, the unique 7 amino acid tail (FIDAVWK) may also contribute to STAT3β’s features, since it is reported to prolong the nuclear retention of phosphorylated STAT3β (Huang et al., 2007).", "One possible reason that might account for these phenomena is that the STAT3β’s hydrophobic tail protects STAT3β from dephosphorylation or keeps it from degradation by proteasome (STAT1β protects STAT1α from degradation (Zhang et al., 2017; Baran-Marszak et al., 2004)), and thus keeps the STAT3β dimers (e.g., STAT3α/β, STAT3β/β) constitutively phosphorylated and exhibiting a stable DNA binding ability by combining with other co-activators.", "For the phosphatase of STAT3, TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in the rapid dephosphorylation of STAT3 (Yamamoto et al., 2002; Kim et al., 2010; Sharma et al., 2016; Lee et al., 2017).", "Initially, researchers thought that the absence of the interaction of the phosphatase (e.g., TC45) with STAT3β, due its different STAT3β C-terminal sequence, might contribute to the prolonged Y705 phosphorylation and nuclear retention of STAT3β.", "However, subsequent studies suggested otherwise, because either isoform interacted with TC45 (Ng et al., 2012).", "Another point of view is that STAT3β may have to cooperate with other activators (Schaefer et al., 1995; Ivanova et al., 2004), thereby showing a diverse transcriptional pattern in contrast to STAT3α, since STAT3β lacks the transactivation domain (especially the S727 site, which is shown to enhance transcriptional activities (Schuringa et al., 2001)).", "Moreover, one paradoxical phenomenon is that the enforced expression of STAT3β substantially increases the level of pSTAT3αY705, which is considered an oncogenic signal.", "However, STAT3α is retarded in the presence of sufficient STAT3β, which indicates that whether pSTAT3αY705 level is oncogenic or carcinostatic is largely dictated by the expression statue of STAT3β (Ng et al., 2012; Zhang et al., 2016).", "All in all, further exploration in this field has implications for relevant issues.", "Considering that STAT3β lacks the transactivation domain (TAD), it may be short of transcriptional activities in contrast to STAT3α.", "In 1995, however, Schaefer et al. identified that STAT3β (but not STAT3α) and c-Jun were capable of cooperatively activating a certain promoter containing an IL-6 responsive element in the absence of added cytokines or growth factors (Ivanov et al., 2001).", "Another group also identified that STAT3β associates with the HLH and the C-terminal regions of STRA13, co-expression of STRA13 with STAT3α or STAT3β modulated the transcriptional outcome indicating a repressing rather than activating potential for the STAT3β complexes (Ivanova et al., 2004).", "These may indicate that the transcriptional activity of STAT3β is quite similar to the different transcriptional activities of the two STAT1 isoforms (known as STAT1α and STAT1β) (Shuai et al., 1993).", "In another study, the activities of three promoters (2-macroglobulin, c-fos, and p53) in Stat3β-deficient MEFs (mouse embryonic fibroblasts) was tested using a transient reporter assay, and their activities were substantially reduced.", "Furthermore, they explored the effects of Stat3β-deficiency on the expression of endogenous transcripts using an oligonucleotide array (283 genes exhibiting differential expression, with 36 genes showing a greater than 2-fold differential expression) and an in-depth identification by RT-PCR (Crip, Tfpi, Ptn, and Scya2 RNA were elevated in Stat3-deficient MEFs, while the Sdf1 and Igfbp5 transcripts were elevated in the WTs) (Yoo et al., 2002).", "Similarly, Ng et al. examined the impact of the reconstitution of the STAT3−/− MEFs with either isoform on gene expression and found 651 genes unique for the re-expression of STAT3α, 1331 genes unique for STAT3β and 506 genes shared between STAT3α and STAT3β, with statistical significance (Ng et al., 2012).", "Recently, morpholinos (one alternative splicing modulator) were applied to specifically promote a physiological α-to-β splicing shift in one type of breast cancer cell line, revealing a unique STAT3β signature, with the downregulation of specific targets (including lens epithelium-derived growth factor, p300/CBP-associated factor, CyclinC, peroxisomal biogenesis factor 1, and STAT1β), which are distinct from that canonical STAT3 targets that are typically associated with total STAT3 knock-down (Zammarchi et al., 2011).", "Moreover, mice specifically lacking STAT3α but still expressing STAT3β (STAT3α-/-) do not die during embryogenesis, which indicates the transcriptional compensatory role of STAT3β (Maritano et al., 2004).", "All of these findings reveal that STAT3β functions as a transcriptional regulator and specifically regulates genes by cooperating with other factors (e.g., c-Jun, STRA13), since it lacks the transactivation domain.", "Continuous inflammation is a common feature of the tumour microenvironment and plays a crucial role in both the occurrence and development of many malignancies (Balkwill and Mantovani, 2012; Mantovani, 2010; Hanahan and Weinberg, 2011; Hainaut and Plymoth, 2013).", "STAT3 was initially identified as acute-phase response factor (APRF) (Wegenka et al., 1993) and was considered as a key player in mediating inflammation-related tumourigenesis via constitutively activating and participating in a positive feedback loop with IL-6 and NFκB (a pro-oncogenic transcription factor) (Yu et al., 2009; Grivennikov and Karin, 2010).", "For STAT3α, it acts both as a pro- and anti-inflammatory factor depending on the activating signal (Hutchins et al., 2013; Hodge et al., 2005).", "For STAT3β, on the one hand, it appears to be a suppressor of systemic inflammation.", "Two Stat3β−/− mice studies show a hyper-responsiveness to endotoxic shock and a diminished recovery from that (Maritano et al., 2004; Yoo et al., 2002).", "Stat3β−/− mice develop exacerbated atherosclerosis in the absence of ApoE (Lee et al., 2013a).", "Peritoneal macrophages from Stat3β−/− mice produce significantly more TNF and IL-6 than Stat3+/+ control mice and have reduced IL-10 (an anti-inflammatory factor) when treated with LPS (lipopolysaccharide) (Maritano et al., 2004), indicating that STAT3β may directly or indirectly participate in the regulation of IL-10 expression to function as an inflammatory regulator.", "On the other hand, STAT3β upregulates the expression of pro-inflammatory cytokines in B16 melanoma cells, and in supernatants from STAT3β-transfected B16 melanoma cells, it induces the activation of macrophages, granulocytes and dendritic cells, indicating the antitumoural aspect of STAT3β (Wang et al., 2004).", "Additionally, in an animal model, restoring STAT3β re-induces acute phase response genes in hepatocytes (Alonzi et al., 2001).", "The same protein gives rise to two apparently opposite results, and this may also indicate the diversity of STAT3β’s co-activators.", "In addition, the cell-specific expression of STAT3β in macrophages also exhibits antitumour effects in mouse breast cancer, indicating that STAT3β may also play an important role in the cells from the tumour microenvironment (Dang et al., 2015).", "In addition to regulating the expression of inflammatory factors, STAT3β may attenuate the secretion of factors that suppress the activity of immune cells, thereby indirectly activating dendritic cell maturation (Wang et al., 2004), which is a process that involves MHC class II and co-stimulatory molecule expression (Park et al., 2004; Kitamura et al., 2005).", "A major role of STAT3 is self-renewal.", "Recently, increasing studies reveal that STAT3 also plays a critical role in the regulation of the stemness of cancer stem cells (Sherry et al., 2009; Guryanova et al., 2011; Marotta et al., 2011; Kim et al., 2013; Liu et al., 2013; Tu et al., 2012).", "For example, STAT3 isoforms have distinct roles in myeloid cell proliferation, survival and differentiation, indicating that STAT3β may not act as a dominant negative regulator in these processes.", "STAT3β has a strong cell and tissue specificity, and the ratio of the STAT3α:STAT3β mRNA and protein levels ranges from 4:1 to 10:1 and 1:3 to 10:1 (Bharadwaj et al., 2014).", "According to these studies, the ratio of STAT3α:STAT3β is highly regulated in myeloid cells and is consistently decreased during cell maturation and activation (Biethahn et al., 1999; Hevehan et al., 2002; Chakraborty et al., 1996).", "In another study, in normal human CD34+ bone marrow cells and HL60 cells, both reported to differentiate upon G-CSF stimulation, G-CSF does not activate STAT3α but only an 83 kD form of STAT3 (STAT3β) (Chakraborty et al., 1996).", "However, only STAT3α (but not STAT3β) generates a markedly higher number of neutrophils in response to G-CSF when it is over-expressed in the 32Dcl3 myeloid cell line (Redell et al., 2007).", "Our research showed that STAT3β overexpression significantly decreased the clonogenic capacity and increased the sensitivity to 5-FU and cisplatin in a STAT3β dose-dependent manner (Zhang et al., 2016).", "Similar to the above, STAT3 also plays a role in the stemness of other cells (Lomada et al., 2016; Sherry-Lynes et al., 2017; Ma et al., 2015; Lee et al., 2013b), however, whether STAT3β plays a regulatory role in these process remain to be explained.", "The cytoskeleton and focal adhesions are two main aspects of cancer metastasis, a multiple process in which tumour cells leave their original location and go to new tissues through the blood vessels.", "Various reports have shown that increased STAT3 activity can enhance intercellular contact and up-regulate the expression of genes related to tumor cell invasion and metastasis, suggesting that STAT3 may be a sensor for tumor cell contact.", "(Tu et al., 2012; Gatsios et al., 1996; Pansky et al., 2000; Peng et al., 2016; Zhou et al., 2015; Lee et al., 2010).", "Sano et al. first described that STAT3 possessed a pivotal role in cellular movement and wound healing processes in cultured keratinocytes (Sano et al., 1999).", "STAT3 cooperates with stathmin and modifies microtubule dynamics and the migration of cells, such that microtubule depolymerization starts when an oncoprotein 18-stathmin binds to α/β-tubulin heterodimers (Ng et al., 2006).", "Additionally, the loss of STAT3 displays some changes in randomized cellular migration, while STAT3 indirectly controls Rac-1 activity to sustain migration (Teng et al., 2009).", "Interestingly, in an in vitro study using SKOV3 cells, STAT3 is critical for cell motility, and the knock-down of STAT3, using siRNA, reverses the situation, such that cellular migration is repressed, and it was further indicated that ph-STAT3 co-immunoprecipitated with ph-paxillin and focal adhesion kinase and required paxillin and Src for its localization to the focal adhesions (Silver et al., 2004).", "In other studies, STAT3 activation can reduce the expression of tumor suppressor gene E-cadherin in human skin squamous cell carcinoma (Hillmer et al., 2016) and prostate epithelial cells (Azare et al., 2007), activate ErbB2/integrin β4 signaling pathway in breast cancer (Guo et al., 2006), and increase the level of ICAM-1 /CD54 in human glioma cells (Kesanakurti et al., 2013), so that cell invasion and metastasis ability enhanced.", "However, STAT3β does exactly the opposite role compared to STAT3 (STAT3α) (Niu et al., 2001; Xu et al., 2009).", "This is an interesting area indicating that STAT3β may participate in these regulations, considering that STAT3β antagonizes STAT3α by inducing or inhibiting the expression of genes associated with cell motility.", "Additionally, our group indeed found that STAT3β disrupted the rhythm of ESCC movement (unpublished data).", "To date, the dominant negative role of STAT3β has been reported in various types of cancer, including melanoma, breast cancer, oesophageal cancer, lung cancer and colonic cancer (Zammarchi et al., 2011; Ivanov et al., 2001; Zhang et al., 2016; Niu et al., 2001; Xu et al., 2009; Niu et al., 1999; Ivanov et al., 2009; Rivat et al., 2005).", "One group found that the overexpression of STAT3β induces cell death in B16 melanoma cells in vitro (Niu et al., 1999).", "Additionally, in U266 myeloma cells, which inherently express elevated Bcl-XL, STAT3β also promotes programmed apoptosis (Catlett-Falcone et al., 1999) by inducing soluble necrosis factors (Niu et al., 2001).", "Similar results are also confirmed in lung cancer, which show the downregulation of Bcl-XL and Cyclin D1 (Xu et al., 2009).", "STAT3β also efficiently upregulates DR5 (the tumour necrosis factor-relate apoptosis-inducing ligand receptor) surface expression and downregulates cFLIP (caspase-8 inhibitor) levels in melanoma cells in vitro and in vivo (Ivanov et al., 2009).", "Another group revealed that the overexpression of STAT3β downregulates the VEGF receptor Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation by using DNA microarrays and a gene differential expression analysis (Rivat et al., 2005).", "Our lab’s studies revealed that a moderate/strong expression of STAT3β significantly was correlated with a longer overall survival and recurrence-free survival and was less likely to have lymph node metastasis in ESCC (Zhang et al., 2016).", "These findings indicate that STAT3β is an independent protective factor for patient survival.", "STAT3β can form more stable dimer (STAT3α/STAT3β, STAT3β/STAT3β), it occupies STAT3α stably and weakens the transcriptional ability of STAT3α/STAT3α thus is suggested to have a promising future in gene therapy, since there are no specific small-molecule inhibitors have entered the clinical stage only targeting STAT3α (Schust et al., 2006; Hong et al., 2015; Li et al., 2015b; Huang et al., 2018).", "Altogether, high-level STAT3β levels in cancer cells indeed lead to favourable results.", "However, among the published clinical studies, researchers rarely differentiate the relative levels of the two STAT3 isoforms, and its prognostic significance has also rarely been identified.", "As we can see from the above mechanisms, STAT3β exerts its negative roles mainly because STAT3β lacks the TAD domain and forms a transcription complex with STAT3α or other co-activators, thereby playing its unique role or repressing STAT3α’s role.", "Thus, the surveying of STAT3β independently of STAT3α is meaningless.", "This molecular mechanism is depicted as a “spongy cushion” (Fig. 3), which cushions the transient and intense transcriptional role of STAT3α, thus avoiding the excessive activation of STAT3α.", "Meanwhile, the relatively high level STAT3β amplifies its role in the regulation of inflammation, immunity, apoptosis, etc.", "Thus, in the exploration of STAT3 that is carried out to this extent, a careful distinction between STAT3α and STAT3β in different cell types and cancers is required.", "To date, there are four recognized subtypes of STAT3, including STAT3α (92 kDa), STAT3β (83 kDa), STAT3γ (72 kDa) and STAT3δ (64 kDa), while STAT3α and STAT3β are generated by alternative splicing, and STAT3γ and STAT3δ are derived from proteolytic processing and exhibit no transcriptional role (Hevehan et al., 2002; Nakajima et al., 2003; Hendry and John, 2004; Kato et al., 2004).", "The oncogenic role of STAT3α has long been recognized, but its spliceform-STAT3β has not yet been given adequate attention.", "STAT3β is mainly distinguished from STAT3α by its truncated terminus and exhibits unique features by cooperating with STAT3α or other co-activators.", "In analyses from reports over the past thirty or forty years, many studies reveal the dominant negative role of STAT3β (Caldenhoven et al., 1996; Niu et al., 2001; Xu et al., 2009; Niu et al., 1999; Epling-Burnette et al., 2001; Karni et al., 1999; Sinibaldi et al., 2000).", "However, the concrete reasons for this are unknown and are only combined with surface gene expression differences.", "A STAT3β/3β homodimer may exist, and a few reports also confirm that STAT3β directly functions with other co-activators (Schaefer et al., 1995; Ivanova et al., 2004).", "In addition to the interactions between STAT3α and STAT3β, STAT3 also interacts with STAT1, which is quite similar in homology but exerts diametric effects.", "In addition, no one can deny the existence of STAT1β/3β and STAT1α/3β heterodimers, and their functions are even less known.", "All of these areas increase the complexity of STAT3’s function.", "Moreover, recently, some groups have concentrated on mitochondrial STAT3 and found that STAT3 enhances the activity of the electron transport chain (Garama et al., 2016; Yang and Rincon, 2016; Meier et al., 2017; Huang et al., 2016).", "However, whether STAT3β binds to mtDNA and such functions as its multiple nuclear roles need further verification.", "Finally, several studies have revealed the possibility of STAT3, especially STAT3β, as a regulator of tumour cell invasion and migration, but the exact mechanism needs further study.", "The adversity that we are faced with is the relative protein levels of STAT3α:STAT3β at approximately 4:1 (Bharadwaj et al., 2014).", "It is precisely because of this ratio that many current studies directly ignore the role of STAT3β and choose STAT3α as the major object.", "However, growing evidence demonstrates that STAT3β does play an irreplaceable role accompanied by STAT3α, and its “spongy cushion” effect is notable.", "The past cognition does not affect STAT3β, and it might be a good drug target and independent prognostic marker in cancer.", "Interestingly, U.Bharadwaj et al. (Bharadwaj et al., 2014) developed monoclonal antibodies that specifically recognize the unique CT7 epitope and do not cross-react with Stat3α and “STAT3β-deg” (proteolytic cleavage forms of Stat3α), which brings many conveniences to the in-depth study of STAT3β.", "Overall, we believe the exploration of STAT3β will yield new insights into cancer therapy and provide new directions for STAT3 studying.", "No conflicts of interest." ]	[ "STAT3β reveals a unique transcriptional role by forming heterodimer with STAT3α or ternary complex with other co-activators.", "STAT3β exerts a “spongy cushion” effect through cushioning the transient and intense transcriptional role of STAT3α.", "STAT3β plays a suppressive role in cancer through regulating inflammatory factors, tumor microenvironment and immune cells.", "STAT3β can reduce resistance to chemotherapy, induce apoptosis and be an independent prognostic factor." ]
A pharmacological mouse model suggests a novel risk pathway for postpartum psychosis	Postpartum psychosis (PP) is a severe psychiatric disorder affecting a small proportion of new mothers shortly after childbirth. The molecular pathophysiology underlying the disorder is currently poorly understood, and there are no amenable animal models for the condition; maternal deficiency for the enzyme steroid sulfatase has been proposed as a potential risk mechanism. Here we show that inhibition of steroid sulfatase with 667-COUMATE (10 mg/kg p.o.) in new mouse mothers results in behavioural abnormalities that can be partially alleviated by the administration of the clinically-efficacious antipsychotic ziprasidone (0.3–1.0 mg/kg i.p.). The pattern of behavioural abnormalities in 667-COUMATE-treated mice implicated a genetic substrate at 21–23 cM on chromosome 15; of the 17 genes within this chromosomal interval, only one (Nov/Ccn3) was significantly differentially expressed in the brains of vehicle and 667-COUMATE-treated mice. Two additional members of the Ccn family (Ccn2/Ctgf and Ccn4/Wisp1) were also significantly differentially expressed between the two groups, as were three further genes co-expressed with Nov/Ccn3 in brain (Arhgdig) or previously implicated in disorder risk by clinical studies (Adcy8 and Ccl2). The expression of Nov/Ccn3, but not of the other differentially-expressed genes, could be normalised by ziprasidone administration (1.0 mg/kg). NOV/CCN3 lies directly under a linkage peak for PP risk at 8q24, and the associated protein possesses numerous characteristics that make it an excellent candidate mediator of PP risk. Our data suggest the 667-COUMATE-treated mouse as a model for PP with some degree of face, construct, and predictive validity, and implicate a novel, and biologically-plausible, molecular risk pathway for PP.	[ "BIOC", "MEDI", "NEUR" ]	[ "667-COUMATE", "Elevated plus maze", "Nephroblastoma-overexpressed", "Startle response", "Steroid sulfatase" ]	[ "1310", "2712", "2738", "2803", "2807" ]	[ "Postpartum psychosis (PP) is a severe psychiatric disorder occurring shortly after childbirth in 1–2 out of every 1000 mothers (Sit et al., 2006).", "The disorder is characterised by hallucinations, delusions, cognitive disorganisation and mood problems, and is associated with an increased risk of maternal suicide or infanticide (Sit et al., 2006).", "The pathophysiological basis of PP is poorly understood due, in part, to the lack of an amenable animal model.", "The identification of biomarkers associated with increased risk is a key goal for ensuring early clinical intervention.", "Increased PP risk is associated with a personal or family history of psychotic disorder (notably bipolar disorder), with precipitous drops in circulating oestrogens following childbirth, with obstetric complications including pre-eclampsia, and with psychosocial stressors (Sit et al., 2006).", "Small genetic linkage and association studies have implicated regions of chromosome 16p13 and 8q24 (Jones et al., 2007) and serotonergic abnormalities (Kumar et al., 2007) respectively, but have not identified robust candidate genes.", "Recently, immune system (Bergink et al., 2013) and tryptophan-kynurenine pathway (Veen et al., 2016) disruptions have been demonstrated in PP, whilst regular smoking is associated with reduced risk (Di Florio et al., 2015).", "Maternal deficiency for the enzyme steroid sulfatase (STS) may predispose to PP (Davies, 2012); STS converts sulfated steroids to their non-sulfated forms (e.g. dehydroepiandrosterone sulfate, DHEAS, to DHEA) which may act as precursors for oestrogens and androgens (Davies, 2012).", "Maternal STS deficiency is associated with obstetric complications (Fernandes et al., 2010), whilst in healthy individuals, DHEAS serum levels positively correlate with postpartum psychoticism (Marrs et al., 2009); decreased postpartum DHEA levels are associated with activation of the maternal immune system (Tagawa et al., 2004).", "In the first part of the study (Experiment 1), we tested whether acute inhibition of steroid sulfatase in new mouse mothers would elicit behavioural and gene expression changes of relevance to PP.", "STS was inhibited using 667-COUMATE (also known as STX64), a drug that systemically and irreversibly inhibits enzyme activity by >90% in rodents at the dose used here (Purohit et al., 2000).", "STS inhibition was indexed indirectly by measuring serum levels of DHEA(S), and levels of the stress hormone corticosterone were also measured.", "The behavioural tasks assayed aspects of emotional reactivity, activity and sensorimotor gating that are commonly perturbed in psychotic disorders; pup maltreatment or infanticide, which may represent a surrogate measure of PP in animals (Quilter et al., 2007) was also recorded.", "In Experiment 2, we tested whether 667-COUMATE-induced behavioural and gene expression changes could be reversed by administration of clinically-relevant doses of the atypical antipsychotic drug ziprasidone (Sit et al., 2006).", "Virgin C57BL/6JOlaHsd female mice (aged 12–26 weeks, originally obtained from Envigo UK and bred within Cardiff University School of Psychology), were housed in trios with males of the same strain.", "3–5 days before giving birth, females were housed individually and were monitored closely until birth.", "Mice were maintained on ad libitum food and water, in a temperature, humidity and light-controlled room (21 ± 2 °C, 50 ± 10% humidity, lights on at 0700hr for 12 h) and were regularly inspected for signs of ill health.", "Experiments were performed according to the UK Animal Scientific Procedures Act (1986).", "<12 h after giving birth, mothers were injected per os (p.o.) with either vehicle solution (tetrahydrofuran:polyethylene glycol 400: distilled water in a 1:6:3 ratio (Ireson et al., 2004)), n = 14) or 667-COUMATE (10 mg/kg, Sigma-Aldrich, UK) in the same vehicle (n = 17) in a pseudo-randomised manner.", "Mothers were administered the same treatment 48 h after this first injection.", "The 667-COUMATE administration regime was based upon previously-published pharmacokinetic data in rodents (Ireson et al., 2004; Purohit et al., 2000) and was intended to provide maximal enzyme inhibition across the postpartum period whilst minimising off-target effects.", "Injections were performed between 09:00–10:00 h. Behavioural testing was carried out 24 h after the second injection between 09:00–13:00 h.", "<12 h after giving birth, mothers were injected per os (p.o.) with 667-COUMATE (10 mg/kg) as above.", "24 h after the first injection, mice were injected intraperitoneally (i.p.) with either vehicle solution (1% methylcellulose in 0.9% saline) or one of two doses of ziprasidone hydrochloride (0.3 mg/kg or 1.0 mg/kg (free-base concentrations) Sigma-Aldrich, UK) in the same vehicle in a pseudo-randomised manner.", "24 h later, mice received a second injection of 667-COUMATE (10 mg/kg), and 23 h after this, mice received a final injection of vehicle, 0.3 mg/kg, or 1.0 mg/kg ziprasidone.", "Injections were performed between 08:30–10:00h.", "The three experimental groups were: mice which received 667-COUMATE (10 mg/kg) with ziprasidone (0 mg/kg)(CVCV group, n = 16), mice which received 667-COUMATE (10 mg/kg) with ziprasidone (0.3 mg/kg)(CZCZ0.3 group, n = 16), and mice which received 667-COUMATE (10 mg/kg) with ziprasidone (1.0 mg/kg)(CZCZ1.0 group, n = 8).", "Ziprasidone doses were selected to have minimal effects on activity (Kalinichev and Dawson, 2011).", "Behavioural testing was carried out 1hr after the final injection between the hours of 09:30–13:00 h.", "Prior to injections, mother/pup health, litter sizes and weights, and maternal weights were recorded; pup deaths or signs of maternal aggression towards the pups were noted.", "These measures allowed us to assess whether the drug regimes were adversely affecting gross maternal and/or pup health, and/or pup maltreatment or infanticide; we were particularly concerned that inhibition of STS in the mother’s mammary gland may affect provisioning of her pups.", "Only mothers who gave birth to at least one live pup were included in the study.", "Mothers were initially behaviourally tested on an elevated plus-maze (Isles et al., 2004) to assay anxiety-related and exploratory phenotypes.", "Mice were placed in a closed arm, and allowed to explore the apparatus freely for 5mins.", "Their lateral and vertical (‘rearing’) activity was objectively tracked using Ethovision Observer software Version 3.0.15 (Noldus Information Technology, The Netherlands); additional measures of vertical and lateral exploration (head dips from the open arms and stretch attend postures) and emotional reactivity (defecation) were recorded manually.", "Key measures of interest were the ratio of open arm:closed arm time and latency to first entry of the open arms (indexing anxiety), entries into the closed arms (an index of within-maze activity minimally confounded by anxiety), numbers of rears/head dips and stretch-attend postures, and numbers of fecal boli.", "Two seconds per animal were added to the open arm latency measure obtained from Ethovision to account for the time lag between putting the animal on the maze and initiating tracking.", "Following testing on the elevated plus-maze, basal activity level was tested in the dark using locomotor cages (Isles et al., 2004).", "The number of infra-red beam breaks made over a 1hr session (4 × 15 min bins) was recorded.", "Finally, sensorimotor gating was assayed using a startle and prepulse inhibition (PPI) paradigm (Dent et al., 2014); the main measures of interest were startle response over first three pulse alone trials (an index of emotional reactivity), the startle response for four levels of prepulse at 0, 4, 8 and 16 dB above background (P120, PP4P120, PP8P120 and PP16P120 respectively, an index of habituated startle and PPI), and startle response over pulse-alone trials with varying stimulus intensity (an index of auditory acuity).", "The behavioural tests were administered in order of increasing severity such that performance on the latter tests would not be substantially influenced by prior exposure to potential stressors.", "Mice were returned to their homecage for 5–10min between behavioural tests.", "3 h after behavioural testing, subjects were culled by cervical dislocation.", "Trunk blood was collected in BD SST Microtainer Gold tubes (BD Biosciences) and serum extracted according to the manufacturer’s instructions prior to storage at −80 °C.", "Whole brains were removed, bisected sagitally, and frozen on dry ice.", "Serum steroid hormone levels were assayed by ELISA (dehydroepiandrosterone sulfate (DHEAS) and DHEA DRG International; corticosterone, Enzo Biosciences) according to the manufacturer’s instructions, with sample dilution as necessary and samples run in triplicate.", "Samples were taken from vehicle-treated mice (n = 9 for DHEA and corticosterone, and n = 10 for DHEAS), and 667-COUMATE treated mice (n = 15 for all compounds); DHEAS and DHEA levels were both measured in vehicle-treated (n = 8) and 667-COUMATE-treated (n = 15) mice.", "Standard curves were determined using SigmaPlot 11.0 (Systat Software Inc.) according to the equation: y = y0 + (ab/(b + x)) where y0, a and b are constants.", "High-quality total RNA was extracted from the right hemisphere of the brain using RNeasy Universal Midi Kit (Qiagen) according to the manufacturer’s instructions.", "For Experiment 1, RNA was extracted from vehicle-treated mice and 667-COUMATE treated mice (n = 12 per group).", "For Experiment 2, RNA was extracted from mice administered 667-COUMATE with ziprasidone (0 mg/kg)(n = 11), mice administered 667-COUMATE with ziprasidone (0.3 mg/kg)(n = 11) and mice administered 667-COUMATE with ziprasidone (1.0 mg/kg)(n = 6).", "20 μl cDNA solution was synthesised from 4 to 5 μg RNA using RNA-to-cDNA EcoDry Premix with random primers (Clontech), and was diluted 50-fold with distilled water.", "Quantitative PCR analysis was performed using the ΔCt method using Gapdh, Hprt and Rn18 s as housekeeping genes (Trent et al., 2014) (primer sequences available in Supplemental Table S1 in the online version at DOI: 10.1016/j.psyneuen.2016.09.019).", "High-quality total RNA was extracted from the right hemisphere of the brain using RNeasy Universal Midi Kit (Qiagen) according to the manufacturer’s instructions.", "For Experiment 1, RNA was extracted from vehicle-treated mice and 667-COUMATE treated mice (n = 12 per group).", "For Experiment 2, RNA was extracted from mice administered 667-COUMATE with ziprasidone (0 mg/kg)(n = 11), mice administered 667-COUMATE with ziprasidone (0.3 mg/kg)(n = 11) and mice administered 667-COUMATE with ziprasidone (1.0 mg/kg)(n = 6).", "20 μl cDNA solution was synthesised from 4 to 5 μg RNA using RNA-to-cDNA EcoDry Premix with random primers (Clontech), and was diluted 50-fold with distilled water.", "Quantitative PCR analysis was performed using the ΔCt method using Gapdh, Hprt and Rn18 s as housekeeping genes (Trent et al., 2014) (primer sequences available in Supplemental Table S1).", "Data were analysed using SPSS 20 (IBM Corporation, New York).", "Normal data (as determined by Shapiro-Wilk test) were analysed by unpaired two-tailed (unless stated otherwise) t-test/One Way ANOVA or Two Way Repeated Measures or Mixed Model ANOVA, with a between-group factor of DRUG TREATMENT and a within-group factor of TIMEPOINT or PULSE TYPE.", "Startle data were analysed using bodyweight and ‘pressure exerted in the absence of a stimulus’ as covariates.", "Where sphericity assumptions were violated in ANOVA, Greenhouse-Geisser corrected degrees of freedom values are presented.", "Post hoc pairwise comparisons were performed using the Sidak test.", "Non-parametric data were analysed by one or two-tailed Mann—Whitney U test.", "Correlational analyses for normally and non-normally distributed data were performed using Pearson test or Spearman Rank-Order Correlation respectively.", "P-values of <0.05 were regarded as nominally significant.", "Normally-distributed data are reported as mean values ± standard error of the mean, and non-parametric data as median values with 95% confidence intervals (obtained by bootstrapping).", "Initial litter sizes (i.e. prior to the first injection) were comparable between the vehicle and 667-COUMATE treatment groups; the number of pups found dead was equivalently low for both groups (Table 1).", "All mothers built and maintained normal nests.", "667-COUMATE treatment in the mother did not affect gross health or bodyweight (DRUG TREATMENT: F[1,29] = 1.18, p = 0.29, DRUG TREATMENT x TIMEPOINT: F[3,87] = 2.15, p = 0.10).", "Pup health was normal across the course of the experiment; mean pup weight increased equivalently across both experimental groups from ∼1.30 g to ∼1.75 g (DRUG TREATMENT: F[1,29] = 0.42, p = 0.52, TIMEPOINT: F[2.1, 61.1] = 3.39, p = 0.038, DRUG TREATMENT x TIMEPOINT: F[2.1, 61.1] = 0.47, p = 0.64).", "Treatment with 667-COUMATE in new mothers resulted in a significantly reduced latency to enter the open arms of the maze, and a significantly greater propensity towards rearing; these effects occurred in the absence of significant effects on other behavioural measures assessed in this test (Table 1).", "The experimental groups showed an equivalent decrease in activity over the course of the 1hr test session (from ∼515 to ∼305 infrared beam breaks per 15 min bin) (DRUG TREATMENT: F[1,29] = 0.00, p = 0.97; TIMEPOINT: F[1.68,48.7] = 24.6, p < 0.001; DRUG TREATMENT x TIMEPOINT: F[1.68,48.7] = 0.40, p = 0.64).", "There was no significant effect of treatment on initial mean startle response to three pulses of 120 dB (i.e. 55 dB above background)(vehicle: 55 ± 10 arbitrary startle units, 667-COUMATE: 61 ± 15 arbitrary startle units, F[1,27] = 0.01, p = 0.94).", "On the ‘pulse alone’ and ‘prepulse’ trials, there was the expected pattern of reduced startle response with increasing levels of prepulse (PULSE TYPE, F[3,29] = 54.4, p < 0.001); whilst there was no main effect of DRUG TREATMENT (F[1,29.5] = 1.41, p = 0.25), there was a significant DRUG TREATMENT x PULSE TYPE interaction (F[3,29] = 3.05, p = 0.044) with 667-COUMATE-treated mice displaying reduced startle when no, or a minimal, prepulse stimulus was present (Fig. 1A).", "Although there was the expected trend of increased startle inhibition with increasing volumes of the prepulse (F[1.66,48.1] = 86.5, p < 0.001), there was no significant main effect of DRUG TREATMENT (F[1,29] = 0.39, p = 0.85) nor any significant DRUG TREATMENT x PULSE TYPE interaction (F[1.66,48.1] = 0.70, p = 0.48) (Fig. 1B).", "Increasing stimulus volume from background (70 dB) to 120 dB was associated with increased startle response in both groups consistent with normal hearing and motor reactivity (DRUG TREATMENT (F[1,29.2] = 1.70, p = 0.20, PULSE TYPE F[5,29] = 17.39, p < 0.001, DRUG TREATMENT x PULSE TYPE (F[5,29] = 1.68, p = 0.17)(Fig. 1C); on average, startle in drug-treated mice at pulses of 110 dB and 120 dB was lower than in vehicle-treated mice, consistent with the data presented above.", "Serum DHEAS levels showed the expected trend towards higher levels in the drug-treated group, whilst serum DHEA levels were, on average, lower in the drug-treated group (Table 1).", "For mice in which both DHEAS and DHEA levels were measured, the ratio of median DHEAS:median DHEA levels was 189 for the vehicle-treated group and 620 for the 667-COUMATE treated group (i.e. ∼3.3fold higher in drug-treated mice than in vehicle-treated mice).", "Consistent with our previous data in adult male MF1 mice (Trent et al., 2013), there was a significant inverse correlation between serum DHEA levels and 1hr locomotor activity in postpartum females (rs = −0.416, p = 0.043).", "Serum corticosterone levels did not differ between groups (Table 1).", "Our behavioural data indicated drug-induced effects on elevated plus-maze rearing and latency to enter the open arms in the same task; intriguingly, these two measures are strongly influenced by a small quantitative trait locus between 21–23 cM on mouse chromosome 15 (Henderson et al., 2004), a region syntenic with human chromosome 8q24.", "Thus, we compared the expression of 17 genes with known products within this interval (http://www.informatics.jax.org/marker, last accessed 2nd September 2016) in vehicle and 667-COUMATE-treated hemibrains.", "Only one between-group comparison (for Nov, also known as Ccn3) was significant, with higher expression in drug-treated tissue (t[22] = −2.19, p = 0.039, Fig. 2A) (p ≥ 0.12 for other comparisons).", "The nominally-significant effect of 667-COUMATE treatment on Nov/Ccn3 expression would not survive correction for multiple testing.", "We considered that if significant effects of 667-COUMATE treatment on the expression of one or more of the other five members of the Nov/Ccn gene family were to be found, this would provide additional confidence that the original finding was a true effect.", "Both the expression of Ctgf/Ccn2 and Wisp1/Ccn4 were significantly increased in 667-COUMATE-treated tissue (t[22] = −2.15, p = 0.043 and t[17.27] = −2.47, p = 0.024 respectively), whilst expression levels of Cyr61/Ccn1, Wisp2/Ccn5 and Wisp3/Ccn6 did not differ between the two conditions (t[22] = 0.01, p = 0.99, p = 0.11, and t[22] = 0.67, p = 0.51 respectively)(Fig. 2B).", "We then assessed the expression of three immune-related genes (Ccl2, Cxcl1 and Il33) whose activity is thought to be regulated by Nov/Ccn3 (Le Dreau et al., 2010; Perbal, 2006), and that have previously been implicated in PP (Ccl2) or bipolar disorder (Il33) pathogenesis (Barbosa et al., 2014; Bergink et al., 2013).", "Brain Ccl2 expression levels were significantly elevated in 667-COUMATE-treated mice (t[22] = −2.28, p = 0.033), whilst Cxcl1 and Il33 levels were equivalent between the two groups (t[22] = −0.10, p = 0.92 and t[22] = 0.30, p = 0.77 respectively)(Fig. 2C).", "We also examined the expression of: a) murine orthologues of three genes suggested as positional candidates for PP (Jones et al., 2007) (Abat and Grin2a at 16p13, and Adcy8 at 8q24), b) two genes within 16p13 whose expression correlates with CTGF/CCN2 or NOV/CCN3 expression in developing human brain tissue (Li et al., 2016) (Hba-a1/a2 and Arhgdig), and c) two genes suggested by genomewide association studies as candidates for bipolar disorder (Cacna1c and Odz4/Tenm4)(Harrison, 2016).", "Adcy8 expression was significantly higher in the brains of 667-COUMATE-treated mice (t[22] = −2.17, p = 0.041), consistent with previous evidence that adenylate cyclase (Adcy) activity might be involved in the mechanoregulation of the Ccn genes (Schild and Trueb, 2004); Arhgdig expression was also increased in 667-COUMATE-treated brain relative to vehicle-treated brain (t[22] = −2.375, p = 0.027)(Fig. 2C), and Nov/Ccn3 and Arhgdig expression (as indexed by ΔCt values) was significantly positively correlated across both experimental groups (r = 0.51, p = 0.01).", "There was no effect of DRUG TREATMENT on the expression of Abat, Grin2a, Hba-a1/a2, Cacna1c or Odz4/Tenm4 (t[22] = −0.29, p = 0.78, t[22] = −0.67, p = 0.51, t[20.2] = −1.04, p = 0.31, t[22] = −1.13, p = 0.27 and t[22] = −1.09, p = 0.29 respectively) (Fig. 2C).", "Finally, we examined the expression of two serotonergic system genes: Htr2a (encoding a primary target for antipsychotic drug action) and Htr2c (the expression of which may be affected by Sts deficiency, Trent et al., 2012).", "The expression level of neither of these two genes was affected by DRUG TREATMENT (Htr2a: t[22] = 0.08, p = 0.94, and Htr2c: t[22] = 0.27, p = 0.79)(Fig. 2C).", "Overall, our gene expression data indicate a high degree of specificity to the effects of 667-COUMATE.", "Initial live litter sizes (i.e. prior to any injections) did not differ between the CVCV, CZCZ0.3 and CZCZ1.0 groups; the number of pups found dead was equivalently low for all groups (Table 2).", "All mothers built and maintained normal nests.", "Maternal health and bodyweight was not significantly differentially affected by DRUG TREATMENT (F[2,37] = 1.83, p = 0.18, DRUG TREATMENT x TIMEPOINT: F[1.4, 76.0] = 0.34, p = 0.83).", "Pup health also appeared normal across the course of the experiment; mean pup weight increased equivalently across all groups (from ∼1.30 g to ∼1.80 g) (DRUG TREATMENT: F[2,37] = 0.47, p = 0.63, TIMEPOINT: F[1.5, 57.0] = 2.02, p = 0.15, DRUG TREATMENT x TIMEPOINT: F[3.1, 57.0] = 0.40, p = 0.76).", "We hypothesised that, in 667-COUMATE-treated mice, ziprasidone administration would attenuate rearing in the elevated plus-maze and increase latency to enter the open arms in the absence of minimal effects on activity.", "Overall, rearing levels were low and equivalent across the three groups (Table 2).", "Whilst median first open arm entry latencies did increase across the three groups, this was not significant (Table 2).", "The interpretation of the rearing and open arm entry data is complicated by the fact that both doses of ziprasidone tended to reduce activity in the aversive elevated plus-maze, as indexed by closed arm entries (Table 2).", "In contrast to the closed arm entry data in the elevated plus-maze, we found no significant difference between groups in terms of their overall number of beam breaks in the relatively non-aversive locomotor activity paradigm (Table 2), indicating that the reduced activity in the antipsychotic-treated groups in the elevated plus-maze test may have been partially due to the anxiogenic effect of ziprasidone in 667-COUMATE treated mice.", "We further hypothesised that, in 667-COUMATE-treated mice, the administration of ziprasidone would enhance the startle response, particularly when a prepulse stimulus was absent or small.", "Ziprasidone did dose-dependently enhance startle, though for all values of pulse-prepulse pairings (DRUG TREATMENT: F[2148] = 9.44, p < 0.001, PULSE TYPE: F[3148] = 3.29, p = 0.023, DRUG TREATMENT x PULSE TYPE: F[6148] = 0.08, p = 1.00, Fig. 3A).", "We compared the brain expression of genes whose expression differed significantly in Experiment 1 (i.e. Nov/Ccn3, Ctgf/Ccn2, Wisp1/Ccn4, Ccl2, Adcy8, and Arhgdig in mice treated with 667-COUMATE plus one of three doses of ziprasidone (0, 0.3 or 1.0 mg/kg)(Fig. 3B).", "Nov/Ccn3 expression was significantly affected by DRUG TREATMENT, with significantly lower expression in the CZCZ1.0 group relative to the other two groups (F[2,25] = 5.07, p = 0.014, p < 0.05 for pairwise comparisons with 1.0 mg/kg group).", "Adcy8 expression was significantly affected by DRUG TREATMENT, with higher expression in the CZCZ1.0 group relative to the other two groups (p < 0.01, p < 0.005 for pairwise comparisons with CVCV and CZCZ0.3 groups respectively).", "The expression of Ctgf/Ccn2, Wisp1/Ccn4, Ccl2 and Arhgdig genes did not differ as a function of DRUG TREATMENT (F[2,25] = 0.90, p = 0.42, F[2,25] = 0.54, p = 0.59, F[2,25] = 3.36, p = 0.051, and p = 0.44 respectively).", "The molecular pathophysiology of PP is poorly defined, partially as a consequence of having no amenable animal models; the main animal model for PP currently is the ‘porcine infanticide model’ (Quilter et al., 2007) which, although valuable, is impractical for large-scale experimental studies and does not accurately reflect the PP phenotype (most mothers with PP are not infanticidal).", "We used pharmacological techniques to produce an experimentally-amenable mouse model of PP with some degree of face, construct and predictive validity that may indicate underlying pathogenic processes.", "Briefly, acute postpartum inhibition of the steroid sulfatase enzyme in mouse mothers, predicted on the basis of theory to give rise to PP-related phenotypes (‘construct validity’)(Davies, 2012), elicits phenotypes of relevance to PP.", "In terms of behavioural face validity, 667-COUMATE-treated mice exhibited: a) a reduced startle response with preserved PPI (a phenotype seen in bipolar disorder and dependent upon levels of pro-inflammatory cytokines (Beck and Catuzzi, 2013)) and b) alterations in elevated plus-maze behaviour, likely to be influenced by a region of chromosome 15 (syntenic to the PP candidate region 8q24 in man).", "These behavioural phenotypes were not confounded by the effects of 667-COUMATE on maternal/pup health, or maternal activity.", "In terms of endocrinological face validity, 667-COUMATE treated mice, like women with PP (Epperson and Ballew, 2006), exhibited normal corticosterone/cortisol levels.", "In terms of molecular face validity, our model showed alterations in the brain expression of a gene previously implicated in bipolar disorder and PP (Adcy8) (Jones et al., 2007) and a gene encoding a pro-inflammatory cytokine (Ccl2) whose levels are elevated in serum of individuals with PP (Bergink et al., 2013); the abnormal expression of these genes may contribute towards the behavioural profiles of the drug-treated mice.", "As one 667-COUMATE-induced behavioural phenotype (reduced startle response) could be reversed by the administration of an antipsychotic used to treat PP, the model may also have some degree of predictive validity.", "The extent to which the model recapitulates other features of PP remains to be established.", "Moreover, whilst 667-COUMATE is an effective inhibitor of STS, it may have off-target effects, notably on carbonic anhydrase II (Ho et al., 2003); these off-target effects have no gross effects on maternal health, but their contribution to the more subtle changes in drug-treated mice is difficult to quantify.", "In support of steroid sulfatase inhibition-mediated effects on the gene expression differences reported here, DHEA influences Ctgf/Ccn2 (Zhang et al., 2013) and Ccl2 (or MCP-1)(Wang et al., 2011) expression and secretion in females.", "Our gene expression work indicated altered expression of several members of the Ccn gene family in 667-COUMATE treated brain.", "This gene family, which encodes a number of secreted extracellular matrix-associated proteins expressed in the brain (Malik et al., 2015), represents an interesting candidate for effects on PP risk given: a) its known involvement in female reproductive function (Winterhager and Gellhaus, 2014), b) its dynamic brain expression throughout pregnancy and the puerperium (Ray et al., 2015), c) its modulation of Notch and Wnt signalling pathways (Winterhager and Gellhaus, 2014) that are disrupted in bipolar disorder (Pedroso et al., 2012) and cases of postpartum psychiatric disturbance (Pantoni et al., 2005) and d) the known role of extracellular-matrix dysfunction in the pathophysiology of mood disorders (Lubbers et al., 2014).", "Additionally, Ccn gene expression may be altered by the administration of psychotomimetic agents (Sakuma et al., 2015), by social stress (Stankiewicz et al., 2015), and by small molecules including cytokines and serotonin (Chen et al., 2014).", "Using methods agnostic to gene function, we have specifically implicated NOV/CCN3, encoding a biologically-relevant protein: a) its overexpression is associated with abnormal maternal behaviour which can be reversed through antipsychotic administration (present study), b) it is located ∼138 cM on human chromosome 8, below the linkage peak indicated from a sample of individuals with bipolar affective puerperal psychosis (137.5–147.5 cM) (Jones et al., 2007), c) the associated protein is thought to modulate intracellular calcium signalling (Lombet et al., 2003) a process that goes awry in both bipolar disorder (Harrison, 2016) and PP (Riley and Watt, 1985), d) its expression is repressed by oestrogen (Vendrell et al., 2004), e) it encodes a regulator of placental angiogenesis and its expression is perturbed in cases of pre-eclampsia (Winterhager and Gellhaus, 2014), f) it is highly expressed in the cortex and limbic system of the adult human brain (Malik et al., 2015), g) it can regulate axonal outgrowth of callosal projection neurons (Park et al., 2015) consistent with corpus callosum abnormalities in PP cases (Udaya et al., 2015), h) it lies adjacent to a polymorphism associated with smoking cessation (Argos et al., 2014) and its expression is downregulated in female tissues exposed to cigarette smoke (Gueugnon et al., 2016), and i) small genomic duplications encompassing NOV/CCN3 have been associated with bipolar disorder (Macayran et al., 2006).", "Given possible thyroid dysfunction in PP (Bergink et al., 2011), it is also interesting that cortical Nov/Ccn3 and Adcy8 expression is regulated by 3,5,3′-triiodo-l-thyronine (T3) (Berbel et al., 2014).", "Finally, recent work has shown that wildtype mouse mothers carrying pups with genetic mutations perturbing placental function exhibit abnormal behaviour and significantly increased hippocampal Nov/Ccn3 gene expression (Creeth et al., 2015 and Hugo Creeth, pers.", "comm.).", "The study has two main limitations.", "First, in the absence of data on behaviour or gene expression in non-postpartum mice administered 667-COUMATE, we cannot ascertain whether STS deficiency and its sequelae confer risk of abnormal maternal phenotypes in the postpartum period only, or confer risk of abnormal maternal behaviour though mechanisms operating both within, and outside, the postpartum period.", "Second, for the gene expression analyses we used a hemibrain dissection given that no individual brain region has robustly been shown to function abnormally in PP cases, and to capture large gene expression changes within one brain region or smaller gene changes throughout multiple brain regions; however, this approach may be insensitive to small gene expression changes in specific brain regions.", "Our results indicate that acute postpartum steroid sulfatase deficiency results in abnormal maternal behaviour (at least in mice), and begin to define a brain pathway that may feasibly be disturbed in women diagnosed with PP i.e. signalling from the extracellular matrix to the cytoplasm via Ccn proteins, transmembrane proteins such as integrins and Notch, and intracellular molecules.", "Further details of the molecular pathway(s) by which steroid sulfatase inhibition influences postpartum behaviour may be revealed by hypothesis-free gene expression analyses in our mouse model.", "Our data offer support for STS (Xp22.3), NOV/CCN3, WISP1/CCN4 and ADCY8 (8q24) and ARHGDIG (16p13) as functional and positional candidate genes for PP risk, and indicate that analyses comparing genetic variation within these genes, or their physiological correlates (e.g. serum DHEA(S) levels), in samples of healthy nulliparous and parous controls and PP cases is warranted.", "The work was funded by the Wellcome Trust (105216/Z/14/Z) the Medical Research Council UK (MR/L010305/1), an Erasmus grant from University of Barcelona, and Cardiff University.", "The authors have no competing financial interests in relation to the work described.", "The authors declare no conflict of interest.", "The funding bodies did not play any role in the design of the study, in data collection or analysis, or in the decision to publish." ]	[ "Postpartum psychosis (PP) is a severe psychiatric disorder of unknown cause.", "Steroid sulfatase (STS) deficiency may influence PP risk.", "Postpartum inhibition of STS in mice results in behavioural and genetic abnormalities.", "These abnormalities can be alleviated with an antipsychotic drug.", "The study suggests a new mouse model and a biological risk pathway for PP." ]
Enteroaggregative Shiga toxin-producing Escherichia coli of serotype O104:H4 in Belgium and Luxembourg	In 2011, a large outbreak of infections caused by Shiga toxin-producing Escherichia coli (STEC) O104:H4 occurred in Germany. This exceptionally virulent strain combined virulence factors of enteroaggregative E. coli (EAggEC) and STEC. After the outbreak only a few sporadic cases of infection with this rare serotype were reported, most of which were related to travel to the Middle East or North Africa. Here we describe two cases of enteroaggregative STEC (Agg-STEC) O104:H4 infection that occurred in Belgium in 2012 and 2013 respectively. In both cases travel in a Mediterranean country preceded the infection. The first strain was isolated from the stool of a 42-year-old woman presenting bloody diarrhoea, who had travelled to Tunisia the week before. The second case involves a 14-year-old girl who, upon her return from Turkey to Belgium, suffered from an episode of bloody diarrhoea and haemolytic uraemic syndrome. Extended typing of the isolates with pulsed field gel electrophoresis revealed that the strains were closely related, though not exactly the same as the 2011 outbreak strain. This report supports the previously made hypothesis that Agg-STEC has a human reservoir and might be imported by travellers coming from an area where the pathogen is endemic. Furthermore, it emphasizes the concern that these bacteria may cause future outbreaks as evenly virulent O104:H4 isolates seem to be widespread.	[ "IMMU", "MEDI" ]	[ "Enteroaggregative Escherichia coli", "Enterohaemorrhagic E. coli", "Gastrointestinal disease", "Haemolytic uraemic syndrome", "Shiga toxin-producing E. coli", "Travel" ]	[ "2404", "2725" ]	[ "O157:H7 is the most common serotype in Shiga toxin- or Vero(cyto)toxin-producing Escherichia coli (STEC/VTEC).", "A key property of these strains is the production of “Attaching and Effacing” (AE) lesions.", "STEC of different serotypes can cause severe disease in humans, including haemorrhagic colitis and haemolytic uraemic syndrome (HUS).", "For this reason, a widely used acronym for these E. coli strains is ‘EHEC’ (enterohaemorrhagic E. coli).", "As there is no agreement on the definition of enterohaemorrhagic strains, the acronym ‘AE-STEC’ will be used throughout this paper to refer to Shiga toxin- and AE-lesion-producing E. coli strains.", "AE-STEC O157:H7 have the highest incidence and are the most frequently involved in outbreaks (seropathotype A).", "AE-STEC of serotypes O26:H11, O103:H2, O111:H-, O121: H19 and O145:H- are also involved in severe human disease and outbreaks, though less frequently (seropathotype B) [1].", "In 2010, 40.7% of all reported STEC serogroups in Europe were O157.", "O26 was the most commonly reported non-O157 serogroup (11%) [2].", "From May to July 2011, a large outbreak of STEC infections with a serotype that was reported only very rarely before, O104:H4, occurred in Germany.", "This outbreak differed from previous STEC outbreaks by its sheer size, the large percentage of patients with HUS and the high proportion of adults, mainly women.", "This exceptionally virulent strain combined virulence factors of enteroaggregative E. coli (EAggEC) and STEC making it a mixed enteroaggregative and Shiga toxin-producing E. coli (designated here as ‘Agg-STEC’) [1,3].", "At the same time, a smaller outbreak of Agg-STEC O104:H4 took place in France.", "Comparison of the back tracing information from the French and German outbreaks led to the conclusion that fenugreek seeds imported from Egypt were the most likely source of infection although the outbreak strain could not be isolated from the seeds [4].", "After the 2011 outbreak, only a few sporadic cases of Agg-STEC of serotype O104:H4 were reported.", "In this paper, we describe two imported cases of Agg-STEC O104:H4 infection that occurred in Belgium after the 2011 outbreak and compare their phenotypic and genetic profiles with the German outbreak strain recovered from a Luxembourg resident who had travelled to the Hamburg area.", "The first case is a 42-year-old woman who presented on the first day of September 2012 to the emergency department of the Universitair Ziekenhuis Brussel (Belgium) with complaints of heavy abdominal cramps, nausea and watery diarrhoea.", "She mentioned the presence of blood in the faeces.", "She had returned 3 days before from a 1-week journey in Tunisia where diarrhoea started in the morning, just before leaving.", "She was not aware of other cases.", "A blood analysis was normal, without signs of HUS (creatinine 0.62 mg/dL, haemoglobin 13.4 g/dL, platelets 393 000/mm3).", "At physical examination only discrete signs of dehydration were present.", "She was discharged with dietetic advice and a prescription of EnterolTM (Saccharomyces boulardii) and domperidone.", "She did not consult anymore, as she recovered uneventfully.", "The second case involves a 14-year-old girl who was referred from the regional hospital AZ Vesalius to the Universitair Ziekenhuis Leuven (Belgium) in the third week of July 2013 because of abdominal pain, bloody stools, fever and general malaise for 4 days.", "Her blood examination revealed signs of HUS (anaemia with low haemoglobin 11.3 g/dL, increased levels of lactate dehydrogenase and bilirubin total 10.21 mg/dL and thrombocytopenia with platelet count 27 000/mm3), with presence of schistocytes in peripheral blood smear and decreased kidney function (oliguria, creatinine 1.07 mg/L, urea 68 mg/dL).", "She was treated supportively with spontaneous recovery of her kidney function.", "No dialysis was needed.", "The patient had travelled to Turkey during the first 2 weeks of July where she developed watery diarrhoea that spontaneously resolved after 5 days.", "Upon her return to Belgium she suffered from a new episode of diarrhoea, this time with the presence of blood in stools, and she developed HUS 6 days later.", "The strains isolated from these patients were compared with an isolate related to the German outbreak.", "A 51-year-old woman residing in Luxembourg travelled for several days during the second week of May 2011 in the Hamburg region in Germany.", "Hamburg was the German state with the highest incidence (10 cases per 100 000 population) of reported HUS during the outbreak [3].", "A few days after her return, she developed diarrhoea, which gradually worsened until she noticed blood in her faeces and consulted a physician who prescribed a stool culture.", "The patient recovered without developing HUS.", "In both Belgian cases a STEC O104:H4, referred to as EH2211 and EH2303, respectively, was recovered out of the stools by using methods described elsewhere [5].", "Slide agglutination was performed with the K9 antiserum (Statens Serum Institut, Copenhagen, Denmark), indicative of the O104 serogroup [6].", "The full serotype was confirmed by a positive PCR result for O104 (wzxO104) and H4 (fliCH4) [7].", "Both strains grew on sorbitol–MacConkey agar with cefixime and tellurite (SMAC-CT), fermented sorbitol and were ß-glucuronidase positive.", "By contrast with EH2211, EH2303 did not ferment lactose.", "A PCR was positive for aaiC and aggR genes [8], but negative for the STEC virulence genes intimin (eaeA) and enterohaemolysin (hlyA) [9].", "Both strains possessed the Shiga toxin 2a (stx2a) subtype, as described by Scheutz et al. [10].", "Disc diffusion susceptibility testing according to EUCAST recommendations (or CLSI when no breakpoints were available from the former) was performed using antibiotics selected by the Programme for Food- and Waterborne Diseases and Zoonoses (FWD) and revealed that strain EH2211 had an intermediate susceptibility to nalidixic acid and was resistant to ampicillin and trimethoprim.", "EH2303 on the other hand was resistant to ampicillin, amoxicillin/clavulanic acid, cefazolin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefepime, aztreonam, sulfonamides, trimethoprim and nalidixic acid.", "No extended-spectrum ß-lactamase (ESBL) was detected for EH2211 according to CLSI screening and confirmatory tests; but EH2303 tested ESBL positive, further characterized by the National Reference Centre for multiresistant Enterobacteriaceae as CTX-M-15; a TEM-1 ß-lactamase was also present.", "Pulsed-field gel electrophoresis using XbaI was performed using the PulseNet standard operating procedure [11] to compare these two Agg-STEC O104:H4 strains with C110638, an Agg-STEC O104:H4 from Luxembourg linked to the 2011 German outbreak, and EH1651, an unrelated STEC O104:H2 strain included in the study of Miko et al. [12] (Table 1).", "Strain EH2303 clustered closest to the 2011 outbreak strain with 91.9% similarity.", "Using a cut-off value of 80% similarity the three O104:H4 strains could be divided in the same cluster A, whereas strain EH1651 showed only 69.7% similarity and formed a separate group B (Fig. 1).", "Although O104:H4 is a rare serotype in humans, a few sporadic infections caused by E. coli O104:H4 took place before 2011.", "STEC O104:H4 caused a case of HUS in South Korea (2004), but this strain was not enteroaggregative [13].", "EAggEC O104:H4 has been associated with human diarrhoea in South Africa (2007, 2009 and 2010) and Canada (2010) [14,15].", "Agg-STEC O104:H4 have been isolated from patients with HUS in Germany (2001), France (2009) and Italy (2009); from two patients with bloody diarrhoea in the Republic of Georgia (2009); from a patient with haemorrhagic colitis in France (2004); and from patients in Norway (2006) and Sweden (2010) [16–20].", "After the German and French outbreaks, cases of Agg-STEC occurred in France, Germany and Denmark [21,22].", "HUS caused by Agg-STEC other than O104:H4 has been reported in France (1992), Japan (1999) and Ireland (2012).", "The strains belonged to serotypes O111:H2, O86:HNM and O111:H21, respectively [23–25] (Table 2).", "Common characteristics of EH2211, EH2303 and the German outbreak strain include: ability to grow on SMAC-CT agar; positive PCR results for aaiC, aggR, stx2a, wzxO104 and fliCH4; negative PCR results for eaeA, hlyA and stx1; agglutination with K9 antiserum; fermentation of sorbitol and production of ß-glucuronidase.", "The outbreak strain and strain EH2303 were both multiresistant and produced an ESBL CTX-M-15 while EH2211 was susceptible to most tested antibiotics [6,22,26].", "A remarkable characteristic of strain EH2303 is the fact that it did not ferment lactose.", "To the best of our knowledge, this is the first lactose-negative Agg-STEC O104:H4 strain ever reported.", "Strain EH2303 shows more similarity to the strain from Luxembourg than EH2211 based on their pulsed field gel electrophoresis profiles.", "Closely related, but not identical, pulsed field gel electrophoresis XbaI patterns of E. coli O104: H4 strains isolated before and after the German 2011 outbreak have already been described [12,21].", "In the cases described in this paper, travel to Turkey and Tunisia preceded the infection with Agg-STEC O104:H4, suggesting that the infection was acquired in these countries.", "Indeed, other sporadic cases of infection with E. coli O104:H4, as well as an outbreak associated with travel to Turkey or North Africa, have been reported in Europe.", "According to the information reported to European Centre for Disease Prevention and Control, four of the STEC O104 cases in the EU Member States in the period 2004–2010 were travel-related; the countries of origin of infection were Afghanistan, Egypt, Tunisia and Turkey.", "One of those was of the serotype O104: H4; a Finnish case with infection acquired in Egypt [26].", "Infections of Agg-STEC O104 were noted in Italy (2009) and Sweden (2010).", "As in the present case, they were linked to travel to Tunisia [19,20].", "In September 2011, an outbreak of HUS due to Agg-STEC O104:H4 occurred among French tourists returning from Turkey.", "Around that same period, two patients from Germany and one from Denmark were diagnosed with an Agg-STEC O104:H4 infection, also after travel to Turkey [21].", "Out of four sporadic isolates of E. coli O104:H4 from children with HUS in France, one was associated with recent travel to North Africa [22].", "Whereas AE-STEC is clearly a zoonosis, mainly related to ruminants, no animal reservoir has been reported for EAggEC nor Agg-STEC.", "Studies conducted in cattle in Germany and France during the 2011 outbreak were not able to detect the presence of the outbreak strain, indicating that cattle are not the reservoir of Agg-STEC [27,28].", "As no other natural reservoir has been established, it has been hypothesized Agg-STEC has a human reservoir and might be imported into the European Union by travellers or migrants coming from an area where the pathogen is endemic [29].", "Without an animal reservoir, it is not expected that Agg-STEC strains can be maintained in countries with high standards of hygiene.", "Comparative genomics of E. coli O104:H4 strains isolated before, during and after the 2011 outbreak provided us with better insight into the evolution of this serotype.", "Genomic analysis of a number of post-outbreak O104:H4 strains has shown that they are not derived from the 2011 outbreak but share a recent common ancestor [22].", "A nearly identical Shiga toxin-carrying prophage was found in several of the historical O104:H4 strains, which suggests that the prophage was transferred into the O104:H4 population at least a decade ago.", "On the other hand, acquisition of the antibiotic resistance plasmid pESBL and gene cassettes for antibiotic resistances seems to be one of the most recent events in the evolutionary history [20,22].", "The two cases reported here had differing antibiotic resistance profiles.", "Strain EH2211 was susceptible to most antibiotics, whereas EH2303 was multiresistant, as was the 2011 outbreak strain, and carried a CTX-M-15 type ESBL.", "In conclusion, the cases described here support the previously expressed concern that these bacteria may cause future severe outbreaks as evenly virulent, though not exactly the same, O104:H4 isolates seem to be widespread.", "Part of this work was performed in the frame of the Belgian National Reference Centres for Shiga toxin- or Vero(cyto) toxin-producing Escherichia coli and extended-spectrum ß-lactamase- and carbapenemase-producing Enterobacteriaceae supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System.", "We gratefully acknowledge Nadine Botteldoorn (Scientific Service Foodborne Pathogens, NRL VTEC in Food – NRL Foodborne Outbreaks, Scientific Institute of Public Health, Brussels, Belgium) and Catherine Ragimbeau (Surveillance & Epidemiology of Infectious Diseases, National Health Laboratory, Dudelange, Luxembourg) for providing the 2011 outbreak strain isolated in Luxembourg." ]	[]
Public perceptions of established and emerging mining technologies in Australia	Technology plays a central role in mining activities throughout Australia and is critical to achieving greater economic and environmental sustainability. Our choices about which technologies to develop, adopt and deploy in the landscape reflect one of the most critical interfaces between mining and society. There have been numerous reviews and studies of the social licence of the mining industry, which have examined the way in which public perceptions influence the broad acceptance and approval of mining activities. However, very few studies have examined public perceptions of the technologies and extractive methods used by the mining industry. This paper therefore contributes to expand the scope of this mining-society scholarship by understanding the drivers that shape public perceptions in relation to established and emerging mining technologies. We present findings from a survey of Australian citizens (N = 476) that tested their general awareness of and response to different types of mining technologies and extraction methods that are currently in use. These included comparisons between three broad methods of resource extraction technologies including open cut, underground and in situ leach mining. Hydraulic fracturing, a technology that is used in conjunction with some forms of resource extraction, was also included. In this paper, we examine the relationships between the public's self-rated knowledge of these four mining technologies, their perceptions of the environmental and safety impacts of those technologies, and their level of acceptance of each mining technology using descriptive statistics and path analysis. Our research found that higher levels of overall acceptance were expressed for established technologies such as open cut and underground mining. However, our results also reveal a nuanced role for the type of knowledge that citizens may have about novel and emerging technologies in determining their acceptance of these technologies.	[ "ECON", "ENVI", "SOCI" ]	[ "Acceptance", "Hydraulic fracturing", "In situ leach", "Open cut", "Social licence", "Underground" ]	[ "2002", "2308", "3308", "3312" ]	[ "In Australia, mining has long been and continues to make a substantial contribution to the national economy and the standard of living (Hajkowicz et al., 2011; Moffat et al., 2017).", "In 2015–16, more than half of Australia's goods and services exports were generated by the mining and energy sectors with a total value of AUD $157 billion (DIIS, 2016a).", "The economic contribution of mining also positively influences growth in a number of other production and services sectors such as retail and wholesale trade, construction, agriculture, and information and telecommunications (Bashar, 2015).", "However in recent years, the global mining industry has also experienced the challenges of falling commodity prices and the rising costs associated with mining lower grade ores and accessing resources that are buried deeper below the subsurface (Mudd, 2010).", "In response to these challenges, it has been recognised that the Australian mining sector needs to develop innovative solutions to increase productivity, lower the costs of production, identify new ore bodies, reduce the impacts from mining, and enhance its social licence to operate with the public (DIIS, 2016b).", "New mining technologies that can both demonstrate improved economic, technical and resource efficiencies along with an increased capacity in responsible environmental stewardship and the creation of social value are a central focus of mining innovation (IGF, 2018).", "Increasingly, the future of mining is characterised by the role of new technologies and innovations such as the automation of high integrity mining equipment, the use of sensor networks, and increasingly sophisticated systems of data-driven analysis and decision-making (Cosbey et al., 2016; WEF, 2017).", "In examining the role of technologies, the Australian Government has also committed to a ten-year plan designed to enhance the competitiveness of the mining sector by: (1) applying new technologies for mining; (2) expediting the pace of innovation; and (3) communicating the national contribution of mining to the public as a way of enhancing its social licence (DIIS, 2016b).", "However, the development, adoption and deployment of technologies in the mining industry increasingly take place in a contested and complex social, environmental and political context (Bice et al., 2017).", "Numerous studies over recent years have examined the social licence of the mining sector, and it is now well established that public perceptions of mining can directly influence its trajectory (Mason et al., 2014; Moffat et al., 2018; van der Plank et al., 2016).", "Much of this work has been characterised by efforts to understand and document the social licence of mining activities at local and national scales (Frantál, 2016; Lacey et al., 2017; Matlaba et al., 2017; Moffat and Zhang, 2014; Wright and Bice, 2017).", "However, most of these studies have focused on the broad acceptance or approval afforded to mining activities generally.", "There has been comparatively little attention paid to how citizens perceive and respond to the technologies and extractive methods used by the mining industry.", "While we may be most interested in understanding the drivers of social acceptance for future mining technologies, it is also instructive to examine how public perceptions are currently being shaped by existing mining technologies.", "There is a gap in the current research that assesses public perceptions of a range of existing mining technologies and what shapes these perceptions.", "Undoubtedly, scientific and academic debates are key to informing policy decisions, but questions pertaining to public perceptions are fundamental to the design of socially responsible technologies, and the policies and regulations associated with them (Costa et al., 2017; Evensen and Brown-Steiner, 2017; Lacey et al., 2019; OECD, 2010).", "This paper presents empirical findings from a national survey of Australian citizens (N = 476) that was designed to test their general awareness of and responses to four different types of mining technologies and extraction methods, collectively called mining technologies for the purpose of this paper, and that are currently in use in Australia.", "Broadly, all mining activities are aimed at extracting raw resources from the earth for processing and use in society.", "However, there are multiple ways to extract resources and this tends to be determined by the nature of the resource itself, its geographical location, how accessible it is, and the technologies available for its extraction.", "In Australia, while there is evidence that Indigenous Australians have long accessed mineral resources, industrial-scale mining commenced soon after European arrival in the 1800s, was characterised by the Gold Rush of the 1850s, and grew steadily until the resources boom of the 1960s (Geoscience Australia, 2015; McKay et al., 2001).", "Industrial mining is a large-scale operation that can involve the movement of tens of thousands to hundreds of thousands of tonnes of material at a single operation per day.", "There are three broad and established methods of industrial mining, which include surface mining, underground mining and in situ mining (Rankin, 2011).", "Alongside these mining methods, technologies such as hydraulic fracturing are increasingly being used to prepare the subsurface for resource extraction, most commonly in relation to the extraction of unconventional gas but not exclusively for this purpose.", "The four mining technologies in this research can further be defined as established or emerging in the Australian context.", "Established mining technologies are identified based on the current and well established history of their use over a long time period.", "Emerging technologies in this study are those that have been used over a comparatively shorter time period in Australia and there are existing commitments or research to explore new or extended applications of these technologies1 (Finkel et al., 2017; Kuhar et al., 2018).", "It should be noted, the emerging technologies in this study are proven technologies that have been used in Australia over several decades.", "Each of four technologies and how they are used in our research is described in more detail below.", "In Australia, open cut mining and underground mining have been and continue to be used extensively to mine a wide range of commodities.", "They are well established mining technologies in the Australian mining industry.", "Surface mining is the most well established form of mining with over 80% of all ore and rock extracted from surface mines around the world (Rankin, 2011).", "Surface mining variously includes strip mining, placer mining, mountaintop removal and open pit mining.", "For the purpose of our research, surface mining was characterised as open cut mining and described as a form of surface mining technique used to extract rock or minerals from an open pit.", "Open cut mining tends to be used when deposits of commercially useful resources are found near the surface or the geography is unsuitable for tunnelling.", "This type of mining requires removal of the surface vegetation, dirt, and if necessary, layers of bedrock in order to reach buried ore deposits.", "Open cut mining is widely used to recover resources including iron ore, coal, copper and gold in Australia (Britt et al., 2017).", "Underground mining may be characterised by unsupported, supported and caving methods (Rankin, 2011).", "Their depth can also vary greatly depending on the location of the ore beneath the surface; the world's deepest underground mine is currently Mponeng gold mine in South Africa, which extends 4 km below the surface (Boffard, 2016).", "In our research, underground mining was characterised as a form of sub-surface mining which consists of digging tunnels or shafts into the earth to reach buried resources.", "Resources for processing and waste rock for disposal are brought to the surface through tunnels and shafts.", "Underground mining is used to recover resources including coal, copper, uranium, gold and diamonds in Australia (Austrade, 2016; Britt et al., 2017).", "Alongside established mining technologies, we also included two emerging mining technologies in our study.", "In situ leach mining and hydraulic fracturing are characterised in this study as emerging technologies as they are being examined for new or extended application in Australia.", "While both are proven technologies and have been used in Australia and globally over several decades, research is currently underway to explore their application to a greater range of resource extraction.", "In situ leach (or solution) mining is a technique that involves leaching minerals from an ore body without removing the ore body from the deposit (Geoscience Australia, 2010).", "It is frequently explored as an alternative to mechanical mining and tends to have lower capital and operating costs.", "The nature of in situ leach mining is however only suitable to specific kinds of commodities and rock formations as it is important to protect groundwater from contamination (Mudd, 1998, 2001).", "In this research, in situ leach mining was characterised as a technique used to extract resources through boreholes.", "Solution is injected into a hole that is drilled into a resource deposit.", "The liquid dissolves the mineral it comes into contact with underground and is pumped back up to the surface where dissolved elements are recovered from the liquid.", "There is little disturbance and no waste rock as major excavation is not required.", "In situ leach mining is currently used to recover uranium in Australia and there is potential to apply this technique to gold and copper mining (Kuhar et al., 2018; Martens et al., 2012).", "Finally, hydraulic fracturing was also included in our research.", "Of the four technologies under consideration, hydraulic fracturing is not a form of resource extraction itself.", "It is however a technology that is increasingly used to prepare subsurface structures in order to enable resource extraction.", "For this reason, it is relevant to explore perceptions and attitudes toward this technology in comparison to others.", "There are also numerous, recent studies on public perceptions of hydraulic fracturing (e.g. Boudet et al., 2014; Clarke et al., 2016; Costa et al., 2017; Macnaghten, 2017).", "Hydraulic fracturing is most commonly used to extract unconventional gas resources such as tight and shale gas, and in some cases, coal seam gas.", "In our research, hydraulic fracturing is characterised as a technique that is used to improve the flow of gas resources from between rock formations that are difficult to access due to depth and composition.", "Highly pressurised liquid is used to fracture or break rock so that gas resources are released.", "As noted, the decision to include hydraulic fracturing in this research was also due to the fact that this technology was attracting increasing public scrutiny in relation to the development of unconventional gas resources both in Australia and around the world (Boudet et al., 2014; Northern Territory Government, 2018; Western Australia Government, 2015; Williams et al., 2015).", "The scope of this research seeks to assess the general public's perceptions of established and emerging mining technologies that are currently in use in Australia.", "This research does not seek to examine these perceptions at the localised, site-specific scale or in relation to specific mining operations.", "In our research, a quantitative survey was designed to broadly assess the perceptions of citizens in relation to different types of mining technologies in the Australian context.", "The survey design incorporated three variables which were central to our analysis of public perceptions.", "These variables are: (1) the public's self-rated knowledge of each mining technology; (2) perceived impacts of each technology; and (3) the level of acceptance of the use of each technology in Australia.", "As described above, data related to these three variables were collected with respect to the four following mining technologies: open cut, underground, in situ leach, and hydraulic fracturing.", "For the reasons described in the section below, we identify perceived impacts as an intermediate variable that mediates the relationship between self-rated knowledge and acceptance (Fig. 1).", "Additionally, we hypothesise that there is a non-linear relationship between self-rated knowledge and perceived impacts (shown as a curved arrow), and a linear relationship between perceived impacts and acceptance of technologies (shown as a straight line).", "To test the hypothesised model depicted in Fig. 1, a path analysis was run using the structural equation modelling package in Stata.", "Prior knowledge is considered a determinant of people's behaviour and can be classified into objective and subjective knowledge (Kerstetter and Cho, 2004).", "Objective knowledge denotes the knowledge a person has about something, while subjective knowledge is the knowledge that a person perceives themselves to have (van Rijnsoever and Farla, 2014).", "The latter is influenced by the former and the degree of confidence the person has about the subject (Park and Lessig, 1981).", "Both types of knowledge can play an important role in shaping preferences over technologies (Payne et al., 1999).", "To examine the role people's knowledge about these four mining technologies plays in the acceptance or rejection of mining technologies, we collected information about people's subjective knowledge by asking them to self-rate their knowledge of each technology on a scale from 1 = no knowledge to 7 = extensive knowledge.", "This occurred prior to receiving an explanation of each technology and is henceforth referred as self-rated knowledge.", "Although the relationship between self-rated knowledge and acceptance of a technology may seem linear, a recent study has exemplified a non-linear relationship between the two variables (Walton and McCrea, 2017).", "It is hypothesised that such a relationship may be mediated through people's perception of impacts.", "There may be a curvilinear relationship between self-rated knowledge and perceived impacts such that people may be less concerned about impacts if they have little knowledge of them; more concerned if they have some knowledge of the impacts (i.e. enough knowledge to become concerned); and a tendency to be less concerned about impacts again when they feel that they have good knowledge about the technology/industry in question (i.e. when their concerns have been addressed to some extent).", "In this research, it is likely that such a curvilinear relationship between self-rated knowledge and perceived impacts may be stronger for emerging mining technologies because people know less about these technologies compared with their knowledge of established mining technologies.", "Perceived impacts of mining technologies is hypothesised as an intermediate variable between self-rated knowledge and acceptance.", "Various studies have demonstrated that citizen perceptions of the negative impacts of mining on the environment, the economy, livelihoods and well-being have a direct influence on the degree to which mining is accepted (Moffat et al., 2017, 2018).", "Similar findings have emerged from studies examining citizen perceptions and acceptance of specific technologies in industry other than mining.", "For example, Huijts et al. (2012) and Volken et al. (2017) have found that people's acceptance of energy technologies is directly affected by how they perceive the impacts of those technologies.", "Therefore, the relationship between perceived impacts and acceptance of technologies is hypothesised to be linear.", "To measure perceived negative impacts of different mining technologies, three broad impact categories were included in the survey: (1) water, incorporating groundwater contamination and other impacts on groundwater such as drawdown, and surface water pollution; (2) air quality, incorporating noise pollution, and surface disturbance; and (3) the health and safety of mine site employees and communities living near mine sites.", "Perceived impacts were also measured on a 7-point scale where respondents were asked how much they agreed that each mining technology had negative impacts on various items in each impact category, from 1 = strongly disagree to 7 = strongly agree (please see full description of scale used in Section 3.2 below).", "The need to manage and mitigate the potential impacts of mining on water, air quality, surface disturbance and health and safety is well established in the literature.", "For example, a number of studies have characterised the impacts of mining on water (e.g. Northey et al., 2018; Northey et al., 2016), on air quality and surface disturbance (e.g. Fugiel et al., 2017; Gastauer et al., 2018), and on the health and safety of those working in and living near mining operations (e.g. Shandro et al., 2011; Smith et al., 2016).", "While we acknowledge that positive social and economic impacts substantially influence social acceptance of or opposition to mining (Hajkowicz et al., 2011; Haslam McKenzie and Hoath, 2014; Kitula, 2006), this study has focused specifically on potential negative impacts to environment and human safety.", "Finally, the level of acceptance of each technology was measured with a single item asking respondents how much they agreed or disagreed with each type of mining technology continuing to be used in Australia.", "Acceptance of each technology was also measured on a 7-point scale from 1 = strongly disagree to 7 = strongly agree.", "Many factors can affect the social acceptance of different forms of mining in Australia.", "For example, perceived benefits, distributional and procedural fairness, governance, and trust in the industry have been found to predict social acceptance of mining activities (Moffat et al., 2017; Moffat and Zhang, 2014; Walton and McCrea, 2017).", "However, this paper focuses on the role of self-rated knowledge on acceptance of different mining technologies and whether acceptance is mediated by perceived impacts.", "In this paper, we hypothesise that: (H1) perceived impacts mediate the relationship between self-rated knowledge and acceptance of each technology; (H2) there is a curvilinear relationship between self-rated knowledge and perceived impacts; and (H3) there is a linear relationship between perceived impacts and acceptance of each technology.", "The survey was developed and commissioned in 2014.", "As described above, it was designed to test the relationship between self-rated knowledge, perceived impacts and level of acceptance of four mining technologies.", "After asking respondents to rate their level of general knowledge about each type of mining or resource extraction, a brief description and a photograph were provided to respondents for each mining technology.", "As per Volken et al. (2017), this is an established approach in survey design that helps to provide a common base for an understanding of the technologies under consideration and answering questions about them.", "All descriptions and images were approved by technical mining experts with detailed knowledge of the technologies being assessed to ensure an objective and factually correct description was provided.", "With the exception of the respondents’ demographic information, all responses were provided on a 7-point Likert scale (1 = strongly disagree, 7 = strongly agree where 4 = neither agree/disagree) unless otherwise stated.", "This is an established approach in the social sciences for assessing perceptions and has been used in several similar studies (e.g. Costa et al., 2017; Fleishman et al., 2010; He et al., 2018; Leonard et al., 2016; Zhang et al., 2013).", "Respondents to the survey were recruited by a market research provider.", "This company was engaged to ensure that respondents to the survey were as close as possible to a nationally representative sample of the Australian population at the time of collection stratified by age, gender and location (i.e. citizens living in regional and metropolitan areas).", "Given we were targeting a total of 500 respondents within a national population of 23.5 million Australians (i.e. the total national population at the time of data collection; see World Bank, 2019), Australian census data was used to establish target quotas for each state and territory Respondents received an email invitation to participate in the survey from the market research provider, which included a link to the online survey.", "All respondents were informed that their responses were anonymous and confidential, and that the research study had been approved by the lead author's institutional human research ethics committee.", "Respondents were also provided with contact details of an ethics officer and the lead researcher if they wished to follow up with questions or concerns.", "A total of 500 responses were received to the survey over the course of four weeks but data cleaning reduced the total useable responses to a total of 476.", "For the purposes of our analysis, there were 239 male (50.2%) and 237 female respondents (49.8%) to the survey.", "The age of the respondents was 18–24 years (11.6%), 25–34 years (15.5%), 35–44 years (18.1%), 45–54 years (18.5%), 55–64 years (16.2%), and 65 years and over (20.2%).", "Regarding the education level of respondents, 15.3% had an education of Year 11 or lower, 18.9% had completed Year 12, 23.5% had post-secondary education, 24.8% had an undergraduate degree, 16% had a postgraduate degree, and 1.5% chose not to disclose their level of education.", "The results of the study are divided into two types of analysis, descriptive and path analyses.", "For descriptive analysis, frequency and means were computed, which are described in further detail below.", "For path analysis, structural equation modelling (SEM) was run using Stata 15.", "In the presentation of the results, the following abbreviations are used: open cut (OC); underground (UG); in situ leach (ISL); and hydraulic fracturing (HF).", "The level of knowledge of each mining technology as self-rated by respondents is displayed in Table 1.", "The results clearly show that respondents considered that they have more knowledge of established mining technologies (OC and UG) than emerging technologies (ISL and HF).", "Only 14.9% and 6.1% of respondents indicated that they have no knowledge of open cut and underground technologies respectively, with the majority of respondents indicating that they have at least some form of knowledge of open cut (85.1%) and underground mining technologies (93.9%).", "By contrast, 57.8% and 33.2% respondents indicated that they have no knowledge of in situ leach and hydraulic fracturing technologies respectively, with the remaining respondents indicating that they have at least some level of knowledge of in situ leach (42.2%) and hydraulic fracturing technologies (66.8%).", "Respondents were asked to rate to what extent they agreed that each mining technology contributed significantly to a range of potentially negative impacts on the environment and human safety.", "Table 2 shows the percentage of respondents who thought that each technology contributed significantly to those negative impacts.", "The table shows the percentage of respondents who scored above 4 on the 7-point scale, indicating higher levels of perceived negative impacts.", "The bolded numbers in Table 2 shows the highest percentage for each perceived impacts category for each technology.", "The results revealed that the majority of respondents perceived that open cut technology has the most negative impacts on air quality and surface disturbance, underground technology has the most negative impacts on health and safety, and in situ leach and hydraulic fracturing technologies are both perceived to have the most negative impacts on water.", "The mean scores of perceived impacts of all three impact categories combined were then calculated for each technology (Table 2).", "The majority of respondents (51.2%) agreed that open cut technology had high negative impacts while far fewer respondents perceived negative impacts for the other three technologies.", "On average, similar percentages of respondents rated impacts negatively for underground mining, in situ mining, and hydraulic fracturing (36.7%, 31.0%, and 42.6% respectively).", "Table 3 shows the percentage of respondents willing to accept each mining technology.", "That is, the number of respondents who rated above 4 on the 7-point scale.", "The acceptance of established technologies (OC and UG) is higher than the acceptance of emerging technologies (ISL and HF), even though there was more concern over the impacts of open cut technologies.", "More specifically, 69.1% and 67% respondents accepted underground and open cut technologies respectively.", "Alongside this, 51% and 38% respondents accepted in situ leach and hydraulic fracturing technologies respectively.", "We also examined non-linear relationships between self-rated knowledge and perceived impacts (Fig. 2).", "We found a curvilinear relationship between these two variables such that perceived impacts tended to increase as respondents perceived they have some knowledge, though declined again as they felt they have a lot of knowledge.", "Note that the confidence intervals (the shaded part in Fig. 2) expanded as people's self-rated knowledge increased, particularly for emerging technologies.", "This indicates more divergent views in perceived impacts among those with higher self-rated knowledge of in situ leach and hydraulic fracturing technologies.", "On the other hand, we found linear relationships between perceived impacts and acceptance of technologies (Fig. 3), which showed an inverse relationship between these variables, i.e. an increase in the perception of impacts decreases the acceptance of the mining technologies.", "Table 4 shows the mean, standard deviation and linear correlation values (N = 476).", "The means for self-rated knowledge of emerging technologies were lower than established technologies, as were acceptance levels.", "This is despite the perceived impacts of emerging technologies being about the same or lower than established technologies.", "This suggests a reticence to accept emerging technologies.", "It is noteworthy that there was more variation in self-rated knowledge and acceptance of mining technologies (SDs between 1.43 and 1.89) than between perceived impacts of established and emerging mining technologies (SDs between 1.12 and 1.28).", "There is a negative and weak linear correlation between self-rated knowledge and perceived impacts for underground technologies (UG), though it was not significant for the other established technology of open cut mining (OG).", "On the other hand, there is a positive, weak and significant linear association between self-rated knowledge and perceived impacts for both emerging technologies (ISL and HF).", "This suggests that perceived impacts tend to decrease or stay about the same, on average, with increasing self-rated knowledge of established technologies, while they tend to increase for emerging technologies.", "However, this is only a tendency and may be qualified by the curvilinear relationships described above, which are tested later with path analysis.", "All correlations between self-rated knowledge and acceptance of technologies were positive except for hydraulic fracturing which was negative (r = −0.18).", "The more respondents felt they knew about hydraulic fracturing, the less accepting they were of this technology, whereas high self-rated knowledge of other technologies was associated with more acceptance.", "This suggests that higher self-rated knowledge of mining technologies does not necessarily lead to higher acceptance.", "This highlights the complexity of the relationship between self-rated knowledge and acceptance.", "Additionally, all correlations between self-rated knowledge and acceptance were significant except for in situ leach technology (r = .09).", "However, all relationships between self-rated knowledge and acceptance of technologies were also hypothesised to be meditated by perceived impacts, which are also tested later with path analysis.", "The correlations between perceived impacts and acceptance of technologies were all found to be negative and significant.", "Higher perceived impacts of different mining technologies were associated with lower acceptance of them.", "The strength of the correlation between these two variables ranged from moderate to strong.", "The correlation values of emerging technologies were stronger than those of established technologies and highest for hydraulic fracturing (r = −0.62).", "Thus acceptance of emerging technologies seems to be more sensitive to their perceived impacts than acceptance of established technologies.", "Path analysis was used to test the hypothesised mediated model (Fig. 1) from self-rated knowledge to perceived impacts, and then from perceived impacts to acceptance for each mining technology.", "In other words, we hypothesised that the effects of self-rated knowledge on acceptance is mediated by perceived impacts.", "Model fit statistics for our hypothesis were then examined to determine the fit of this model with the data.", "Then additional paths were explored which may improve the model's fit with the data, resulting in a revised model.", "To test the significance of the hypothesised curvilinear relationships between self-rated knowledge and perceived impacts, a squared term for self-rated knowledge was added to the model, while self-rated knowledge itself was used to test for significant linear relationships (Table 5).", "Both self-rated knowledge and its squared term were centred on the mean to reduce multicollinearity with each other.", "Table 5 shows the beta coefficients for predictors of the dependent variables (DV) perceived impacts and acceptance for each mining technology.", "Three of the four curvilinear relationships between self-rated knowledge and perceived impacts in the hypothesised model were significant, as indicated by asterisks.", "An exception was in situ leach mining where the squared term, or the slight curve shown in Fig. 1, was not significant.", "Thus the slight curve can be adequately approximated by a weak positive linear relationship (0.18), which indicates that perceived impacts generally increase as people feel they know more about this type of mining technology (ISL).", "The negative beta coefficients for the squared terms for the three other types of mining indicated a concave relationship.", "That is, as self-rated knowledge increases from low levels, perceived impacts increase, but then as self-rated knowledge increases from medium levels, perceived impacts start declining (Fig. 3).", "However, as mentioned in the descriptive statistics, perceived impacts vary more between respondents with high confidence in their knowledge of emerging mining technologies like HF and ISL (see Fig. 2).", "While interesting, these relationships between self-rated knowledge and perceived impacts for all four mining technologies were relatively weak, explaining between 2 and 4 percent of perceived impacts, as shown by R-squared (R2) in Table 5.", "Turning to the other path in the hypothesised model, the direct linear effects of perceived impacts on acceptance for each mining technology were all significant, being stronger for emerging technologies (ISL = −0.52; HF = −0.62), and moderate in strength for established mining technologies (OC = −0.39; UG = −0.44).", "Perceived impacts is a strong driver of mining technology acceptance.", "The negative beta coefficients mean that as perceived impacts increase, acceptance of these mining technologies decrease.", "Perceived impacts explained between 27 and 39 percent of acceptance for emerging technologies (ISL and HF respectively) compared to established technologies at between 15 and 19 percent (OC and UG respectively) (see R-squared statistics).", "Despite the hypotheses being mostly confirmed (with the exception of not finding a significant curvilinear relationship between self-rated knowledge and perceived impacts for in situ leach technology), the fit statistics for the hypothesised model were quite poor (Table 6), suggesting that the model may need to be modified.", "Thus, modification indices were examined to suggest any additional paths that may be added to improve model fit.", "For each type of mining technology, the modification indices suggested an additional path between self-rated knowledge and acceptance would significantly improve model fit.", "This made theoretical sense because self-rated knowledge can include knowledge about other aspects of the mining industry (apart from perceived impacts) that may also influence the acceptance of different mining technologies.", "Therefore, the hypothesised model was revised by adding an additional path from self-rated knowledge about different mining technologies to acceptance of each mining technology.", "This revised model was a very good fit with the data for all four technologies, as indicated by the Chi-squared statistics (Chi2) not being significant, the root mean square error of approximation (RMSEA) being 0.08 or less, and standardised root mean squared residuals (SRMR) being 0.05 or less (Table 6).", "In the revised model (Fig. 4), the added path (the dotted line) from self-rated knowledge to acceptance was significant and positive for all mining technologies (except for hydraulic fracturing which was significantly negative), such that higher self-rated knowledge of different types of mining was associated with higher acceptance.", "This direct negative relationship for hydraulic fracturing (−0.08) shows that higher levels of self-rated knowledge lowers levels of acceptance for that mining technology.", "These direct effects of self-rated knowledge on acceptance were moderately strong for established technologies (OC = 0.31; UG = 0.25); however, they were weaker for emerging technologies (ISL = 0.15; HF = −0.08).", "Presumably, more is known about other aspects of established technologies that can directly affect acceptance.", "This effect is less or even negative for emerging technologies.", "The effects of the other paths were much the same as in the hypothesised model (Table 5).", "The direct paths between self-rated knowledge and acceptance for each of the four mining technologies mean that self-rated knowledge is only partially mediated by perceived impacts.", "This suggests that knowledge about different mining technologies that is not related to perceived impacts can also affect acceptance of these technologies.", "It is also noteworthy that the modification indices did not suggest adding curvilinear relationships between perceived impacts and acceptance would further improve model fit.", "That is, the curvilinear relationships with self-rated knowledge were fully mediated by perceived impacts.", "The finding that self-rated knowledge can have indirect curvilinear effects on acceptance, via perceived impacts, as well as direct linear effects show that the effects of self-rated knowledge on acceptance of mining technologies is complex.", "In this paper, we hypothesised that: (H1) perceived impacts mediate the relationship between self-rated knowledge and acceptance of each technology; (H2) there is a curvilinear relationship between self-rated knowledge and perceived impacts; and (H3) there is a linear relationship between perceived impacts and acceptance of each technology.", "These hypotheses were confirmed for the most part.", "First, our findings demonstrated that perceived impacts partly mediated the relationship between the public's self-rated knowledge and their acceptance of each technology (H1).", "Our findings also showed that overall acceptance of established mining technologies was higher than that of emerging technologies.", "Second, there was a curvilinear relationship between the public's self-rated knowledge and their perception of impacts of established and emerging mining technologies.", "This suggests that perceived impacts initially increase as people with little self-rated knowledge begin to gain some knowledge, and then decrease again when they feel they have a high level of knowledge (H2).", "And third, there were also direct linear relationships between perceived impacts and acceptance of each mining technology (H3).", "Our analysis also shows both direct and indirect relationships between perceived impacts and acceptance of technologies, highlighting the importance of understanding how self-rated knowledge is formed, and how it influences people's perceived impacts of technologies and social acceptance in relation to novel and emerging technologies.", "The findings of this research show that respondents have higher levels of self-rated knowledge of established mining technologies (OC, UG) than emerging technologies (ISL, HF) (Table 1).", "Similar to this finding, a study by the OECD (2010) on public perceptions of nuclear energy shows that in countries with existing nuclear energy plants, citizens demonstrate higher levels of knowledge and awareness than citizens in countries without nuclear energy plants.", "However, alongside high self-rated knowledge of established technologies, the respondents also perceived negative impacts of established technologies on the environment and human health to be about the same or higher than emerging technologies (Tables 2 and 4).", "The higher perceived impacts and high self-rated knowledge of established mining technologies may be because these technologies are more widespread.", "Nonetheless, perceived impacts from emerging mining technologies had a larger effect on their acceptance than perceived impacts from established technologies.", "That is, acceptance of emerging technologies seems more sensitive to perceived impacts.", "The association between self-rated knowledge and perceived negative impacts was examined further.", "As hypothesised in Fig. 1, curvilinear relationships between self-rated knowledge and perceived impacts were found (Fig. 2).", "Additionally, for emerging mining technologies (in situ leaching and hydraulic fracturing), perceived impacts diverged as people became more confident in their knowledge, as indicated by increasing confidence intervals in Fig. 2.", "In other words, there were more divergent views about accepting emerging mining technologies among those with the highest levels of confidence in their knowledge about those emerging technologies.", "This potentially reflects contested sources of knowledge around emerging technologies.", "The path analysis tested and confirmed that both established and emerging mining technologies are subject to curvilinear relationships between self-rated knowledge and perceived impacts, though not always.", "Increasing self-rated knowledge about in situ leaching generally led to higher perceived impacts.", "These curvilinear relationships for the other three mining technologies revealed that having some knowledge of established and emerging technologies can lead to higher perceived impacts, though having high levels of knowledge reduces perceived impacts, indicating that perhaps ‘a little bit of knowledge can be a dangerous thing’.", "The path analysis also showed that perceived impacts only partly mediated relationships between self-rated knowledge and acceptance of different technologies.", "This suggests that self-rated knowledge also includes aspects of mining technologies apart from perceived environmental and safety impacts, which can also affect acceptance of different mining technologies.", "Many factors influence acceptance of different mining activities: perceived benefits, distributional and procedural fairness, governance, and trust in the mining operating industry (Moffat et al., 2017; Moffat and Zhang, 2014; Walton and McCrea, 2017).", "To fully understand the nature and sources of knowledge that influence acceptance of established and emerging mining technologies (beyond factors such as environmental and safety impacts) needs further research.", "There is already evidence that even information provided in the form of a letter by a local industry operator to local communities around community engagement, benefits, and governance arrangements can improve a local mine's social licence to operate (McCrea et al., 2018; Zhang et al., 2018).", "Overall, more self-rated knowledge about mining technologies generally leads to increased acceptance.", "Perceived impacts around environmental and human health may initially increase as self-rated knowledge increases, but then decrease again with higher levels of self-rated knowledge.", "There are also other aspects of self-rated knowledge, unrelated to perceived environmental and safety impacts that may directly affect acceptance of different mining technologies, both positively and negatively.", "To some extent, this will depend on the specific mining technology being examined.", "The picture is also more complex for emerging technologies.", "Perceived impacts tend to vary more, the more confident people are in their knowledge about emerging technologies, which suggests there may be contested knowledges in relation to emerging technologies.", "These findings have important implications for how new mining technologies might be developed and deployed in the future.", "These findings may also have broader relevance for understanding how the public understands and accepts the presence of existing mining activities versus proposed new mining activities (i.e. acceptability of brownfield versus greenfield mining developments).", "It is already evident that concepts like shared values are becoming central to discussions about how the mining sector will evolve and innovate into the future (Cosbey et al., 2016).", "While established technologies enjoy a certain amount of established acceptance, emerging technologies may have to better negotiate a multiplicity of values, contested knowledges, and uncertainty in the mind of the public.", "This requires an increased focused on reducing harm associated with new and existing operations, decreasing the use of water, energy and other resources, and delivering value for the sector and their host communities, regions and countries.", "At the same time, we cannot assume that simply providing some information about reduced impacts and increased benefits will lead to acceptance of emerging technologies.", "Relatively high levels of knowledge confidence about reduced impacts and increased benefits may be needed before the general public broadly accepts an emerging mining technology.", "However, with new technologies, it may be the case that the information presented is more likely to be contested as various stakeholder interests and groups grapple with and make sense of the roles, functions, meanings and impacts of these technologies.", "In short, emerging technologies will need to work harder for acceptance than in days gone by and this will require increased engagement with the range of contested sources of knowledge.", "The mining sector is also grappling with increasing challenges in relation to maintaining productivity, efficiency and the recognition that new deposits are likely to be increasingly remote, deep and difficult to access (IGF, 2018).", "Thus, emerging technologies will have a key role to play in demonstrating changes to extraction methods in the future which ensure that economic, social and environmental benefits of innovation are realised.", "However, what sort of knowledge the public has will be important for understanding how they will perceive the impacts of that technology.", "It is important for the mining industry and governments to be proactive in informing and addressing public and stakeholder concerns around emerging mining technologies.", "Addressing public and stakeholder concerns relates to more than simply providing information about relevant mining technologies per se.", "Generating trust in the mining industry generally and in local industry operators in particular is an important driver of social acceptance in mining (Moffat et al., 2017; Moffat and Zhang, 2014; Walton and McCrea, 2017).", "This trust is also based on meaningful consultation and engagement, which is procedurally fair (Mercer-Mapstone et al., 2017).", "Further, it is recognised that it is important to provide information about how mining projects will be governed and how local community views can be incorporated into local mining operating decisions (Zhang et al., 2018).", "Additionally, a number of studies have also documented that public perception of mining is influenced by media reports (Edwards et al., 2019; Lyytimäki and Peltonen, 2016).", "Therefore, in introducing new technologies, simply understanding what impacts the public perceive of the technology will matter little if we do not also understand how their impact perceptions were formed and how their knowledge confidence was established.", "There is clearly a need for additional research on the formation and confidence in knowledge around established and emerging mining technologies.", "Having an understanding of how the public's knowledge can affect perceived impacts and social acceptance of various mining technologies is useful for building a picture of possible responses to particular technologies and their use (Crowe et al., 2015; Lacey et al., 2017), but it is also important to be able to determine what kind of knowledge the public has and how their views are formed.", "In our research we did not examine the source or nature of self-rated knowledge, which may be critical to the determination of perceived impacts of established and new mining technologies.", "For example, trusting information from pro- or anti-mining sources for self-rated knowledge of a technology would presumably result in different perceived impacts of that technology.", "As a result, there are some limitations in our understanding of the nature of the self-rated knowledge of the respondents in this study.", "The relationships we found between self-rated knowledge and perceived impacts from mining technologies were significant though moderately weak.", "This can be partly explained by the general nature of the self-rated knowledge survey question.", "It was a single item question asking them to rate their level of general knowledge about different types of mining technologies currently practiced in Australia.", "The relationships may also have been weakened by asking this question before providing information about each of the different mining technologies, which was necessary before asking about the perceived impacts of little known emerging technologies.", "In future research, it may be better to use a multi-item measure of different aspects of self-rated knowledge for a mining activity (e.g. extractions processes, community impacts, etc.) and a community already familiar with particular emerging mining technology (e.g. an affected local community).", "Further, additional research is also needed on the effects of actual knowledge of mining technologies on perceived impacts and acceptance, especially actual knowledge of emerging and contested mining technologies.", "For example, this could be done by including a measure which tests survey respondents’ knowledge of the technology (i.e. by marking answers to questions as correct or incorrect) or perhaps the depth of their understanding could be assessed by asking whether they understand each term in a list of increasingly technical terms associated with a particular technology and its potential impacts.", "To conclude, the mining industry is increasingly acknowledging the importance of the public's acceptance of their activities for their initial approval and successful on-going operations.", "However, the idea of simply providing information to the general public on mining technologies will not necessarily increase their acceptance of those technologies.", "A little information can initially raise concerns about environmental and health impacts, and quite high levels of public understanding may be needed to alleviate those concerns and increase overall acceptance, especially for emerging mining technologies.", "Acceptance of emerging mining technologies appears more sensitive to perceived impacts than established technologies, even though the perceived impacts of established technologies are high, and there is more variation in the perceived impacts of emerging industries by those confident in their knowledge about those technologies.", "That is, knowledge of emerging technologies is more contested by people, who are very confident in their views.", "However, high levels of self-rated knowledge about mining technologies generally increases acceptance of those technologies, though results vary between different mining technologies and more research is needed into how and where knowledge perceptions of different mining technologies are formed.", "In this paper, we have aimed to contribute to the mining-society scholarship by providing evidence that will enhance our understanding of the drivers that shape public perceptions of a range of mining technologies." ]	[ "Mining technologies are critical to achieving triple bottom line outcomes.", "Social attitudes to mining technologies are poorly understood.", "We examine social attitudes to emerging and established mining technologies.", "Findings reveal higher levels of overall acceptance of established technologies.", "Findings reveal knowledge type may determine acceptance of emerging technologies." ]
Fuel reactor modelling for chemical looping combustion: From micro-scale to macro-scale	Chemical looping combustion (CLC) is a promising technology able to convert energy whilst managing CO2 emissions. The CLC system is composed of two fluidised bed reactors working in different hydrodynamic regimes. Transition metal particles circulate between the two reactors to carry out the oxidation and reduction reactions with air and fuel, respectively. This technology, which might be embedded into a fuel power plant for electricity generation, would lead to higher thermal plant efficiency than other technologies for carbon capture. The main concern about the technology is related to the total solid inventory needed to achieve full gas conversion which is believed to count for most of the cost of a CLC power plant. Thus, noteworthy attention is given to the modelling of the reactors to optimise the solid inventory and thus the plant's cost. In this work, a 2D computational fluid dynamics (CFD) analysis of the fuel reactor is carried out. The results, in terms of the effect of the different kinetic and hydrodynamic conditions on the outlet gas conversion, are compared with the results using a macro-scale model implemented in Aspen Plus. Based on the micro scale (CFD) outcomes, the macro scale model is enhanced to capture the main physics influencing the performance of the fuel reactor. The latter is considered more suited as power plant simulator for thermal efficiency and cost estimations.	[ "CENG", "CHEM", "ENER" ]	[ "Aspen Plus", "CFD", "Chemical looping combustion", "Fluidised bed reactors", "MFIX" ]	[ "1500", "1605", "2102", "2103" ]	[ "CO2 capture from fuel power plants is a commitment involving industry and academia to reduce the CO2 emissions in atmosphere and thus mitigate the global temperature rising.", "Chemical looping combustion (CLC) is a promising technology, initially patented by Lewis and Gilliland [1], to capture pure CO2 streams with low energy penalties [2–4].", "The CLC process is based on metal oxide solid particles that circulate between two reactors to carry out the fuel combustion.", "Air reacts with metal solid particles in the air reactor (riser) to produce metal oxide particles that are conveyed from the air reactor to the fuel reactor where a reaction with the fuel occurs (Fig. 1).", "The outlet stream from the fuel reactor is composed by CO2 and H2O easily separable by condensation; the outlet stream from the riser is mostly nitrogen and un-reacted oxygen at high temperature.", "A well-studied design of the CLC system assumes a riser working in fast fluidisation regime to convey the solid particles from the oxidiser to the reducer and a fuel reactor working in bubbling bed regime [5–7].", "The overall reaction system is merely a fuel combustion reaction with the following main features: air is never mixed with the fuel (thus reducing consistently NOx emissions), energy penalties for CO2 capture are much lower compared to other technologies [3,4,8,9].", "Nickel, iron and copper metal oxides with different inert supports are the most tested materials when reacting with gaseous fuel such as methane, hydrogen and carbon monoxide [10–14]; they should be cheap and environmentally sustainable.", "The enhancement of durability, mechanical strength and long term high reactivity of these materials represents the key to make the CLC technology viable.", "Kinetic and hydrodynamic phenomena occurring in both reactors affect largely the CLC system, specifically in terms of reaction volumes, total solid inventory into the fluidised beds and consequently the make-up of the solid oxygen carrier.", "Those phenomena have to be investigated carefully since the main issue influencing the competitiveness of the CLC technology is related to the cost of the solid particles [15].", "In this scenario, a reactor model that helps to predict optimal operating conditions of the CLC system plays a crucial role in reducing the capital and operating cost of the whole fuel power plant.", "Aspen Plus software is largely used by industries to simulate a variety of chemical and petrochemical processes.", "The software provides operational blocks to implement most of the units characterising engineering processes.", "A whole plant made of blocks connected by mass and energy streams can be implemented: mass and heat macro balances are solved across the blocks.", "Thermal efficiency estimation and cost analysis can be readily undertaken for the whole process.", "The disadvantage of this macro level approach is that some unit operations, such as fluidised bed reactors, cannot be simulated with a good degree of reality.", "For this reason, several efforts were carried out in the past [16–19] to mimic the real behaviour of fluidised bed reactors.", "Computational fluid dynamics (CFD) simulations for chemical looping combustion purposes [20–27] have been employed to understand at the particle level how the kinetics and the hydrodynamics influence the motion of the solid particles into a fluidised bed, the gas–solid contact efficiency and therefore the outlet fuel conversion.", "The disadvantages of this micro level approach are related to the computational cost, the long runs of simulation and in particular the small domain that can be processed (few or most commonly just one unit processed).", "The present work aims to model the fuel reactor of the CLC system using a CFD approach to achieve a better understanding of the kinetic and hydrodynamic phenomena involved.", "The results from the micro scale approach are analysed and compared with those obtained from a macro scale approach using Aspen Plus model.", "The latter results are improved to increase the accuracy of the macro scale model making it useful for future thermal efficiency and economic evaluations of a fully integrated CLC power plant.", "The metal oxide reduction rate is lower than its oxidation rate under the same temperature conditions [5,10,11,13]; consequently, a higher solid inventory into the fuel reactor than into the air reactor is expected to get full gas conversion.", "The present work focuses on a better understanding of the bubbling fuel reactor behaviour at the macro and micro scale to get useful information about the minimum/optimal solid inventory needed into the reducer (the fuel reactor).", "The system under investigation is a fuel reactor sized for 10 MW of fuel power working at 950 °C under atmospheric pressure.", "The reduction reaction involves pure methane that reacts with nickel oxide to produce CO2, H2O and nickel oxide to be regenerated in the air reactor.", "The kinetic data for the NiO reduction and the physical properties of the solid particles come from Abad et al. [15] as summarised in Table 1.", "Computational fluid dynamics (CFD) is carried out using an open source code called MFIX (Multiphase Flow with Interphase eXchange [29]).", "A 2D reactor is filled up with solid particles in a packed bed manner at time equal zero.", "At the beginning of the simulation, nitrogen flows uniformly from the bottom to the top of the bed fluidising the solid particles.", "After 2 s of simulation, the feed is switched from nitrogen to methane and the simulation runs until approximately constant values of flue gas concentrations are achieved (pseudo stationary condition).", "Nitrogen is used at time equal zero (with no reaction occurring) to help the convergence at the beginning of the simulation.", "The initial conditions are shown in Table 3.", "More details of the CFD model employed are given in Section 3.", "The size of the reactor must be decreased compared to the system implemented in Aspen Plus to reduce the computational costs.", "The hydrodynamic conditions simulated in Aspen Plus are kept to guarantee a right comparison between micro and macro model.", "The reactor is scaled to a fuel power of 10 kW just reducing the area to 34.8 cm2 that means 300 g/kW of solid inventory in the bed at the same height of fixed bed Lm modelled in Aspen Plus.", "As reported by Mahalatkar et al. [25], the fuel reactor can be simulated over the time by decoupling it from the air reactor for the following reasons:", "The amount of solid material inside the bed is high compared to the reacting gas flow-rate, implying that the metal oxide conversion does not appreciably change during the simulation time (of the order of few seconds).", "Few seconds of simulation are enough to reach pseudo stationary conditions at approximately constant metal oxide conversion; consequently, such the solid flow-rate is not needed to be simulated for replacing the converted metal oxide particles.", "In MFIX environment, the initial NiO mass fraction (i.e. 36%) is chosen equal to the NiO mass fraction in the inlet solid flow-rate simulated in Aspen Plus (XMO_exit is assumed to be 0.9).", "This assumption does not influence greatly the comparisons between the micro model (CFD) and the macro model (Aspen Plus) since in either CFD or Aspen simulations this value is almost constant, so its influence on the kinetic rate is the same.", "Indeed, in CFD the metal oxide conversion does not appreciably change during the simulation time due to few seconds of simulation; in Aspen, the metal oxide conversion changes slightly, regardless the degree of gas conversion, since the solid flow-rate applied for models comparison is 8 times higher than the stoichiometric one (Fs = 75 kg/s).", "The comparison between the steady state case implemented in Aspen Plus and the pseudo stationary case implemented in MFIX under the same operating conditions is run for two different case studies called “high” and “low” kinetics.", "High kinetics refers to the value of ki obtained from the data reported by Abad et al. [15] and shown in Table 1, whereas low kinetics refers to a fictitious low value of ki equal to 0.1.", "The different kinetics (with two orders of magnitude difference) affect largely the hydrodynamics of the system allowing for a better understanding and comparison between the macro scale model (Aspen Plus) and the micro scale model (CFD).", "The 2D model implemented in MFIX code is based on the Eulerian–Eulerian approach.", "Gas and solid phases are considered as continuum phases in the domain under investigation.", "The solid phase is characterised by uniform spherical particles of constant mean density and diameter.", "Continuity and momentum equations in two directions (x and y) are solved for both phases and the exchange of mass and momentum between them is taken into account.", "No energy equations are solved because the reduction reaction is slightly endothermic and the drop in temperature under few seconds of simulation is negligible.", "All the equations come from the well known theory embedded in the MFIX code [29] and they are listed in the Appendix A.", "No-slip boundary condition for the gas phase and the free-slip condition for the solid phase are applied at the wall.", "At the inlet section of the bed the Dirichlet boundary condition is applied (with constant inlet mass flux) whereas at the outlet section of the bed the Neumann boundary condition is assumed (with constant outlet pressure).", "The domain is divided into 7000 cells (see Table 3) and for each cell the continuity and momentum equations of each phase are solved simultaneously with linear solvers (finite volume method).", "The algorithm applied to solve the set of partial differential equations (PDE) is an extension of SIMPLE algorithm [30] introduced for the solution of pressure–velocity coupled PDE.", "The scalar variables are positioned at the cell centre whereas the vector variables at the cell faces (staggered grid arrangement).", "The convection terms of PDE are discretised using a second upwind order called Superbee to improve the numerical accuracy and the bubble shape resolution [29].", "Finally, MFIX code uses a variable time step and the range selected is between 10−4 and 10−8 s.", "As mentioned, all the CFD simulations run in a computational domain divided in 7000 cells.", "A grid independence test was carried out increasing the number of cells up to 28,000.", "No appreciable effects on the flow behaviour and main variables were found.", "Low and high kinetics (see Section 2) are implemented to study their effect on the hydrodynamics and the outlet gas concentrations.", "High kinetic simulations run for 10 s whereas low kinetic simulations run for 15 s since longer times are needed to achieve pseudo stationary condition in terms of gas outlet concentrations (Fig. 2).", "A pure nitrogen flow-rate is fed at 36 cm/s up to t = 2 s. For t > 2 s, the feed flow-rate is witched from pure nitrogen to methane (Figs. 2 and 3) and the heterogeneous reaction with NiO occurs.", "The delay in the methane mass fraction detection after t > 2 s is due to the time that the methane feed flow-rate needs to cross the whole fluidised bed.", "Further delay in CO2 and H2O mass fraction detections is related to the very low kinetics of reaction.", "Indeed, this effect is not noticeable at the high kinetics (Fig. 3).", "Fig. 4 shows the void fraction in the dense phase versus X axis averaged over Y axis between 12.95 and 16.45 cm.", "The low kinetics case is averaged over time between 13 and 15 s whereas the high kinetics case is averaged over time between 8 and 10 s.", "The reaction of methane with NiO is characterised by an increase of the number of moles in the gas phase (with an expansion factor ζ equal to 2).", "Thus, high kinetics, which lead to high methane conversion, produce an increase in the gas volume fraction.", "Inversely the solid volume fraction shows higher values in the low kinetics case (Fig. 5).", "Bubbles formation occurs at the bottom of the fluidised bed and they rise and coalesce along the bed height mostly far from the walls.", "Thus, higher solid volume fractions are expected to be found close to the walls (Fig. 5).", "Fig. 6 shows the axial component of the gas velocity Vg for the two cases under investigation.", "Two main features can be noticed: the applied no-slip condition for the gas phase (with Vg equal to zero at the walls) and higher values of Vg in the centre of the bed due to the bubble tendency to rise mostly towards the centre.", "The rising of the bubbles creates recirculation of the solid phase in the so called “emulsion phase” that leads to negative value of Vg close to the walls.", "As previously explained, high methane conversion produces a considerable increase in the gas volumetric flow-rate and thus in the axial gas velocity.", "Fig. 7 shows the axial component of the solid velocity, Vs, for both high and low kinetics cases.", "The solid free slip condition is verified (with Vs ≠ 0) at the walls.", "The bubbles’ rising pushes the solid particles upward in the centre of the bed and subsequently they fall down following the recirculation motions.", "Fig. 8 shows the change in axial gas velocity, Vg, versus Y axis averaged over time and X axis.", "The double effect of the heterogeneous reaction and the reduction of the void space (εg < 1) produces an abrupt increase in Vg in the dense phase, which is particularly pronounced for high kinetics.", "The value of Vg at the bed exit follows the increase in the number of moles due to the methane conversion.", "Figs. 9 and 10 show the gas volume fraction and the mass fraction of the gas species along the bed for low and high kinetics, respectively.", "The main features charactering the bubbling regime can be observed: the bubbles’ formation takes place few centimetres above the gas inlet; bubbles rise mostly at the centre of the bed increasing in volume and coalescing along the bed height.", "At high kinetics, bubbles are larger and their motion approaches the turbulent regime (Fig. 10) due to the high methane conversion that leads to high increase in number of moles in the gas phase.", "This phenomenon can be ideally explained with the transfer of the gas exceeding the minimum fluidisation velocity Umf from the emulsion phase at high solid content to the bubble phase at high gas content [31].", "Due to larger and faster bubbles and their burst at the interface dense phase–freeboard, at high kinetic conditions higher bed expansion is detected.", "At low kinetics (Fig. 9), high values of un-reacted methane mass fraction are observed, mainly in the centre of the bed where most of the bubble phase takes place.", "The reaction occurs mainly in the bottom section of the bed characterised by an average high solid volume fraction, high methane mass fraction and no well defined bubble phase; far from the bottom region, bubble phase formation determines gas by-pass along the bed that is subtracted from the high reacting regions (Fig. 11); thus poor gas conversion is expected to occur.", "At high kinetics, the reaction occurs mainly in the bottom phase and gas by-pass apparently has no effect in lowering the gas conversion.", "This outcome is affected by the assumed condition of inlet uniform gas flow-rate.", "Modelling the gas distributor (preferably in a 3D simulation) is expected to show bubble formation after few millimetres above the distributor reinforcing the negative effect of gas by-pass on the gas conversion.", "Being aware that the gas distributor (not modelled in our work) can affect the development of the bubbles in terms of location and percentage of occupied volume and thus the gas conversion, the simulations carried out at two different kinetics conditions show that high reacting solid materials are important to achieve full gas conversion in a fluidised bed operating under bubbling regime.", "No freeboard effect in the kinetic reaction is detected [20,22,32].", "The influence of the freeboard region, characterised by low solid content on the gas conversion, was experimentally demonstrated [31,33,34].", "As reported by Wang et al. [32], the zero solid–gas contact efficiency in the freeboard might be related to the failure of the Eulerian–Eulerian approach to describe the gas/solid mechanisms in regions at very low solid particles concentrations.", "An Eulerian–Lagrangian approach might help to estimate the reduction of the reaction in the freeboard region.", "At the macroscopic scale, a bubbling bed can be ideally thought as made of two phases: a so called “emulsion phase”, characterised by good mixing of gas and solid particles, and a so called “bubble phase” at low content of solid acting as gas by-pass along the bed.", "This representation comes from the Davidson and Harrison theory [35] that states how the excess of gas to keep the minimum fluidisation condition in the emulsion phase is transferred to the bubble phase.", "Several authors [16–18,36] modelled the bubble phase as a plug flow reactor (PFR) and the emulsion phase as a perfect mixing reactor (CSTR).", "The bed is axially divided into stages; each stage is composed by two sub-reactors: PFR to mimic the bubble phase and CSTR to mimic the emulsion phase (Fig. 12).", "Gas mass transfer occurs between the exit streams of the two sub-reactors at the end of each stage.", "Isothermal conditions within the bed are assumed and the radial mass solid gradient is neglected.", "In this work, the hydrodynamics (see Table 4) is implemented using subroutines written in Excel which are linked to the Aspen Plus environment through the so called “calculator blocks”.", "In the “Feed Calculator” block (Fig. 12), a split of the inlet volumetric flow-rate to enter the two phases, bubble and emulsion, has to be accomplished.", "The difference Uo − Umf is the term driving the split.", "The approach proposed by Johnson et al. [37] and applied also by Adanez et al. [38] is taken into account.", "Johnson et al. [37] reported an expression of the volumetric bubble fraction independent of the height of the bed.", "Combining the two phase theory with the dependence of the bubble velocity, Ub, from the bubble diameter and applying the Darton correlation for the bubble size [39], Johnson et al. [37] first defined a function f2 and, after some manipulations, they expressed the volumetric fraction of the bubble phase, σ, as a function of f2 and the difference Uo − Umf (see Table 4).", "σ is the volumetric fraction of the bubble phase in the whole bed and its value is independent of the height in the bed.", "Given σ, the rise bubble velocity, Ub, is calculated from the gas mass balance.", "Thus, the inlet volumetric gas flow-rate can be split into two streams entering the first PFR and CSTR reactors to run the bubbling bed model.", "The kinetic expression (Eq. (1)) is introduced in each sub-reactor through subroutines written in FORTRAN language.", "Porrazzo et al. [17] reported how the suitable number of stages to model the fluidised bed depends on kinetic and hydrodynamic considerations.", "It was found that 5 stages provide outcomes of reasonable accuracy in terms of gas conversion at the bed exit under different operating conditions [17].", "A number of authors applied the concept of dividing axially the bubbling bed in stages and considering a number of stages of the same length [16–18,36].", "Previous CFD simulations showed how most of the heterogeneous reaction occurs at the bottom of the fluidised bed since the reacting gas concentration is higher and the bubbles’ diameter at the bottom of the bed is at its minimum.", "Furthermore, the incoming gas, which encounters the solid particles, creates turbulent motions that enhance the gas–solid contact efficiency promoting an increase in the gas conversion.", "The mass transfer between the bubble and the emulsion phase decreases significantly along the bed due to the increase in the bubble diameter that reduces the mass transfer coefficient Kbe.", "Moreover, the increase in the bubble volumetric fraction, σ, along the bed, due to the gas expansion, reduces the emulsion volume fraction and thus the portion of the bed where most of the reaction takes place.", "Consequently, a new concept of stages is introduced.", "Porrazzo et al. [17] reported a multi stage model where the whole bed was divided into 5 stages of the same length.", "The new concept of stages refers to a different split of the bed length according to the following strategy: 4 stages are used to model the bottom of the bed whereas the last (fifth) stage is used to model the rest of the fluidised bed length.", "Based on the increment in the bubble diameter per unit bed height (Fig. 13), as an indicator of the mass transfer effectiveness between the two phases, 4 stages are employed to model the first 10 cm of the bed above the inlet and the last stage is employed to model the rest of the bed length.", "The trend of the mass transfer coefficient Kbe follows the same trend of the incremental bubble diameter per height unit.", "In this way, we assure a good utilisation of the limited number of stages employed since we mainly focus on the modelling of the bed zone where most of the phenomena of interest occur.", "The two fuel reactor models (MFIX and Aspen Plus) were tested under different operating conditions: various superficial gas velocity, solid inventory and kinetics of reaction.", "Fig. 14 shows the variation of the methane mass fraction along the bed height for different superficial gas velocities at constant solid inventory and low kinetics.", "The values of the CH4 mass fraction in the Aspen Plus model are detected at the end of each reaction stage.", "The CH4 mass fraction in the CFD model is averaged over X axis and over time between 13 and 15 s.", "In both models, the reduction in the superficial gas velocity Uo determines a decrease in the methane mass fraction and thus an increase in the gas conversion; indeed, values of Uo closer to the minimum fluidisation velocity lead to a reduction of the gas by-pass through the bubble phase promoting the reaction in the emulsion phase.", "The increase in methane conversion is amplified from the fact that keeping constant the area of the reactor, a decrease in Uo leads to a decrease in the fuel power and thus the same amount of solid inventory reacts with less methane flow-rate driving the gas conversion to higher values.", "As mentioned previously, most of the reaction occurs in the bottom of the bed where gas and solid show high contact efficiency.", "The Aspen Plus model assumes an initial split of the superficial gas flow-rate between the two phases based on the amount of gas exceeding the minimum fluidisation conditions [17]; thus, part of the gas flow-rate is initially subtracted to react with the solid particles leading to lower values of gas conversion compared to the CFD model.", "In the CFD model, we assume uniform inlet gas flow-rate and the modelling of the gas distributor is not taken into account.", "The gas distributor plays a role in affecting the bubble phase in terms of bubble diameter and distance of initial formation from the bottom of the bed [31,40].", "For this reason, it is believed that the modelling of a gas distributor in a 3D CFD simulation could give a better match between the macro-scale and micro-scale models.", "The mass transfer term plays a negligible role far from the inlet, determining almost flat profiles of methane mass fraction above 10 cm from the inlet.", "This aspect justifies the implementation of just one stage to model the rest of the bed height in Aspen Plus.", "Fig. 15 shows the methane conversion at the bed exit for the two models at different initial superficial gas velocities.", "As mentioned, the Aspen model provides lower results of the methane conversion compared to the CFD model due to the initial split of the gas flow-rate at the bed inlet.", "The CFD trend is slightly affected by fluctuations in the axial velocity component (pseudo stationary condition).", "The difference in the outlet gas conversion between the two models ranges from 1% to 6%.", "Fig. 16 shows the variation of the methane mass fraction along the bed for different solid inventories at initial superficial gas velocity equal to 36 cm/s and low kinetics.", "The reduction in the solid inventory, expressed as height of fixed bed Lm, determines lower gas residence time into the bed leading to lower gas conversion.", "The Aspen model captures this feature in the last stage of reaction that is smaller in terms of volume of reaction at lower solid inventories.", "Explanation of the effect of the change in the solid inventory on the gas conversion with the change in the gas residence time fails by looking at the CFD trends.", "In this case, the change in solid inventory affects the solid volume fraction distribution in the bottom of the bed and thus the kinetic rates: higher solid inventories lead to higher average solid volume fraction in the bottom zone given that statistically more solid particles participate to the recirculation motions into the bed; consequently, higher solid volume fraction lead to higher kinetic rates and thus lower methane mass fractions.", "The comparison in gas conversion at the bed exit between the two models is good (Fig. 17).", "The difference in the outlet gas conversion between the two models is below 3%.", "The same analysis on the methane mass fraction under different superficial gas velocities and solid loading is carried out for the case at high kinetics.", "Fig. 18 shows the change in gas mass fraction along the bed for different superficial gas velocities.", "The effect of the reaction kinetics, in reducing steeply the methane mass fraction at the bottom of the bed is more evident for high kinetics.", "The effect of changing the superficial gas velocity on the gas conversion is much more appreciable from the Aspen model calculations.", "Also, in this case, lower values of Uo determine higher gas conversion.", "From Fig. 19 it is noticeable how the difference in the gas conversion between the two models decreases at lower Uo.", "At lower Uo values, less gas flow-rate goes to the bubble phase and the effect of the initial gas splitting in the Aspen model is reduced: decreasing Uo, the gas conversion values from the two models tend to converge.", "The difference in the outlet gas conversion between the two models ranges from 0.05% to 15%.", "At high kinetics, a change in solid inventory in a range 20–45 cm of height of fixed bed does not influence appreciably the methane gas conversion in both models.", "The work compared two models for CLC bubbling bed, one at macro-scale (Aspen Plus) and one at micro-scale level (CFD).", "The two models were compared under different operating conditions.", "Based on CFD simulations, a new division of the number of stages in Aspen Plus was introduced to capture the phenomena occurring at the bottom of the bed.", "The methane mass fraction trends along the bed and outlet gas conversions were evaluated at different kinetic rates, inlet superficial gas velocities and solid inventories.", "Despite the construction of the multi-stage macro-scale model is based on idealised hydrodynamic and kinetic phenomena, the trends and the overall results from the two models are comparable.", "The multi-stage model implemented in Aspen Plus can capture the influence of the kinetics, superficial gas velocity and solid inventory on the gas conversion.", "A new division of the stages that models better the bottom of the fuel reactor reduces the gap between the macro-scale and micro-scale model.", "It is believed that a 3D modelling of a gas distributor at micro-scale level might enhance the match between the two models since bubbles’ formation and diameter are affected by the distributor design.", "The difference in the outlet gas conversion between the two models can be evaluated with good confidence for engineering purposes.", "Consequently, the macro-scale model of the fuel reactor, embedded into a fuel power plant simulation, can lead to a reasonable estimation of the effect of the reaction operating conditions on the overall thermal efficiency, capital and operating cost of a fuel power plant based on the CLC technology." ]	[]
Determination of critical epitope of PcMab-47 against human podocalyxin	Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.	[ "BIOC" ]	[ "Epitope mapping", "Monoclonal antibody", "Oral cancer", "PODXL", "Podocalyxin" ]	[ "1303", "1304" ]	[ "Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated and has a molecular weight of 150,000–200,000 [1–5].", "PODXL expression has been reported in several human cancers, including oral [6], breast [7], and lung cancer [8,9] and leads to tumor growth, invasion, and metastasis [7,10].", "Several studies have developed anti-PODXL monoclonal antibodies (mAbs) [11,12].", "We previously produced a novel anti-PODXL mAb, PcMab-47 (IgG1, kappa) [13].", "PcMab-47 reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis.", "We engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC).", "We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a, to augment its ADCC.", "Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration than the former.", "In vitro analysis showed that 47-mG2a-f exhibited a much stronger ADCC than 47-mG2a against OSCC cells.", "In vivo analysis revealed that 47-mG2a-f, but not 47-mG2a, exerted an antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times.", "Although 47-mG2a and 47-mG2a-f exerted antitumor activities in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG2a-f showed a higher antitumor activity than 47-mG2a.", "These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.", "Although engineered mAbs of PcMab-47 show high antitumor activities against cancer cells, the critical epitope of PcMab-47 remains to be identified.", "In this study, we clarified the binding epitope of PcMab-47 using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemistry.", "CHO-K1 was obtained from the American Type Culture Collection (ATCC, Manassas, VA).", "SAS (oral squamous carcinoma cell line from tongue) was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).", "CHO-K1 cells were transfected with PA-tagged PODXL deletion mutant plasmids using Lipofectamine LTX (Thermo Fisher Scientific Inc., Waltham, MA).", "PODXL deletion mutants were cultured in an RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and SAS was cultured in Dulbecco's Modified Eagle's Medium (DMEM; Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B (Nacalai Tesque, Inc.), and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.", "The cDNA encoding the full-length open reading frame (ORF) of PODXL was obtained by PCR using cDNA derived from the LN229 cell line (ATCC) as a template.", "Appropriate oligonucleotides were used as primers to make each deletion mutants.", "PCR products were subcloned into pCAG vector (FUJIFILM Wako Pure Chemical Industries Ltd., Osaka, Japan) with signal sequence and PA tag using the In-Fusion PCR Cloning kit (Takara Bio, Inc., Shiga, Japan).", "All amino acid number were consistent with the NCBI Reference Sequence, NP_005388.2.", "Synthesized PODXL peptides (PEPScreen; Sigma-Aldrich Corp., St. Louis, MO) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at 1 μg/ml for 30 min.", "After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the plates were incubated with purified PcMab-47 (1 μg/ml), followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies Inc., Santa Clara, CA).", "The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.).", "Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).", "These reactions were performed at 37 °C with a total sample volume of 50–100 μl.", "Cells were harvested after brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.).", "After washing with 0.1% bovine serum albumin in PBS, the cells were treated with PcMab-47 (1 μg/ml) or PcMab-47 (1 μg/ml) plus peptides (1 μg/ml) for 30 min at 4 °C, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA).", "Fluorescence data were acquired using the Cell Analyzer SA3800 (Sony Corp., Tokyo, Japan).", "Histological sections of 4-μm thickness were directly autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min.", "Sections were then incubated with PcMab-47 (5 μg/ml) or PcMab-47 (5 μg/ml) plus peptides (5 μg/ml) for 1 h at room temperature, treated using an Envision+ kit (Agilent Technologies Inc.) for 30 min.", "Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Industries Ltd.).", "We previously developed PcMab-47, a novel anti-PODXL mAb which exhibits a high specificity and sensitivity against human PODXL.", "PcMab-47 was shown to be useful for immunohistochemical analyses using paraffin-embedded tissues [13,14].", "Furthermore, engineered mAbs of PcMab-47 exerted high antitumor activities against cancer cells in mouse xenograft models.", "Therefore, the determination of the binding epitope of PcMab-47 is critical to further develop a molecular targeting therapy against PODXL.", "In this study, eight deletion mutants of PODXL were constructed (Fig. 1A).", "Stable transfections of PODXL-mutant clones were established on CHO-K1 cells, including dN25 (corresponding to 25–526 amino acids [aa]), dN80 (corresponding to 80–526 aa), dN100 (corresponding to 100–526 aa), dN140 (corresponding to 140–526 aa), dN180 (corresponding to 180–526 aa), dN200 (corresponding to 200–526 aa), dN220 (corresponding to 220–526 aa), and dN300 (corresponding to 300–526 aa).", "All deletion mutants of PODXL contained an N-terminal PA tag and were analyzed using flow cytometry for the epitope mapping of PcMab-47.", "NZ-1 (anti-PA tag mAb) detected all deletion mutants of PODXL, including dN25, dN80, dN100, dN140, dN180, dN200, dN220, and dN300 (Fig. 1A, upper panel).", "In contrast, PcMab-47 did not react with dN220 and dN300 (Fig. 1A, lower panel).", "These results indicate that the N-terminus of PcMab-47 epitope exists between the 200th and 219th aa.", "These results are summarized in Fig. 1B.", "We next synthesized a series of PODXL peptides such as 180–199 aa, 190–209 aa, 200–219 aa, 210–229 aa, 220–239 aa, and 230–249 aa (Table 1, Fig. 1B).", "PcMab-47 reacted only with 200–219 aa peptides in ELISA.", "We synthesized a series of point mutations of PODXL peptides of 200–219 aa (Table 1).", "PcMab-47 reacted with T200A, P201A, T202A, S203A, S204A, G205A, H206A, K211A, S213A, S214A, S215A, S216A, S217A, T218A, and V219A.", "PcMab-47 weakly reacted with I212A and did not react with D207A, H208A, L209A, or M210A, indicating that PODXL peptides from the 207th to the 210th aa (207-DHLM−210 sequence) constitute the critical minimum epitope of PcMab-47.", "We further performed a blocking assay using flow cytometry.", "PcMab-47 reacted with the SAS cell line (Fig. 2).", "This reaction was completely neutralized by the wild-type peptide (200-TPTSSGHDHLMKISSSSSTV-219), H206A, and K211A.", "In contrast, D207A, H208A, L209A, and M210A did not block the reaction of PcMab-47 with SAS, confirming that the (207-DHLM-210) sequence is a critical epitope of PcMab-47.", "Finally, we performed a blocking assay using immunohistochemistry.", "PcMab-47 reacted with oral cancer tissues (Fig. 3A) and the reaction was completely neutralized by the wild-type peptide (200-TPTSSGHDHLMKISSSSSTV-219) and H206A.", "D207A did not block the reaction of PcMab-47, and this confirmed the results of the flow cytometric blocking assays.", "Taken together, our results indicate that the critical epitope of PcMab-47 is Asp207, His208, Leu209, and Met210.", "Our findings could lead to the production of more functional anti-PODXL mAbs, which would be advantageous for antitumor effects against PODXL-expressing cancers." ]	[ "PODXL is involved in cancer growth, invasion, and metastasis.", "PcMab-47 mAb against PODXL shows high ADCC/CDC and antitumor activities.", "PcMab-47 epitope was determined to be Asp207, His208, Leu209, and Met210.", "A blocking peptide completely neutralized PcMab-47 reaction by FCM and IHC." ]
Structural and functional hepatocyte polarity and liver disease	Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canalicular bile. They have a uniquely organized polarity with a basal membrane facing liver sinusoidal endothelial cells, while one or more apical poles can contribute to several bile canaliculi jointly with the directly opposing hepatocytes. Establishment and maintenance of hepatocyte polarity is essential for many functions of hepatocytes and requires carefully orchestrated cooperation between cell adhesion molecules, cell junctions, cytoskeleton, extracellular matrix and intracellular trafficking machinery. The process of hepatocyte polarization requires energy and, if abnormal, may result in severe liver disease. A number of inherited disorders affecting tight junction and intracellular trafficking proteins have been described and demonstrate clinical and pathophysiological features overlapping those of the genetic cholestatic liver diseases caused by defects in canalicular ABC transporters. Thus both structural and functional components contribute to the final hepatocyte polarity phenotype. Many acquired liver diseases target factors that determine hepatocyte polarity, such as junctional proteins. Hepatocyte depolarization frequently occurs but is rarely recognized because hematoxylin-eosin staining does not identify the bile canaliculus. However, the molecular mechanisms underlying these defects are not well understood. Here we aim to provide an update on the key factors determining hepatocyte polarity and how it is affected in inherited and acquired diseases.	[ "MEDI" ]	[ "Canalicular diseases", "Cholestasis", "Hepatocyte biology", "Hepatocyte polarity", "Inherited liver diseases" ]	[ "2721" ]	[ "A defining feature of metazoans is the existence of polarized layers of epithelium which give rise to the three dimensional shapes of body parts and types.", "The formation and maintenance of a polarized epithelium is complex and requires specific cell adhesion molecules, cytoskeletal factors and intracellular trafficking components [1].", "These give rise to apical and basolateral plasma membrane domains which separate interior from external environments and permit directional absorption and secretion of proteins and other solutes.", "Most epithelial cells, such as intestinal and renal tubular cells, are polarized in the plane of the tissue [2].", "In contrast, hepatocytes have a unique polarization arrangement in which each of two adjacent cells contributes an apical plasma membrane that form one or more capillary-like structures, the bile canaliculus (BC), which is the smallest branch of the bile ductal system (Fig. 1) [3].", "The BC is functionally sealed by tight junctions (TJs) and, with its microvilli, constitutes ∼13% of total hepatocyte plasma membrane [4].", "Defects in hepatocyte polarization leads to major pathophysiological consequences.", "Hepatocyte polarity mechanisms may be divided into structural and functional components.", "Structural polarity includes morphologic integrity of TJs and apical plasma membranes with their microvilli, and BC network formation.", "In cultured hepatocytes and cell lines, reversion to planar polarity phenotype and loss of BC has been demonstrated as a result of deletion or inhibition of individual components of the complex polarization machinery [5–7].", "In contrast, functional polarity is predominantly defined by the action of canalicular ATP binding cassette (ABC) transporters.", "The scope is expanding to include acquired diseases although pathologists rarely comment on hepatocyte polarization because BC cannot be visualized by hematoxylin and eosin staining and require imaging of canalicular proteins such as 5’nucleotidase, ABCB1, ABCB11 or others [8].", "In the presence of functional polarity defects canalicular morphology is initially retained but eventually can be damaged as a consequence of intracellular retention of biliary components, particularly bile acids [9].", "Although most basic studies of components and mechanisms of polarity were identified in polarized cell lines, such as MDCK, WIF-B and HepG2; corroborating investigations in mammalian hepatocytes generally support similar mechanisms.", "Cell lines and primary hepatocyte cultures have significant limitations for polarization studies; however, collagen sandwich cultures of mammalian hepatocytes have proven useful because they are non-dividing, do not undergo autophagy, have stable gene expression for about two weeks and, most importantly, sequentially form a canalicular network similar to that seen in vivo (Fig. 2) [6,10].", "Embryologically, hepatoblasts are non-polarized and give rise to hepatocytes on stimulation by Oncostatin M (OSM) and TNF-alpha, and cholangiocytes, which are signaled by NOTCH and TGF-beta [11–13].", "In mice, hepatocytes begin polarization on fetal day 14; however, mature BC do not appear until fetal day 21 [14,15].", "Early canalicular network occurs by day 20 and rapid postnatal network formation occurs within two-three days after birth.", "During development, tight junctional complexes form, and apical and basolateral proteins including transporters become associated with specific plasma membrane domains (Fig. 3) [16].", "These changes are associated with activation of 7-alpha-hydroxylase and synthesis of bile acids which may participate in regulating canalicular network formation similar to effects observed in hepatocyte cultures in which bile acids acting through a cAMP-Epac-MEK-AMPK pathway accelerate canalicular network formation (Fig. 4) [7].", "Acquisition of polarity requires an evolutionarily conserved interconnected network of determinants that have been discovered in Drosophila and which specify future polarization domains in differentiating epithelial cells [17,18].", "Crumbs complex (including Crumbs, Patj, and Pals1) provides apical identity and is linked to Par complex (Par3, Par6, and alpha-PKC), which promotes TJs and apical targeting after phosphorylation of Par3 and Crumbs by alpha-PKC.", "Scribble complex (containing Lg1, Dlg, and Scribble) defines the basolateral domain.", "How these systems work in hepatocytes is unknown.", "The complexity of subsequent processes makes it difficult to identify specific signaling events, which explicitly shape polarization.", "These events, for the most part, involve protein trafficking, which has been extensively studied in canine-derived MDCK cells and to a limited extent in hepatocytes [19].", "In only a few cases have the signaling events involved in canalicular network formation been demonstrated.", "For example, bile canalicular network formation is impaired in HNF-4alpha [20] and LKB1 knockout mice [21,22], and accelerated by STAT3 [23] and OSM [24].", "OSM is an IL-6-related cytokine secreted by the hematopoietic cells in the fetal liver [25] and is able to activate transcription factors STAT3 and HNF-4alpha [23,26] as well as G-protein K-Ras [27].", "OSM promoted cell-cell adhesion and adherens junction formation in cultured embryonic murine hepatic cells and bile canalicular formation in fetal human hepatocytes [28,29].", "It has been shown that OSM functions in protein kinase A pathway by inducing the expression of the cell cycle inhibitor p27KIP that keeps cells in G1 and may couple centrosome-associated signaling to canalicular domain formation [24,30–32].", "Hepatocyte polarization and canalicular network formation require coordinated expression of several key evolutionarily conserved elements each of which consists of many components.", "These elements are extracellular matrix (ECM), adherens and tight junctions, intracellular protein trafficking machinery including recycling endosomes, cytoskeleton and energy production.", "The ECM is a complex macromolecular structural network which forms a scaffold for adhesion and provides a signaling platform by sequestering or releasing cytokines, anchoring processing enzymes and activating hepatocyte surface integrins which trigger intracellular signaling [33,34].", "Chemical and physical properties of the ECM largely determine hepatocyte differentiation.", "Unlike other epithelia, hepatocytes are not attached to a tough basal lamina.", "Instead they are surrounded by a low density ECM, that contains hepatocyte secreted components.", "Mature hepatocytes are embedded in the ECM that lacks laminin which, however, is present during hepatocyte differentiation in liver development and regeneration, suggesting a crucial role for laminin during polarization [35].", "The absence of a basal lamina allows the exchange of macromolecules between the sinusoid and Disse space through endothelial cell fenestra which exclude cells.", "In cirrhosis, excess deposition of ECM into the space of Disse results in distortion of liver architecture and abnormal hepatocyte function [36].", "Collagen sandwich cultures demonstrate in vitro that the trapping of hepatocyte secreted ECM proteins, such as collagen IV, laminin and fibronectin, and possibly growth factors on both non-opposing surfaces is required for the development of hepatocyte polarity phenotype [6,37].", "Experiments in MDCK and WIF-B cells demonstrated that Par1b, a serine/threonine kinase activated by LKB1 (see below), is a crucial determinant of hepatocyte-like polarity phenotype [38].", "MDCK cells with overexpressed Par1b demonstrated hepatocyte-like polarity with interrupted staining of collagen IV and laminin at the basal surface and appearance of collagen IV and laminin at the apical surface [39].", "Interestingly, seeding Par1b overexpressing MDCK cells on high collagen IV concentration ECM reverted the phenotype back to columnar [38].", "In parallel, reduced expression of Par1b in WIF-B cells disrupted hepatocyte-like polarity.", "Studies in HepG2 cells proposed RhoA GTPase as the effector of ECM signaling that determines the position of the lumen [40,41].", "Work in MDCK cells and hepatocyte cell lines established that integrins, cadherins and junctional adhesion molecule A (JAMA) act as transducers that transfer the signal from the ECM to RhoA GEFs and GAPs in order to induce the cytoskeletal changes required to establish a biliary lumen [34,42–44].", "Hepatocytes connect through intercellular tight, anchoring (adherens and desmosomes) and gap junctions [45–47].", "The tight junctional complex consists of several claudins, occludins, TJP (also called ZO) proteins [48], and prevents paracellular flux of molecules.", "Disruption of the TJs in transgenic mice [49], collagen sandwich cultured hepatocytes and tissue sections of various hepatobiliary diseases results in depolarization and eventual hepatocellular injury.", "In non-polarized hepatocytes and other epithelial cells, TJ proteins and some apical transporters relocate to intracellular sites including the microtubular organizing center where they colocalize with Rab11a and Myosin 5b [5].", "Identification of pathogenic mutations causing disruption of TJP2 and Claudin1 in patients with severe liver disease confirms the importance of TJs in maintaining hepatocyte structure and function [50,51].", "Apical protein trafficking in hepatocytes involves at least two distinct pathways (Fig. 3).", "Hepatocytes target polytopic membrane proteins, such as ATP transporters, from the Trans Golgi Network (TGN) either directly to the bile canalicular domain or to the Rab11a recycling endosome (RE) pool from which they cycle to the canalicular membrane [52,53].", "In contrast, hepatocytes target single membrane spanning and GPI anchored bile canalicular membrane proteins to the basolateral plasma membrane from which they transcytose in endosomes through the cell to the canalicular membrane [16,53].", "A specific pathway that targets copper (Cu) transporter ATP7B to the canalicular membrane via lysosome exocytosis has recently been reported [54].", "It was found that in response to increasing intracellular concentrations of Cu, ATP7B is trafficked to a subset of lysosomes where Cu gets stored until the threshold concentration is reached.", "Once the threshold level is reached ATP7B is delivered to the canalicular portion of PM and Cu gets released.", "How cargo for the transport pathways is sorted at the TGN or elsewhere, and whether these pathways intersect in recycling or other endosomes has not been fully established in hepatocytes (Figs. 3 and 5).", "Several studies in HepG2 and WIF-B cell lines identified candidate lipid and protein determinants that contribute to sorting of canalicular proteins into distinct pathways [55–57].", "The presence of different mechanisms may relate to the extensive secretion of proteins from hepatocytes into the circulation in contrast to the selective secretion of bile acids, metabolites, etc. into the bile [1].", "Precise regulation of membrane sorting and of the endosomal recycling system is required to sustain hepatocyte polarity and specificity of function in the various plasma membrane domains.", "Approximately half of the proteins of a typical plasma membrane are endocytosed per hour, whilst only ∼8% of the hepatocyte basolateral plasma membrane is internalized per hour [58].", "The difference in trafficking of different types of canalicular proteins and the importance of the Rab5 dependent endosomal pool to trafficking of GPI anchored proteins, such as DPPIV and proteins trafficked via RE such as ABCB11, was demonstrated in an in vivo knockdown of Rab5 homologues.", "In mice with Rab5 knockdown DPPIV remained at the basolateral membrane, whilst ABCB11 relocated to REs [59].", "In contrast, the localization of ABCC2 was unaffected suggesting an independent trafficking mechanism for this protein.", "Largely based on studies in cell lines and schematized in Figs. 3 and 5, the TGN is the site for release and sorting of proteins destined for the apical plasma membrane.", "The involvement of specific endosomal pools has not been characterized in hepatocytes but has been revealed in pulse chase experiments in liver and in cell lines.", "RE constitutes a large reservoir for ABC transporters which is at least six times greater than the content of those proteins in the canalicular membrane [52].", "The pool is mobilized by bile acids which circulate in the enterohepatic circuit, and by postprandially secreted peptide hormones which increase cAMP production in hepatocytes to cope with the increased demand for bile acid secretion.", "Taurocholate and cAMP activate distinct signaling pathways to mobilize ABC transporters to the canalicular domain [60].", "Regulation of these two responses differs.", "Increase in cAMP concentration results in PKA-mediated stimulation of PI3K but not taurocholate stimulated incorporation of ABCB11 into the canalicular membrane (Fig. 4) [61].", "The RE component of the secretory pathway plays a critical role in apical as well as basolateral localization of various proteins [62].", "This cargo-bearing structure contains a complex including one or more Rab protein GTPases and an actin-associated molecular motor, Myosin 5b, as well as adaptor proteins Rab11a, Fip1 and Fip2 as shown by the work in the MDCK cells [63].", "Inhibition of Rab11a or Myosin 5b prevented polarization in WIF-B cells and primary hepatocytes and, when introduced into polarized cells, prompted depolarization and internalization of apical proteins [5].", "These observations indicated that the RE is a major determinant of polarization not due to its ABC transporter cargo.", "Some of the endosome components which provide cues for apical membrane polarization have been previously characterized [64–68].", "Mutations in MYO5B encoding Myosin 5b, which acts as a molecular motor not only for Rab11a, but also Rab11b, Rab25, and Rab8, cause MicroVillus Inclusion Disease (MVID) in which malabsorption results from the absence of the intestinal brush border [69].", "Recent studies reveal that many patients with MYO5B mutations also manifest cholestasis and progressive liver disease [70].", "Mouse Rab8 conditional knockouts mimic MVID; furthermore, patients with MVID without Myosin 5b defects were found to have mutations affecting Syntaxin 3, an apical membrane SNARE (family of membrane proteins that ensure fusion between opposing membranes), suggesting that Myosin 5b, Rab8, and Syntaxin 3 may be involved in the same trafficking pathway [71,72].", "As the liver disease in mice with Rab8 deficiency or MVID patients with Syntaxin 3 defects has not been described thus far, it is possible that Rab8 and Syntaxin 3 role in this pathway is not as important in hepatocytes as it is in the intestine.", "Discovery of loss of function mutations in genes encoding RE-associated proteins such as Myosin 5b in MVID, VPS33B, and VIPAR in Arthrogryposis, Renal dysfunction and cholestasis syndrome (ARC) [73] supports the importance of the RE in establishment and maintenance of hepatocyte polarity (Fig. 6).", "Proper endosomal trafficking and recycling of proteins to all plasma membrane domains requires an intact actin and microtubular cytoskeletal system [74,75].", "In particular, dynamic microtubules mediate trafficking of secreted and canalicular proteins [76].", "Newly synthesized ABCB11, the canalicular bile acid transporter, and other canalicular ABC transporters traffic from the TGN along microtubules [77].", "However, microtubules do not attach to the canalicular membrane and their cargo endosomes are transferred to the pericanalicular actin system (Fig. 5).", "The complete mechanism for cargo transfer is not known; however, microtubules become associated with actin through a pericanalicular actin-binding complex containing CLIP170, IQGap, APC, Hax-1, and cortactin proteins [78].", "Live cell imaging studies reveal that selective plasma membrane localization of transporter proteins is predominantly due to the localization of specific docking proteins.", "In polarized WIF-B cells, ABCB11 and ABCB1 were shown to traffic along microtubules throughout the cell but only attach to sites on the canalicular membrane [77].", "The docking site has been proposed to be Syntaxin 3 that facilitates fusion of protein sorting vesicles with the inner leaflet of the canalicular membrane [79,80].", "Radixin also participates in this process and links some cargo molecules such as ABCC2, to the pericanalicular actin system [81].", "Radixin knockout mice manifest impaired ABCC2 localization to the canalicular domain which becomes progressively devoid of microvilli resulting in hepatocyte injury [82].", "Assembly and disassembly of short actin filaments involved in endosomal transport are under the control of formin [83].", "Work in HepG2 cell line demonstrated the requirement for INF2, CDC42 and transmembrane protein MAL2, for trafficking of canalicular membrane proteins in the transcytotic pathway.", "Hepatocyte polarization is energy-dependent but the mechanism is unclear.", "AMPK, a serine threonine kinase containing a catalytic alpha subunit and a regulatory beta and gamma subunits, controls energy metabolism within cells by sensing the cellular AMP to ATP ratio [84].", "Activation of AMPK by phosphorylation of the alpha subunit Thr172 decreases energy consumption and increases energy production during cellular stress such as hypoxia, glucose deprivation and ischaemia, and has an important role in hepatic metabolism through effects on glucose, lipid and protein homeostasis and mitochondrial biogenesis (Fig. 4) [85].", "Long-term effects involve regulation of the glycolytic and lipogenic pathways [86].", "In collagen sandwich hepatocyte cultures, AMPK activation by metformin, cAMP activators, 2 deoxyglucose, AICAR or taurocholate increased canalicular network formation [6].", "Phosphorylation of AMPK Thr172 is performed by LKB1, an upstream serine threonine kinase which is activated by various growth factors [87].", "AMPK and LKB1 regulate polarity in Drosophila, polarized cell lines, neurons and hepatocytes [86].", "In collagen sandwich cultured hepatocytes, the stress of isolation resulted in depolarization, ATP depletion and mitochondrial fragmentation [7,88].", "Mitochondrial fusion occurred within two days associated with increased ATP synthesis from oxidative phosphorylation and canalicular network formation.", "Subsequent AMPK activation upregulated glucose uptake, glycolysis and a further increase in ATP.", "These in vitro studies reveal that, after stress, hepatocytes preferentially restore polarity even at low ATP levels, suggesting that polarity is a prime requirement for cellular activity [7].", "Mitochondrial fission and fusion are important in polarity maintenance.", "In other studies, LKB1 conditional liver knockout mice reveal polarity defects, cholestasis and liver injury [21,22].", "Whether LKB1 and/or AMPK enhance polarization through direct effects on mitochondrial bioenergetics and/or specific phosphorylation of downstream polarity components, such as TJs, RE or cytoskeleton, is unknown.", "Typically, diseased liver loses its polarized structure.", "Polarity defects are seen in single-gene rare inherited disorders (Table 1 and Fig. 6) as well as common infections such as hepatitis C, in which damage to hepatocyte polarization can be restored by treatment [89], and multifactorial diseases like cancer, as genes involved in polarity have been implicated in its pathogenesis (Table 2) [90].", "Defects in several apical junction proteins have been associated with inherited human liver diseases (Fig. 6).", "CLDN1 and TJP2 encode integral TJ proteins; mutations in CLDN1 were found in neonatal ichthyosis-sclerosing cholangitis syndrome (NISCH) and TJP2 mutations cause familial hypercholanemia (FHC) and a newly described subtype of Progressive Familial Intrahepatic Cholestasis syndrome (PFIC-4) [50,51,91].", "Patients with NISCH are born with generalized skin, hair and nail abnormalities.", "The liver disease is said to affect both cholangiocytes and hepatocytes and the biopsy appearance varies even in patients with the same mutation, and can demonstrate typical sclerosing cholangitis features or non-specific hepatocellular and canalicular cholestasis with normal bile ducts [92].", "The variability of phenotype expression suggests that variants in other genes may influence liver disease severity.", "The likely mechanism for liver injury is increased paracellular permeability due to abnormal TJ formation, resulting in bile regurgitation [93].", "FHC patients have elevated serum bile acid concentrations, pruritus and fat malabsorption.", "FHC can be caused by defects in several proteins including TJP2 and also enzymes involved in bile acid biogenesis.", "FHC patients have normal liver enzymes apart from increased serum alkaline phosphatase activity, and variable findings on liver biopsy that include canalicular cholestasis and minimally active chronic hepatitis.", "Symptoms usually respond to treatment with ursodeoxycholic acid.", "Some patients have a combination of homozygous missense V48A mutations in TJP2 with a heterozygous BAAT (Bile acid-CoA:amino acid N-acyltransferase) M76V mutation (which in the homozygous state can also cause FHC).", "Frame shift deletions and duplications, and splice site TJP2 mutations identified in all patients with PFIC-4 so far are predicted to result in the absence of the protein product [51].", "The described patients had normal gamma glutamyl transpeptidase (gamma-GT) cholestasis and progressive liver disease course, with most children requiring lifesaving early liver transplantation.", "Two patients with severe TJP2 mutations were reported to have developed hepatocellular carcinoma at 24 and 26 months of age [94].", "Defects in four intracellular trafficking machinery proteins (Myosin 5b, Syntaxin 3, VPS33B, and VIPAR) cause related inherited disorders with polarity defects [69,70,72,73].", "Abnormalities in Myosin 5b and recently described defects in Syntaxin 3 cause MVID which typically present in infants with intractable diarrhea and characteristic features include hypoplastic villous atrophy with intracytoplasmic inclusions of brush border microvilli detected by electron microscopy.", "Many patients with MVID develop cholestatic liver disease similar to that in PFIC-1 and -2 [70].", "Cholestasis becomes particularly prominent when parenteral nutrition is introduced.", "Immunostaining of ABCB11 and Rab11a in liver biopsies from patients with Myosin 5b defects demonstrated abnormal distribution of these proteins, suggesting that mislocalization of ABCB11 due to abnormal trafficking could be responsible for the liver disease, at least in this subgroup of MVID [70].", "Liver disease has not yet been described in patients with Syntaxin 3 defects.", "As mentioned above Myosin 5b is an actin-based molecular motor whose mechanism of action is closely linked with the RE-associated Rab family proteins, which regulate polarized epithelial protein trafficking.", "Syntaxin 3 is an apical membrane SNARE that may act as a hepatocyte docking site for the vesicles delivering canalicular membrane transporter proteins from the REs.", "ARC is an autosomal recessive multisystem disorder caused by mutations in VPS33B and VIPAS39 encoding VPS33B (vacuolar protein sorting 33 homologue B) and VIPAR (VPS33B interacting protein, apical-basolateral polarity regulator).", "Characteristic presentation of ARC includes neonatal cholestatic jaundice, renal tubular acidosis, arthrogryposis and severe failure to thrive.", "Patients have normal gamma-GT but significantly increased alkaline phosphatase activity, consistent with the canalicular damage [95].", "Patients’ liver biopsies show evidence of giant cell hepatitis, bile duct hypoplasia and accumulation of intrahepatocyte lipofuscin granules.", "Most patients described so far carry severe nonsense, splice site or frame shift mutations resulting in the absence of protein product, and fail to survive past the first year of life.", "However, affected children with an attenuated ARC phenotype have been identified.", "A splice site c.1225+5 G>C mutation in VPS33B results in production of an abnormal protein transcript that retains some function and appears to confer the mild phenotype [96].", "VPS33B and VIPAR form a stable complex and interact with Rab11a suggesting a role for the VPS33B-VIPAR complex in RE trafficking pathway.", "Moreover, localization of bile salt export pump ABCB11, which is trafficked to canalicular membrane via Rab11a positive RE was predominantly cytoplasmic in the liver of ARC patients [73].", "Localization of other canalicular membrane proteins in ARC patients’ liver was found to be variable.", "For example CECAM5, which is a GPI anchored protein was predominantly localized to the basolateral membrane, while the location of MRP2 in hepatocyte canaliculi was unchanged.", "Furthermore structural and functional abnormalities in the apical junction complex (AJC) was found in mIMCD3 cells with VPS33B and VIPAR knockdown, although the mechanism behind this defect is not clear and is likely to be caused by transcriptional downregulation of some AJC proteins such as E-Cadherin and Caludin-1 [73].", "Defects in hepatocyte transporter proteins are the most common collective cause of inherited forms of cholestasis.", "A scope of severity has been demonstrated with more severe protein defects manifesting in infants, with milder abnormalities conferring susceptibility to drug induced and pregnancy induced cholestasis.", "Progressive Familial Intrahepatic Cholestasis types I and II (PFIC-1 and PFIC-2) are characterized by persistent cholestasis with normal gamma-GT and progressive liver damage that often requires liver transplantation in childhood.", "Reduced concentrations of primary BA are found in bile [97,98].", "A scope of severity exists in PFIC and a range of mutations resulting in the absence or mistrafficking of the proteins has been described.", "The majority of patients with this phenotype have mutations in ATP8B1 (PFIC-1) and ABCB11 (PFIC-2) although patients with a similar phenotype and mutations in TJP2 (PFIC-4) were described recently (see above) [51,99,100].", "ABCB11, also known as bile salt export pump (BSEP) is responsible for the transport of salts of primary bile acids across the canalicular membrane [101].", "Patients with ABCB11 mutations are at increased risk of hepatobiliary malignancy [102].", "ATP8B1 is a member of the type 4 subfamily of P-type ATPases and is present in the apical membrane of many epithelial cells, including hepatocytes and enterocytes.", "It was found to translocate aminophospholipids such as phosphatidylserine (PS), from the outer to the inner leaflet of the plasma membrane bilayer [103–105].", "The extrahepatic manifestations of ATP8B1 deficiency include diarrhea, recurrent pancreatitis, sensorineural deafness, delay in growth and puberty and elevated sweat chloride concentration [106].", "Patients with milder missense mutations in ABCB11 or ATP8B1 that may confer partial instability to the protein display a benign recurrent intrahepatic cholestasis (BRIC) phenotype, in which cholestasis can completely resolve between relapses [107].", "Mutations in ABCB4 encoding ABCB4 are associated with PFIC-3 [108].", "ABCB4, also called multidrug resistance protein 3 (MDR3) is a P-glycoprotein that translocates phospholipids from the internal to the external leaflet of the canalicular membrane [109].", "Unlike PFIC-1, -2, and -4, PFIC-3 patients have high serum gamma-GT values, ductular reaction and early fibrosis on liver biopsy.", "ABCB4 deficiency cause abnormal phosphatidylcholine secretion into bile leading to hepatocyte and cholangiocyte damage due to absent emulsification of bile acids [110].", "A number of cholestatic disorders have been associated with partial ABCB4 deficiency, including neonatal hepatitis and biliary cirrhosis [111,112].", "Mutations in ABCC2 cause Dubin-Johnson syndrome.", "Patients with this condition have recurrent episodes of jaundice without plasma bile acid accumulation.", "The liver biopsy demonstrates intrahepatocyte deposits of dark pigment often without any obvious hepatobiliary injury [113].", "Treatment is recommended only for severe neonatal cases.", "Upregulation of other transporters such as ABCC3 in Dubin-Johnson syndrome patients may explain a mild phenotype [114].", "ABCC2 encodes ABCC2 or MRP2, which is a member of the multidrug resistance protein subfamily that exports anionic glutathione and glucuronate conjugates (including bilirubin) from hepatocytes into canaliculi [115].", "ABCC2 is expressed on the apical membranes of many epithelial cells including hepatocytes, proximal renal tubules, gallbladder, small intestine, bronchi and placenta [116].", "Rotor syndrome is caused by simultaneous recessive mutations in SLCO1B1 and SLCO1B3 genes and phenotypically is similar to Dubin-Johnson syndrome [117].", "It manifests with mild jaundice that can be detected in the neonatal period or childhood.", "In contrast to Dubin-Johnson syndrome there are no intrahepatocyte pigment deposits present and delayed plasma clearance of unconjugated bromsulphthalein can be found [118].", "SLCO1B1 and SLCO1B3 encode organic anion transporting polypeptides OATP1B1 and OATP1B3, which localize to the sinusoidal membrane of hepatocytes and mediate sodium-independent cellular uptake of multiple compounds, including bilirubin glucuronide, bile acids, steroid and thyroid hormones, as well as numerous drugs [119].", "MicroRNAs (miRNAs), are small 18–24 nucleotide noncoding RNAs that regulate gene expression by binding to mRNAs and interfering with translation [120,121].", "Typically, miRNAs downregulate expression of their target genes by binding to the 3′ untranslated region (UTR).", "However miRNAs may also upregulate target gene expression when interacting in a non-3′ UTR-dependent fashion.", "More than 1000 mammalian miRNAs are known [122].", "The miRNA-target gene interaction is complex and probably exist as part of carefully regulated transcription factor networks [123].", "More than one miRNA can affect expression of the same gene and each miRNA can influence dozens of gene transcripts.", "The exact roles of miRNAs in the establishment and maintenance of hepatocyte polarity are not known, but evidence is emerging that miRNA influence expression of adherens and TJ proteins and cytoskeleton remodelling.", "miR-155 was found to be upregulated after TGF-beta induction in an epithelial cell line NMuMG which resulted in loss of polarity [124].", "Moreover numerous studies identified specific alterations in miRNAs signatures in different liver diseases.", "ZEB1 and ZEB2, the transcriptional repressors of E-cadherin are influenced by members of the miRNA-200 family and lead to epithelial to mesenchymal transition [125,126].", "Upregulation of miRNAs miR-200a and miR-200b in liver fibrosis is consistent with their influence in this disease mechanism [127].", "A role in regulating the PTEN – TGF-beta axis was ascribed to a network of miRNAs (including miR-106a, miR-106b, miR-18a, miR-18b, and others) that results in EMT of hepatocytes thus suggesting potential for this miRNA network to promote neoplastic transformation of hepatocytes [128].", "Cancer cells become depolarized and there is evidence for the role of disturbed polarity pathways in oncogenesis.", "Beta-catenin is a constituent protein of adherens junctions and is critical for establishment and maintenance of epithelial polarity.", "It can dissociate from the junctions and is translocated to the nucleus where it may transmit the contact inhibition signal.", "Mutations that lead to accumulation of intracytoplasmic and nuclear Beta-catenin were identified in more than half of patients with sporadic hepatoblastoma (HB), a malignant childhood liver tumor [129].", "Such Beta-catenin translocation is likely to upregulate the Wnt signaling pathway.", "Furthermore mutations in CTNNB1, which encodes Beta-catenin are associated with increased Wnt signaling were identified in ∼ 20% of patients with hepatocellular carcinoma (HCC), including a patient with primary PFIC-2 diagnosis [130].", "Inactivating mutations in APC cause familial adenomatous polyposis and approximately 10% of patients with HB have germline APC mutations [131,132].", "APC is a tumor suppressor that downregulates the Wnt signaling pathway by decreasing the amount of translocated Beta-catenin.", "APC forms a complex with glycogen synthase kinase 3 beta (GSK3B) and AXIN1 that binds and phosphorylates cytoplasmic Beta-catenin, facilitating its ubiquitination and proteasomal degradation.", "Understanding signaling events underlying HCC lead to development of novel treatments.", "A complete resolution of tumors in an in vivo mouse model of HCC with Beta-catenin mutations was recently demonstrated after treatment with a “locked nucleic acid” antisense approach to inactivation of Beta-catenin driven Wnt signaling [133].", "In another project, MET/ΔN90-β-catenin mutant model of HCC was employed to test the effect of in vivo nanoparticle mediated siRNA inhibition of integrin subunits, which slowed down progression of HCC and was proposed as a possible novel treatment for this tumor [134].", "Germline mutations in PTEN and more recently PIK3CA and AKT1 were found in patients with “Cowden” or “multiple hamartoma” syndrome, an inherited disorder that leads to multiple hamartomas including biliary hamartomas, and predisposes patients to several types of cancer.", "PIK3CA encodes the catalytic subunit of PI3K, which adds a phosphate to phosphatidylinositol-4,5-biphosphate (PIP2) to form phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cellular membrane.", "PTEN dephosphorylates PIP3, which is required for recruitment of AKT1 to the cell membrane where it is phosphorylated.", "The PIP3 pathway is important for apical membrane formation.", "PI3KCA somatic mutations are also occasionally seen in HCCs and other cancers [135].", "Hepatitis B and C both promote hepatocarcinogenesis, which is associated with E-cadherin downregulation and Beta-catenin activation.", "Hepatitis C virus (HCV) enters hepatocytes using the TJ proteins claudin1 and occludin as co-receptors and a tetraspanin CD81 [136–139].", "Hepatitis B virus (HBV) entry into hepatocytes is dependent upon hepatocyte polarization and it is suggested that the putative viral cell receptor is located in the basolateral membrane, although its identity is not yet known [140].", "HBV X protein is thought to activate the Wnt signaling pathway by binding to APC [141].", "The work in hepatoma cell lines demonstrated that HCV infection upregulates VEGF which disrupts tight junction integrity promoting viral transmission [89].", "HCV core protein expression was associated with disrupted apical polarity in MDCK cells, which may be a result of deactivation of PI phosphatase SHIP2 which converts PtdIns(3,4,5)P3 to PtdIns(3,4)P2 [142].", "Furthermore, HCV-induced liver inflammation is associated with upregulation of Wnt signaling and increased miR-155 expression [143].", "Expression of another HCV protein, HCV-NS5A, in primary hepatic precursors and immortalized hepatocyte cell lines led to epithelial to mesenchymal transition (EMT) through activation of Twist2, which regulates EMT [144].", "HCV entry into via hepatocyte TJs was targeted by the novel approach to therapy.", "Inhibition of HCV entry using anti-CLDN1 antibody in human liver-chimeric mice successfully treated chronic HCV infection and provides a novel therapeutic approach to this devastating disease [145].", "Genome wide association studies are helping to unravel pathways involved in the pathogenesis of common human diseases.", "Primary biliary cirrhosis is the most common autoimmune liver disease primarily affecting women over the age of 40.", "Recent studies found significant association between defects in several molecular pathways and primary biliary cirrhosis, including known polarity pathways such as mTOR, PI3K, MAPK, Hh, and Wnt signaling, and adherens junctions [146].", "In cirrhosis and inflammatory hepatic diseases, depolarization of hepatocytes occurs; however its frequency and contribution to pathology have not been studied in depth.", "A recent study has shown that induction in oxidative stress by administration of carbon tetrachloride disrupted Par-3 – alpha-PKC complex formation resulting in disassembly of TJ and depolarization of hepatocytes.", "These changes led to cholestasis and cirrhosis [147].", "The mobilization of fibrogenic cells has been proposed to involve conversion of polarized hepatocytes and biliary epithelial cells into mesenchymal cells with concomitant loss of epithelial polarization and acquisition of a mobile phenotype, but this is controversial [148,149].", "Improved understanding of hepatocyte polarization benefited from the work in cell lines, model organisms and discoveries of inherited mutations in patients with cholestatic diseases.", "The unique role of the canalicular membrane in bile acid secretion and the deleterious intracellular effects of their retention prompt the hypothesis that links polarity, bile acid retention, mitochondrial damage, energy metabolism and cholestasis.", "Hepatocyte polarization and canalicular membrane formation are likely to be dependent upon stimulation by bile acids and LKB1 phosphorylation of AMPK and Par1b, as well as activation of STAT3 and TNFα transcription factors by cytokine OSM.", "The unique importance of ECM composition and localization in hepatocyte differentiation has been further defined by research efforts in the liver regeneration field.", "Hepatocytes have specific trafficking pathways for GPI anchored and single transmembrane domain canalicular membrane proteins that utilise the transcytotic route while polytopic ABC transporters traffic from TGN, either via RE or directly to the canalicular membrane.", "Cholestasis of various etiologies eventually results in inhibition of the direct and transcytotic protein trafficking pathways resulting in bile acid retention within hepatocytes and damage to mitochondria, Golgi and other organelles [150–152].", "Interdependence of polarization and mitochondrial damage may well underlie cholestasis associated with viruses, drugs, shock and other factors.", "Identification of the molecular defects responsible for pathogenesis of rare and common diseases affecting liver polarity provides desirable targets for drug design, and although there are no known therapeutic agents that can restore hepatocyte polarity, the research into drug development has already benefited from the understanding of the involvement of polarity factors in disease.", "Researchers have successfully tested an antisense oligonucleotide approach to treating HCC in an in vivo model with Beta-catenin mutations.", "This treatment can potentially benefit more than 20% of patients with this tumor that is driven by Wnt signaling activation.", "Furthermore blocking HCV entry via claudin1 with a monoclonal antibody approach eliminated chronic HCV infection, which is one of the most common causes of liver cancer.", "Better understanding of the miRNA role in liver polarity and disease will provide new RNA based approaches to treatment.", "For these reasons a better understanding of hepatocyte polarity and the increasing arsenal of potential therapeutic approaches give optimism for future treatment development in liver diseases.", "P.G. is a Wellcome Trust Senior Research Fellow in Clinical Sciences (WT095662MA); P.G. is supported by the ERC Starter Grant 337057 CLOC.", "The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.", "Both PG and IMA contributed to design, writing and revising the manuscript." ]	[]
A multi-level spatial analysis of clinical malaria and subclinical Plasmodium infections in Pailin Province, Cambodia	Background The malaria burden is decreasing throughout the Greater Mekong Subregion, however transmission persists in some areas. Human movement, subclinical infections and complicated transmission patterns contribute to the persistence of malaria. This research describes the micro-geographical epidemiology of both clinical malaria and subclinical Plasmodium infections in three villages in Western Cambodia. Methods Three villages in Western Cambodia were selected for the study based on high reported Plasmodium falciparum incidence. A census was conducted at the beginning of the study, including demographic information and travel history. The total population was 1766. Cross-sectional surveys were conducted every three months from June 2013 to June 2014. Plasmodium infections were detected using an ultra-sensitive, high-volume, quantitative polymerase chain reaction (uPCR) technique. Clinical episodes were recorded by village health workers. The geographic coordinates (latitude and longitude) were collected for all houses and all participants were linked to their respective houses using a demographic surveillance system. Written informed consent was obtained from all participants. Results Most clinical episodes and subclinical infections occurred within a single study village. Clinical Plasmodium vivax episodes clustered spatially in each village but only lasted for a month. In one study village subclinical infections clustered in geographic proximity to clusters of clinical episodes. The largest risk factor for clinical P. falciparum episodes was living in a house where another clinical P. falciparum episode occurred (model adjusted odds ratio (AOR): 6.9; CI: 2.3–19. 8). Subclinical infections of both P. vivax and P. falciparum were associated with clinical episodes of the same species (AOR: 5.8; CI: 1.5–19.7 for P. falciparum and AOR: 14.6; CI: 8.6–25.2 for P. vivax) and self-reported overnight visits to forested areas (AOR = 3.8; CI: 1.8–7. 7 for P. falciparum and AOR = 2.9; CI: 1.7–4.8 for P. vivax). Discussion Spatial clustering within the villages was transient, making the prediction of spatial clusters difficult. Interventions that are dependent on predicting spatial clusters (such as reactive case detection) would only have detected a small proportion of cases unless the entire village was screened within a limited time frame and with a highly sensitive diagnostic test. Subclinical infections may be acquired outside of the village (particularly in forested areas) and may play an important role in transmission.	[ "MULT" ]	[ "Geography", "Infectious disease", "Information science", "Public health" ]	[ "1000" ]	[ "In much of the Greater Mekong Subregion (GMS) malaria case numbers have been decreasing and nations are moving from malaria control to elimination.", "Village-based treatment of malaria is central to elimination strategies [1], however, in many parts of the GMS there is no functioning village health worker network, especially in areas near international borders [2].", "Interventions such as insecticide treated bed nets (LLITNs) are less effective in the GMS than in Africa, since most of the important Anopheles vectors feed outdoors and during dusk and dawn when few people are sleeping [3].", "Within the remaining malaria endemic areas in the GMS, malaria risk is heterogeneously distributed and the complete interruption of malaria transmission has thus far been elusive.", "In many parts of the GMS malaria persists in clusters along international borders as a result of a complex combination of political, environmental, and socio-economic factors that are not completely understood.", "Migration of both mosquitoes and humans can lead to the reintroduction of parasites to regions that might otherwise be malaria free [4].", "A high prevalence of subclinical and submicroscopic Plasmodium infections, even in low transmission areas such as the GMS, undermines elimination efforts as these infections contribute to transmission and are unlikely to be detected and treated through conventional control activities [5, 6, 7].", "Current elimination efforts are focused on Plasmodium falciparum.", "Plasmodium vivax presents particular problems to control and elimination because liver hypnozoites cause periodic relapses.", "The situation is compounded by safety concerns regarding the two-week treatment with primaquine required for the radical cure of hypnozoites [8, 9, 10].", "Micro-geographical analyses can inform how, when, and where clustering occurs and how it relates to transmission patterns [11].", "Clustering can occur at multiple levels: within hosts (individuals with repeated infections over time) and within populations (groups of hosts that share exposure sites such as houses, villages, or work environments).", "Major control and elimination strategies (such as targeted mass treatment with or without screening, reactive case detection, and focused screening and treatment) rely on being able to identify space-time clusters of malaria.", "This clustering can give information about transmission patterns.", "For example, if transmission occurs within villages then cases tend to cluster in particular groups of houses [12].", "Conversely, if transmission occurs outside of the village then the disease tends to cluster in individuals who are exposed to transmission sites [13].", "An understanding of local clustering patterns can be used to help design or select appropriate interventions.", "The main goals of this research were to investigate potential associations between 1) subclinical Plasmodium infections and clinical malaria episodes, 2) travelling to or spending the night in the forest and clinical episodes or subclinical Plasmodium infections, and 3) spatial and spatio-temporal clustering of clinical episodes or subclinical Plasmodium infections within villages in Pailin Province, Cambodia.", "The study area consisted of three villages in Pailin Province, Cambodia, near the international border with Thailand (Fig. 1).", "In 2004 Pailin reported some of the highest annual parasite incidences (API) in Cambodia, with API above 160/per 1000 people per year for both P. falciparum and P. vivax.", "By 2013 this incidence had fallen to around 10/1000 for P. falciparum and 20/1000 for P. vivax.", "The areas of highest transmission were no longer in the west of Cambodia but were now in the north and north east [14].", "The dramatic decline of malaria in western Cambodia may be attributable at least in part to the increased malaria control efforts following the detection of artemisinin resistant P. falciparum [15] with an important scale-up of the village malaria worker programme and improved coverage of insecticide treated bednets [16, 17].", "Deforestation may also play a role [13].", "Despite the fall in clinical incidence, a reservoir of subclinical malaria remains and is an important target for accelerated elimination strategies [5, 6, 7, 18].", "The study villages were selected based on P. falciparum incidence in 2012 and are adjacent to each other, covering an area approximately ten km long and four km wide.", "Detailed demographic and geographic surveys were conducted beginning in June 2013 and ending in December of 2014.", "During the study period, first-line treatment for P. falciparum and for P. vivax was dihydroartemisinin-piperaquine.", "Primaquine was not used for P. vivax due to a lack of availability of G6PD testing.", "In addition radical cure of P. vivax with primaquine was not recommended in the national treatment guidelines at the time of the study.", "Individuals who agreed to participate in the study had their age and sex recorded and were given an identification code.", "The geographic coordinates (latitude and longitude) for all houses in the study villages were recorded and each house was assigned an identification code.", "All participating individuals were linked through a demographic surveillance system to their respective houses.", "All participants provided written informed consent.", "Mass blood screenings were conducted in the villages every three months from June 2013 to June 2014, (M0–M12 [7]).", "During the cross-sectional surveys a mobile clinic was established in a central location within each village, usually in the village school.", "Village volunteers were trained to assist in the surveys and they went house-to-house to inform and invite all residents to participate.", "Surveys took place over several days and were usually followed up one week later in order to survey participants who were absent during the survey.", "Within each survey, house members attended as groups and passed through a series of desks where they provided informed consent, they were registered, weighed, measured, had their tympanic temperature taken, completed a short survey form, provided a venous blood sample and received a consultation with a nurse if necessary.", "During the screenings, 3 mL of blood was collected from each adult participant (500 uL from children six months to five years old), any malaria symptoms (e.g. fever, chills) were noted, and the blood was analyzed using an ultra-sensitive quantitative polymerase chain reaction method (uPCR), with a lower level of detection of 22 parasites per mL (described in detail in [5]).", "Patients without clinical symptoms who had Plasmodium infection detected by uPCR are herein referred to as having “subclinical” infections.", "Anyone with a temperature of 37.5 °C or above had an rapid diagnostic test (RDT) performed by the village malaria worker and, if positive for malaria, was treated according to national guidelines.", "Each village had a village malaria worker (VMW), which is the primary care provider for patients with febrile illness.", "The VMW provided diagnosis and treatment for symptomatic malaria cases.", "Patients who attended the VMW were recorded in a log book and matched to their study identification number.", "Patients were tested for malaria using a RDT (CareStart™ Malaria Pf/Pv Combo test) and treated as per national guidelines if found infected.", "Patient outcomes were recorded and for the present analysis were considered “clinical” episodes of P. falciparum or P. vivax malaria beginning in June 2013 and running through December 2014 (M0–M18).", "Both clinical episodes and subclinical infections were then linked to houses of residence for analysis.", "Brief travel history and bednet usage interviews were also conducted during each of the three-monthly cross-sectional surveys.", "Participants were asked if they had travelled to another location or spent the night in the forest within the last two weeks.", "The original census population was treated as a cohort for this analysis.", "Some individuals moved into the villages during the study period but were not included in this analysis as their data do not account for their previous exposures and environments.", "Participants who moved out of the cohort and villages during the study period [7] were included in this analysis.", "The outcome variables for these analyses were clinical episodes (of P. falciparum or P. vivax, separately) and subclinical infections (also of P. falciparum or P. vivax separately).", "Both clinical malaria episodes and subclinical infections detected by uPCR during surveys can occur multiple times within individual participants and all episodes were included in the analysis.", "It was not possible to determine whether or not recurrent malaria infections in individuals were new infections or recrudescences, as the genetic material was typically insufficient for genotyping.", "Also, relapse infections in P. vivax cannot be distinguished from recrudescent infection.", "Therefore, individuals who had either a confirmed clinical episode or uPCR-confirmed subclinical infection with malaria (for P. falciparum and P. vivax separately) were coded with a “1” regardless of their number of infections.", "Participants who never presented at a clinic with symptomatic malaria and who never had malaria parasites detected by uPCR during the study period were coded as “0”.", "A quantitative variable was included to account for the number of screenings attended by individuals, with some individuals only attending 1 screening and others attending all 5.", "In order to test for house-level clustering of infections, two house-level covariates were also included to indicate whether or not there were secondary infections within the house: one indicating secondary clinical episodes and one indicating secondary uPCR detected infections.", "This covariate excluded the index case.", "Categorical predictor variables were created for participant sex and age, with age being aggregated into groups based on work behaviors of villagers (age groups 0–9; 10–45; and 46 plus).", "Two predictor categorical variables related to human movement patterns were also created, one based on the participant reporting spending the night in the forest (1 = yes; 0 = no) and the other based on spending the night away from the village (1 = yes; 0 = no).", "Participants were asked whether or not they had spent 1 or more nights in the forest or traveling away from the village within the previous two weeks.", "A categorical variable was created for self-reported bednet use (1 = reported having regularly used bednets; 0 = reported not regularly using bednets).", "A continuous spatial variable that measured the distance from a house to the nearest house with a clinical infection (for clinical P. falciparum and clinical P. vivax) was generated (Fig. 2).", "A categorical variable for village was included both to account for unmeasured village-specific factors and clustering at the village level.", "Exploratory analyses investigated outcome and predictor variables with regard to outcome variables as well as the timing between episodes that occurred within house and within individuals.", "Contingency tables were used to look for associations between subclinical infections and clinical malaria episodes, between cases in houses and individuals in those houses, and between spending the night in the forest or away from the village and both clinical episodes and subclinical infections.", "Odds ratios were calculated for binary variables and Chi- squared or Fisher’s exact tests were used where appropriate (for variables with multiple levels).", "Counts of clinical episodes, subclinical infections and population numbers were aggregated at the individual house level for spatial analysis.", "Clinical episodes were aggregated into three-month periods matching the timing of the cross sectional surveys.", "Scan statistics were then used to investigate the timing, location and sizes of potential clusters of either clinical episodes or uPCR detected subclinical infections.", "Discrete Poisson space-time models were used to estimate statistically significant clusters for each of the villages [19].", "Clusters that were statistically significant denote times and areas within study villages in which the relative risk of either a clinical episode or a subclinical infection were higher than expected by chance alone.", "The scan statistics were run on both clinical episodes and subclinical infections, for both P. vivax and P. falciparum, and for each of the villages independently (a total of 12 tests).", "Scan statistics were run using SatScan software (https://www.satscan.org).", "Global Moran’s I statistics and Local Indicators of Spatial Autocorrelation (LISA) statistics were then used as verification for SatScan results.", "When clusters were identified using the scan statistics, a Moran’s I test was used on the same village and time period to look for concordance between the two approaches.", "The Moran’s I statistic provides an indication of spatial clustering, including the absence or presence of clustering and a measure of clustering magnitude, but it does not indicate the location of clustering when it exists.", "LISA statistics were therefore used to identify clusters of houses with high numbers of infections.", "House population sizes were used to standardize infection and clinical episodes counts in houses for both the Moran’s I and the LISA statistics.", "Moran’s I and LISA statistics were calculated using ArcMap 10.5.", "Logistic regression was used to calculate model adjusted odds ratios for predictor variables with regard to clinical episodes and uPCR detected subclinical infections of both P. falciparum and P. vivax (a total of 4 regressions).", "All regressions, bivariate and univariate analyses were done using R CRAN (http://www.R-project.org/).", "This study was approved by the Oxford Tropical Research Ethics Committee (1015–13) and the Cambodian National Ethics Committee for Health Research (0029 NECHR).", "A total of 1,766 villagers were recorded in the demographic surveillance system, of which 1,520 responded to interviews regarding bednet use, travel or forest dwelling behavior, and 1,593 participated in the cross-sectional surveys using uPCR.", "Of the original 1766 individuals recorded in the baseline census, 82% (1447/1,766) participated in the screening at M0 and 33% (575/1,766) participated in all five of the full village surveys (at M0, M3, M6, M9 and M11).", "Migration in and out of the village influenced the proportion of this cohort that participated.", "For example, 95 of the 1,447 who participated in M0 did not participate in M3 but did participate in M6 and 49 out of those 95 who missed M3 continued with surveys from M6 onward.", "The median number of full village surveys in which cohort members participated was 4 (mean 3.14; IQR: 1–5).", "During the study period 21 participants (21/1,766; 1.2%) were diagnosed with clinical P. falciparum episodes and 81 participants (81/1,766; 4.6%) were diagnosed with clinical P. vivax episodes.", "Of those who participated in the blood screening, 3.3% (53/1,593) were found to carry subclinical P. falciparum infections and 8.6% (137/1,593) were found to carry subclinical P. vivax infections, and 19% (308/1,593) had Plasmodium infections that could not be differentiated at the species level because of low parasite densities.", "Most clinical episodes (53/81 for P. vivax and 17/21 for P. falciparum) occurred in one study village (PDB) (Table 1).", "During the travel surveys 39.7% (604/1,520) of participants reported having spent the night away from the village and 8.6% (131/1,520) reported having spent the night in a forest.", "Bednet use during the night previous to the questioning was reported by 99% (1,516/1,520) of interviewed participants.", "Both the prevalence of uPCR detected malaria and the incidence of clinical cases decreased throughout the study period, during which a well-functioning village malaria worker system was maintained.", "No bednet distributions were undertaken during the study period.", "As previously noted, subclinical P. vivax infections in study participants were repeatedly detected through successive uPCR screenings, suggesting relatively long-term chronic infections up to several months [7].", "Some of these P. vivax carriers also presented with clinical episodes during the study period and in between screenings in which they were detected with subclinical infections through uPCR.", "For example, in M1 (July 2014) there were 25 clinical P. vivax episodes of which five had submicroscopic P. vivax infections at M0 (June 2014) and seven at M3 (September 2014; two at both M0 and M3).", "During M4 (October 2014) there were nine clinical P. vivax episodes, with one patient having an uPCR detected subclinical P. vivax infection at the M3 screening (September 2014) and four at the M6 screening (December 2014; one participant carrying P. vivax parasites at both M3 and M6).", "Several individuals experienced repeated clinical episodes of both P. falciparum and P. vivax during the 19 month study period (Table 2).", "Five villagers experienced repeated P. falciparum episodes, with a median (IQR) time between episodes of 29 days (28–37); while 29 villagers experienced repeated P. vivax episodes with a median (IQR) time between episodes of 74 (54.5–109) days.", "There were also repeated episodes of symptomatic malaria episodes within individual houses.", "Three houses had multiple episodes of P. falciparum malaria, with a median (range) time between episodes of 32 (2.5–222) days while 17 houses had repeated episodes of P. vivax malaria with a median (IQR) time between episode of 46.5 (23.5–124) days.", "Two-by-two contingency tables indicated associations between subclinical infections and clinical malaria episodes.", "Participants with a subclinical P. falciparum infection had 13 times the odds (unadjusted odds ratio (UOR):13.0; CI: 4.8–34.9) of having a clinical P. falciparum malaria episode whereas participants with subclinical P. vivax infections had over 18 times the odds (UOR: 18.1; CI: 11.2–29.5) of also having a clinical P. vivax malaria episode during the 19-month study period.", "Individuals who lived in a house with another person who had a clinical P. falciparum malaria episode had approximately 15 times higher odds (UOR: 15.2; CI: 6.2–37.1) of having a clinical P. falciparum malaria episode.", "Participants who had a house member with a clinical P. vivax malaria had three times higher odds (UOR: 2.8; CI: 1.8–4.6) of having a clinical P. vivax malaria episode.", "The UOR for an individual with an episode of P. falciparum who lived in a house with someone who had a subclinical P. falciparum infection was 7.3 (CI: 3.0–17.3).", "The UOR for an individual with a clinical P. vivax malaria episode given that another house member had a subclinical P. vivax infection was 2.1 (CI: 1.3–3.3).", "The results indicated a strong, positive association between having travelled to forested areas and being detected with subclinical P. falciparum and P. vivax (uPCR P. falciparum UOR: 4.1; CI: 2.1–8.0; uPCR P. vivax UOR: 3.40; CI: 2.2–5.4).", "We did not find a statistically significant association between clinical or subclinical infection and a history of travel or between clinical malaria and having spent the night in the forest.", "Bednet usage was uniformly high (99% reported using bednets) and was not further analysed.", "Most clinical P. falciparum episodes, clinical P. vivax episodes, subclinical P. falciparum and subclinical P. vivax infections (both total counts and proportion positive) occurred within one study village: PDB (Fig. 3).", "Conversely, the proportion of villagers reporting having travelled or spent the night in the forest was higher in other villages.", "There was no statistically significant difference between participants with and without clinical falciparum episodes with regard to living near another house with a clinical falciparum infection.", "Participants with a clinical vivax episode lived closer to other houses with clinical vivax episodes (a median distance of 48 meters compared to 103 meters for participants without a clinical vivax episode; Wilcox rank sum test p-value 0.0062).", "The scan statistics indicated clusters of clinical P. vivax episodes in each of the study villages (Fig. 3).", "The relative risk of infection within these clusters was significantly higher in comparison to the population outside of the cluster.", "Single clusters were found in village KL and OK during M6–M8 (December 2013–February 2014) and in village PDB during months M0–M2 (June–August 2013).", "The clusters of clinical P. vivax in KL and OK were not confirmed with the Moran’s I statistics.", "The cluster in village OK consisted of a single household only, with two P. vivax clinical episodes occurring among five house members.", "There were two statistically significant clusters of clinical P. falciparum in village PDB during M0 through M5 (June–November 2013), one of which consisted of a single household with four cases among eight inhabitants.", "Neither cluster of P. falciparum was detected using the Moran’s I statistics.", "PDB was also the only village to have clusters of subclinical Plasmodium infections, with a single cluster of subclinical P. falciparum in M0 and a single cluster of subclinical P. vivax (in M3 and M6 (September and December 2013) combined).", "The cluster of subclinical P. falciparum was not detected by the Moran’s I statistic but the cluster of subclinical P. vivax infections was.", "The cluster of subclinical P. vivax infections (M3 and M6 (September and December 2013)) overlapped but was smaller than the cluster of clinical P. vivax (M0–M2 (June–August 2013)), detected by scan statistics, Moran’s I and LISA statistics.", "The cluster of subclinical P. falciparum (M0 (June 2013)) overlapped with one of the clinical P. falciparum clusters (M0–M5 (June–November 2013)).", "The single house cluster of P. falciparum in the village PDB occurred within the spatial clusters with clinical P. vivax episodes and uPCR detected subclinical P. vivax clusters (Fig. 3).", "The strongest predictor for clinical P. falciparum malaria episodes was living in a house with another person that had a clinical P. falciparum malaria episode.", "Individuals who lived in a house with someone who had a clinical P. falciparum malaria had 7 times the odds (adjusted odds ratio (AOR): 7.15; CI: 2.4–20.", "5) of having a clinical P. falciparum episode (Table 3).", "Having a subclinical P. falciparum infection (AOR: 5.9; CI: 1.5–19.8) and living in a house with someone else who had a subclinical P. falciparum infection (AOR: 4.2; CI: 1.5–12.1) were also strong predictors of clinical P. falciparum malaria during the 19 month study period.", "Participants who lived in village PDB had 6 times the odds (AOR: 6.10; CI: 1.5–42.5) of having a P. falciparum episode compared to participants living in village KL.", "There were no clear sex or age patterns in clinical P. falciparum episodes.", "The strongest predictor for having a clinical P. vivax episode was having a subclinical P. vivax infection.", "Participants who were found to have subclinical infections had over 13 times the odds (AOR: 13.3; CI: 7.7–23.0) of having a clinical P. vivax episode, after controlling for other potentially important factors (Table 4).", "P. vivax malaria episodes also tended to cluster in the 10–45 age group (AOR: 3.4; CI: 1.6–8.1) and in males (AOR: 1.8; CI: 1.0–3.2).", "As with clinical P. falciparum episodes, clinical P. vivax episodes were predominantly clustered in village PDB.", "Participants who were from PDB had an AOR of 2.1 (CI: 1.2–3.9) when compared to village KL.", "Distance to the nearest house with a clinical infection showed no statistical significance for either clinical P. falciparum or P. vivax episodes after controlling for covariates.", "The largest predictors of having a subclinical P. falciparum infection was self-reported traveling to the forest (Adjusted Odds Ratio (AOR) = 3.5; CI: 1.6–7.1) or having a clinical P. falciparum malaria episode during the study period (AOR: 5.7; CI: 1.4–19.6) (Table 5).", "Several factors were statistically significant predictors of subclinical P. vivax infection.", "The strongest predictor was having a clinical P. vivax malaria episode during the study period (AOR: 14.1; CI: 8.2–24.5) (Table 6).", "Individuals with subclinical P. vivax infections tended to be male (AOR: 2.4; CI: 1.6–3.8).", "The risk of being detected with subclinical P. vivax infections increased with age, participants above age nine having 2.6 times the odds (CI: 1.5–4.9) of carrying P. vivax infections and those above age 45 with 2.9 times the odds (CI: 1.4–5.9) of being detected with subclinical P. vivax infection (both in comparison to the 0–9 age group).", "Staying overnight in a forested area also had a strong association with subclinical P. vivax infections (AOR = 2.4; CI: 1.4–4.1).", "Cohort members who participated in more surveys also had a higher odds of being detected with a subclinical P. vivax infection (AOR = 1.5; CI: 1.3–1.8).", "This study found clustering of both clinical cases subclinical parasite carriage at multiple scales within the villages.", "While the study villages are within three to four kilometers from each other, the majority of clinical malaria episodes and subclinical infections occurred in one village (PDB), which was home to 67% (70/102) of all malaria episodes and 46% (88/190) of all subclinical infections.", "This study found cases clustering at levels below the village at specific times during the 19 month study period.", "Clustering of clinical P. vivax episodes was most common, occurring in all three study villages.", "Clustering of clinical falciparum episodes occurred only in one village, and was confined to small geographical areas (one cluster consisted of a single house while the other consisted of four neighbouring houses).", "Clustering of subclinical infections only occurred in one village (PDB), with the subclinical vivax cluster overlapping with a cluster of clinical vivax malaria cases that occurred at the beginning of the study.", "These findings have implications with regard to strategies for elimination and control efforts, especially those that use spatial targeting.", "If an approach of reactive case detection screening surrounding households with a sensitive diagnostic had been used it may have occasionally resulted in the detection of other cases within the same index house.", "This study found that clinical P. falciparum cases and subclinical P. falciparum carriers tend to occur within the same house during the study period.", "However, these within-house clusters account for a small proportion of all cases.", "For example, 43% (9/21) of clinical P. falciparum episodes were in participants who lived in a household with at least one other villager who had a subclinical P. falciparum infection during the study period.", "Under half of all participants who had clinical P. vivax episodes (36/81) shared a house with another villager who had a subclinical P. vivax infection.", "Interventions that targeted index households would therefore have been missed over half of all subclinical infections, even if diagnoses had been made using a highly sensitive approach and if the timing of those subclinical infections had been properly predicted.", "Clinical episodes of P. falciparum malaria occasionally clustered within individual houses (Tables 2 and 3).", "In four out of the seven houses with multiple P. falciparum episodes during the study period, the timing between episodes was within a month of each other (Table 2).", "One house experienced double episodes (with two house members having clinical P. falciparum episodes on the same day) at two different points in time (once in October 2013 and again in November 2014).", "Though there was statistically significant clustering of P. falciparum within houses in the study period, household screening and treatment within a month of the index case would have detected only 20% of the clinical P. falciparum episodes (four out of a total of 21).", "Clinical episodes did not consistently cluster in groups of houses during the study period.", "The logistic regression models indicated that the distance to the nearest neighbor was not a statistically significant predictor of an individual having a clinical infection, for either P. falciparum or P. vivax.", "While the scan statistics did indicate spatio-temporal clustering, the clustering was not consistent across time and less than half (34/81; 42% P. vivax and 9/21; 43% P. falciparum) of all clinical episodes were recorded within these detectable clusters.", "Follow up verification of the scan statistics through the use of Moran’s I statistics could only verify 2 out of the total of 6 statistically significant clusters (referring to the partially overlapping clusters of subclinical P. vivax infections and clinical P. vivax episodes in village PDB).", "This further supports the limitations of identifying and predicting clustering of infections within villages in this setting.", "Conversely, an intervention treating presumptively one entire village (PDB) or using a highly sensitive detection and treatment approach could have accounted for most (but not all) of the cases in the three villages (Table 1).", "Participants who reported spending the night in a nearby forest were more likely to have subclinical infections (both P. vivax and P. falciparum) during the study period when compared to participants who did not report a forest visit.", "This finding, in combination with sporadic and transient clustering of cases within the villages, suggests that transmission also occurred outside of the village, and warrants specific targeting of forest goers.", "These data suggest that a considerable proportion of individuals in villages may have chronic, low-density P. vivax infections [6, 7] and that there is a strong association between subclinical infections and malaria episodes.", "These data also suggest that subclinical infections may eventually become clinical episodes.", "Some participants were found subclinically infected by P. vivax after having presented with a clinical episode.", "Also in village PDB, the cluster of subclinical P. vivax infections occurred after the cluster of clinical P. vivax malaria episodes.", "Since curative doses of primaquine were not used in the villages at the time of the study, it is plausible that these were relapse infections rather than reinfections.", "In the presence of viable mosquito vectors, individuals with subclinical infections over long periods of time may be important contributors to ongoing transmission in this region.", "If P. vivax is targeted for elimination it will be important to address chronic, subclinical infections.", "Our data suggest that spatial targeting at levels smaller than the village may not adequately address this potential reservoir.", "Finally, both clinical and subclinical P. falciparum infections in these study villages decreased to almost zero in all study villages despite the absence of any additional targeted interventions other than sustaining a well-functioning village based malaria worker system providing early diagnosis and treatment.", "P. vivax infections also decreased but the decrease was less pronounced than that of P. falciparum infections.", "Village health workers are relatively easy to implement, cost efficient and can have a profound effect on P. falciparum incidence [17, 20].", "Other factors may have led to the decline in P. falciparum in these study villages, but they occurred in the absence of expensive and labour heavy approaches (e.g. screen and treat approaches).", "In order to see a similar effect on P. vivax incidence it will be necessary to target liver stage parasites with radical treatment, as well as subclinical, chronic infections.", "There are several limitations to this study.", "It is possible that some villagers with febrile illness attended health facilities outside of the village clinics and these cases would have been missed in this analysis.", "Some of the uPCR detected infections could not be identified at the species level.", "Low numbers of P. falciparum infections, especially clinical P. falciparum cases, made sophisticated analyses difficult and led to wide confidence intervals around model predictions.", "However, all significant covariates in the statistical models were also significant in univariate analyses and while large odds ratios may be over-predictions they indicate strong correlations.", "Participation in surveys was associated with increased odds of an individual being detected with at least one subclinical P. vivax infection.", "The numbers of people detected with subclinical P. vivax infections here may therefore be an underestimate.", "Another limitation to this work is reporting bias in bednet use and travel interviews.", "Participants may have forgotten important travel histories or may have been hesitant to admit travelling to specific areas (for example, to forests for illegal logging).", "This type of bias is likely to have a stronger influence with regard to false negatives than to false positives.", "Positive associations are likely to remain despite this bias whereas a finding of no correlation may be the result of under-reporting.", "Finally, the variable for having spent the night in the forest within the previous two weeks may be prone to bias in that no specific definition of “forest” was given by either interviewer or interviewee.", "It is therefore possible that different participants have different understandings of what constitutes a forest.", "Furthermore, the question regarding forest stays and travelling did not specify the amount of time spent either in the forest or away from the village.", "If forests are an important source of Plasmodium infections then it would be valuable to know if longer exposure to forests is associated with higher risk of infection.", "Future research will aim to address this question.", "These data contribute to a growing body of literature that illustrate the inherent difficulties of predicting spatio-temporal clustering of malaria at scales above the household level and below the village level.", "While helpful for directly (human-to-human) transmitted diseases with relatively short incubation periods (e.g. smallpox), predicting the transmission of vector borne diseases is more challenging.", "This is likely the result of complex transmission patterns and frustrates interventions that are based on prediction of space time occurrence of malaria at these scales (such as reactive case detection).", "Finally, elimination of P. vivax will require curative treatment of hypnozoites, with subclinical infections potentially playing an important role in P. vivax transmission.", "Daniel Parker, Richard Maude: Analyzed and interpreted the data; Wrote the paper.", "Rupam Tripura, Thomas Peto: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.", "Mallika Imwong: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data, Wrote the paper.", "Pasathorn Sirithiranont, Jeremy Chalk, Chea Nguon: Performed the experiments, Wrote the paper.", "Lorenz von Seidlein, Nicholas White, Arjen Dondorp: Conceived and designed the experiments; Wrote the paper.", "The authors declare no conflict of interest.", "This work was supported by the Bill & Melinda Gates Foundation and the Wellcome Trust of Great Britain.", "No additional information is available for this paper." ]	[]
Building bridges between agribusiness innovation and smallholder farmers: A review	The mergers of some of the world's largest agribusinesses have led to speculation about what sort of global citizens the new companies will become and whether vulnerable rural populations, especially smallholder men and women farmers, will be negatively impacted. As innovation leaders in the agriculture industry, these new companies will be expected to play key roles in finding solutions for major agricultural challenges facing the world today. The private sector has a unique voice and responsibility to help bridge the innovation gap and ensure that good science reaches those countries where public investment in agricultural research is a low priority. In this paper, we review the obstacles facing agriculture over the next few decades, the role of agricultural innovation in overcoming those obstacles, and the need for greater public funding for agricultural research. We discuss how science-based solutions that drive revenue for industry can also advance agriculture in developing economies. Expediting agricultural innovation as well as increasing access to those benefits requires a different way of thinking about the sharing of technology to improve the lives of smallholder farmers and create a more equitable playing field for women in agriculture.	[ "AGRI", "ENGI", "ENVI", "SOCI" ]	[ "Agribusiness", "Agricultural innovation", "Agricultural productivity", "Public-private partnership", "Smallholder farmer", "Women in agriculture" ]	[ "1106", "2213", "2303", "3311" ]	[ "Where modern, high input agriculture – based on decades of investment, research, and innovation – has been implemented, more food has been produced on less land with fewer resources at lower cost to consumers while also creating greater prosperity.", "In parts of the world where smallholder farmers dominate, investment in agricultural innovation can be life-changing.", "Improvements in agriculture don’t just alleviate poverty, they also enrich lives.", "In Rajasthan, India, for example, higher-yielding pearl millet varieties, developed over a decade by farmers and the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), gave farmers the income stability to replace mud homes with concrete, invest in schools and hospitals, and educate their daughters (Parthasarathy and Chopde, 2001).", "In the Republic of Malawi in Africa, where agriculture accounts for 80% of employment but most farmers are unable to produce enough maize on their small plots of land to feed their own families, adoption of modern maize varieties resulted in a 26% increase in per capita income and an additional month of food security for the poorest farmers and their families, with the greatest gains in food consumption seen in households headed by women (Bezu et al., 2014).", "The need for agricultural innovation has never been more clear, from new and improved varieties of seed to new ways of growing, storing, and marketing that seed.", "Achieving the Sustainable Development Goals put forth by the UN (2017) while a growing global population strives for an improved standard of living will require increased agricultural productivity where rapid population growth is occurring.", "Realizing that level of productivity will be challenging for farmers, especially in the face of obstacles such as climate change, violent conflicts in many areas of the globe, growing refugee populations, societal barriers that lessen the production of women farmers, groundwater depletion, and pests that have become resistant to current control measures.", "The best – and possibly the only – way to bridge that gap is expanded global investment in agricultural research and development (R&D), especially through collaborative efforts such as public-private partnerships (PPPs).", "We contend agribusiness has not only the capability but the responsibility of becoming leaders in this arena.", "Solutions conceived from public-private collaborations could provide effective advances in the pursuit of answers to global agricultural challenges.", "While engaging in these partnerships provides benefits for society, it also helps establish agricultural companies as good global citizens which in turn can benefit agriculture by increasing global acceptance of new technologies.", "Progress has been made toward eliminating food insecurity globally.", "From 1990 to 2016, the percent of undernourished people in the world fell from 18.6% to 11%, meaning that about 214 million people achieved improved diets (FAO, 2017a).", "In the next ten years, this percentage is expected to continue to drop, according to projections from the USDA Economic Research Service, to just 6% of the global population classified as undernourished (Rosen et al., 2016).", "This progress is likely to be short-lived, however.", "By 2050, just over 30 years from now, the global population is projected to grow almost 30% to 9.7 billion (DESA UN, 2015), with highly uneven growth between developed and developing economies.", "Although projections vary widely on the size of the food gap between current agricultural production and the levels needed in 2050, they all include a substantial shortfall, with estimates ranging from 25% to 110% (Alexandratos and Bruinsma, 2012; Hunter et al., 2017; Searchinger et al., 2013; Tilman et al., 2011).", "Part of the uncertainty stems from the fact that demand for food doesn’t just depend on the size of the population, but on how that population lives.", "The percentage of the global population living in extreme poverty fell from 35% in 1990 to 10.7% in 2013 (World Bank, 2017), a trend that is expected to continue.", "At the same time, population projections estimate an additional 3 billion people will enter the middle class by 2030 (Boutabba and Ahmad, 2017; Searchinger et al., 2013), with Africans expected to make up a larger percentage (9%) of the world's middle class than North Americans (7%) (Kharas, 2017).", "As per capita income goes up, so does the per capita demand for calories and protein.", "A study conducted in a district of Hanoi, Vietnam, found that a 10-fold increase in household incomes from 1999 to 2013 led to a 5-fold increase in food expenditures, with the percentage spent on rice dropping from 60% to 33% and the percentage spent on meat jumping from 11% to 24% (Thi et al., 2015).", "During roughly the same period in South Africa, the population classified as poor dropped 80% while consumption of poultry, the most important protein source in South African diets, increased by almost exactly the same percentage (USDA-FAS, 2015), demonstrating a similar drive for more protein-rich diets across cultures.", "Meeting this increased demand for calories and improved nutrition promises to be difficult, but history shows it is not impossible.", "During the last 50 years, the world's population grew by close to 4 billion people and meat consumption and calorie intake both soared (Smith, 2013).", "Science and farming have attempted to keep pace, with production of cereals such as wheat, maize, and rice multiplying 2.7 times and meat production quadrupling.", "But a repeat of the big gains in yields seen in the period between 1962 and 2006 will be challenging, and increasing the amount of land devoted to farming carries environmental consequences, including deforestation, loss of biodiversity, and increased greenhouse gas emissions (Searchinger et al., 2013).", "In fact, most of the expansion in harvested area in the 1980s and 1990s came from clearing tropical forests (Gibbs et al., 2010).", "Climate change may also present a roadblock to efforts to close the 2050 food gap with many predicting lower yields due to higher average temperatures and an increase in frequency of intense storms (Searchinger et al., 2013).", "A major challenge for agriculture is the poverty faced by many of the roughly 2 billion people who work in agriculture globally (Gaffney et al., 2016; Searchinger et al., 2013), a growing percentage of whom are women as men leave impoverished rural areas for urban jobs (FAO, 2017a).", "Overall, for low and middle-income countries, women provide about 43% of farm labor but that percentage rises above 65% in countries like Mozambique, Lesotho, and Libya (FAO, 2017a).", "An analysis of household income data of smallholder farmers in nine countries found that although these small farms produced as much as 85% of their nations’ agricultural output, their poverty rates almost always exceeded the general population and ranged from approximately 25% of smallholder farmers in Tanzania who lived in poverty to greater than 80% in Bolivia (Rapsomanikis, 2014).", "Smallholder women farmers in much of the world generate even less income from their labor than their male counterparts, as societal barriers to land ownership and reduced access to capital for improved seed varieties and technology combine to limit their productivity (FAO, 2017a).", "Regardless of gender, if the goal is to increase agricultural output by 60–70% by 2050, farmers living at subsistence level is not likely sustainable nor should this be acceptable.", "Despite the challenges facing global agriculture, substantially increasing production while simultaneously reducing the environmental impact of farming are achievable goals; the keys are increased investment in agricultural innovation and implementation in those regions most in need.", "The returns on investment are readily visible in the United States where public and private sector commitment to R&D resulted in productivity gains in most agriculture segments (Fig. 1) and an unprecedented 169% growth in total agricultural output between 1948 and 2013 (Clancy et al., 2016).", "This growth in output was achieved despite a 23% reduction in the amount of land devoted to farm production from 1948 to 2015, decreased use of pesticides and fertilizers, decreased levels of ammonia emissions, and only a minor increase in greenhouse gas emissions (OECD, 2016; USDA-ERS, 2017).", "Total Factor Productivity (TFP) growth, which measures changes in the efficiency and intensity of input use, averaged 1.38% annually over the period (USDA-ERS, 2017) and nearly all that growth is attributable to the fruits of agricultural research, including adoption of products of innovation in breeding, pest management tools and practices, nutrient use, and improvements in equipment (OECD, 2016).", "Globally, much of the nearly $1.5 trillion growth in agricultural output between 1961 and 2009 can also be attributed to R&D investments in agriculture.", "During those five decades, average maize, wheat, rice, and soybean yields grew annually by 2.0%, 2.0%, 1.8% and 1.5%, respectively, while the amount of public money invested globally on agricultural R&D grew an average of 3.31% annually, from $5.5 billion to $33.5 billion (Pardey et al., 2012).", "Investment in agricultural innovation plays an especially vital role in developing countries.", "In a world in which nearly 80% of the world's poor still rely on farming for income (World Bank, 2014), analyses of public spending in China, India, Thailand, and Uganda found dollars spent on agricultural R&D offered the greatest return when it came to improving agricultural performance and ranked first or second when it came to reducing poverty (Mogues et al., 2012).", "Studies in sub-Saharan Africa estimate that in aggregate, agricultural research provides a rate of return of 55–93% while decreasing the population of poor by 2.3 million annually (Alene and Coulibaly, 2009; Benin, 2015).", "This equates to roughly one person pulled out of poverty for every $630 spent on research (Carpenter and McGillivray, 2012).", "When smallholder women farmers are provided equal access to agricultural advancements, the gains can be even greater.", "The FAO estimates closing the yield gap between male and female farmers would result in a 12–17% reduction in the number of undernourished globally (Magomere et al., 2016).", "Improvements in agriculture also spark advancements in health, nutrition, education and community development.", "In Bangladesh, Indonesia, and the Philippines, adoption of modern rice varieties by farmers between 1989 and 2009 resulted in an economic benefit of $25 billion across the three countries with nearly 45% of that amount going to individuals living below the poverty line (Raitzer et al., 2015).", "In Ethiopia, a 26.4% drop in production costs for maize farmers who adopted a variety of improved technologies led to economic gains of $140 million a year for farmers and $105 million for consumers, leading to an estimated reduction in the number of poor people by 788,000 annually (Kassie et al., 2017).", "In the Gambia, an organization of predominately smallholder women farmers that planted improved rice varieties as part of an African Development Bank-funded project saw enough profit to open a bank account and send nearly all their children to school (World Bank, 2008).", "Although productivity gains in agriculture have been proven to reduce poverty and increased R&D spending has been proven to increase agriculture productivity, only a fraction of the estimated $69.3 billion in public and private expenditures on food and agricultural R&D globally in 2011 was actually spent on or in the countries most in need (Pardey et al., 2016b).", "In 1980, the per capita spending on public agriculture R&D in low-income countries (including south Asia and sub-Saharan Africa) was $1.73, 13% of high-income country (including the United States and Japan) spending.", "In 2011, the per capita figure had dropped to $1.51, 8.5% of high-income country spending (Pardey et al., 2016a).", "In developed economies, private sector investment has helped fill the void caused by declining public investment, but in developing economies, this funding pool is not nearly as deep, with 15 cents in private investment for every public dollar spent (Pardey et al., 2016a), further widening the research divide (Fig. 2).", "If the world is to meet the challenge of sustainably feeding nearly 10 billion people in 2050, it will be necessary to find the research dollars to unlock the agricultural potential of low-income countries, especially those in sub-Saharan Africa, where more than 600 million people make their living from farming and 70% of the food supply comes from family farms (Moyo, 2016).", "The region has been largely shut out of the gains of the Green Revolution, with TFP growth of less than 1% annually and average yields of about 1–2 t per ha, roughly 20% of yield potential (van Ittersum et al., 2016), with production by female farmers lagging that of male farmers by 20–30% (FAO, 2017a).", "Although the food-insecure population of sub-Saharan Africa decreased from 33.2% in 1990–92 to 23.2% in 2014–2016, population growth means the sheer number of malnourished has actually increased from 176 million to 220 million in 2014–16 (FAO et al., 2015), despite increases in gross cropped area from 52.6 million ha in 1961–97.4 million in 2012 (Moyo, 2016).", "Africa's population is projected to grow by 1.3 billion people by 2050, (DESA UN, 2015), which would lead to a 335% spike in cereal demand (van Ittersum et al., 2016).", "Even if the sub-Saharan Africa region managed to double current agriculture productivity levels, it would still be forced to either import more food – something beyond the means of low-income economies – or increase the amount of land area devoted to cereal production by more than 80% (van Ittersum et al., 2016), leading to increased deforestation, land degradation, and greenhouse gas emissions (Searchinger et al., 2013).", "Alleviating these pressures, however, will require more investment in agriculture research, development, and implementation in the region, as well as government buy-in (Wesseler et al., 2017).", "One country that has done this successfully is Ethiopia, where government funding for agricultural research and extension workers helped propel acceptance of modern maize varieties and fertilizer use, leading to annual yield gains of 68 kg/ha between 1990 and 2013 (Abate et al., 2015).", "Spending on agricultural R&D in Ethiopia grew by 10.9% annually from 1991 to 1996 and escalated to 16.5% annually from 1996 to 2001, helping pay for the training of nearly 63,000 agricultural extension agents who provided boots on the ground support to smallholder farmers on modern maize varieties and production methods (Abate et al., 2015).", "As a result, Ethiopia went from producing 1.8 million metric tons of maize in 1991 to 7.1 million metric tons of maize in 2018 (USDA, 2018).", "Despite these gains, Ethiopia has not quite reached the 10% of total national expenditures commitment to agriculture called for by the Comprehensive Africa Agriculture Development Programme (CAADP) or the follow-up goal by the African Union's New Partnership for Africa's Development (NEPAD) to dedicate at least 1% of agricultural GDP to spending on agricultural research.", "The 10% level is significant because it mirrors the average amount spent by the governments of countries in East and South Asia during the decades of the Green Revolution (Goyal and Nash, 2017) and many believe Africa could undergo a similar transformation if its governments made that level of investment.", "However, despite the recognition that agriculture is the prime driver of African economies, with about a third of the region's GDP derived from agriculture and nearly two-thirds of its workforce dependent on it, public spending on agriculture in Africa still lags far beyond other parts of the world, with spending on agricultural research especially at a disadvantage (FAO, 2018; Goyal and Nash, 2017).", "Greater involvement in public-private partnerships (PPP's) by agribusinesses could help bridge the innovation gap in countries where public investment in agricultural research is a low priority and help drive more agricultural research where it has been historically strong.", "The PPP model has been successful for agriculture, helping to deliver productivity and sustainability-improving technologies developed by agribusiness to improve their revenue-generating products into the hands of small-scale farmers in low-income countries.", "These technologies can be adapted to develop staple crops that are more nutritious and more resistant to drought or pests, improve local agricultural practices, and decrease poverty among local farmers and communities (CropLife International, 2012; Gaffney et al., 2015).", "In one well-known example, Henry Wallace, founder of Pioneer Hi-Bred International, Inc., and vice president under Franklin D. Roosevelt, travelled through Mexico and promised to help poor farmers there.", "Back in the U.S., he found insufficient support for using tax dollars to aid Mexican farmers so he turned to the Rockefeller Foundation to fund the crop improvement research he envisioned (Pardey and Beddow, 2013).", "The result was the creation of what is currently known as the International Maize and Wheat Improvement Center (CIMMYT), one of several international agricultural research centers under the CGIAR umbrella.", "Research coming out of CIMMYT resulted in the development of new varieties of rice and wheat that doubled yields (Pardey and Beddow, 2013).", "CIMMYT has maintained a connection to private seed companies, having worked in 17 countries over 9 years in cooperation with national research programs and private industry to establish extensive hybrid development under abiotic stress conditions (Boyer et al., 2013; Weber et al., 2012).", "This and other efforts with private industry have enabled CIMMYT to build hybrid (Masuka et al., 2017a) and open pollinated (Masuka et al., 2017b) breeding pipelines with well-documented genetic gain in target environments, a model which should be replicated and strengthened for other crops at public research organizations.", "The effort continues with the Seed Production Technology for Africa (SPTA) project with the Bill & Melinda Gates Foundation.", "The SPTA process uses technology from private industry to create maize female inbreds that do not shed pollen.", "CIMMYT, the Agriculture Research Council (ARC) of South Africa, and Kenya Agriculture and Livestock Research Organization (KALRO) are using this technology, royalty-free and at cost of production, to develop the inbreds and make them available to small seed companies.", "The technology eliminates the need for detasseling in seed production and the resulting hybrids are optimized for nitrogen use efficiency for African countries where almost 80% of the soil is low on nitrogen and fertilizer is both expensive and hard to access in some regions (Fox et al., 2017; Loussaert et al., 2017).", "Another example is the Africa Biofortified Sorghum Project, a consortium of academic, agribusiness, government, and NGO resources led by Africa Harvest Biotech Foundation International.", "A high percentage of African preschoolers suffer from Vitamin A deficiency and since sorghum is a staple food crop in Africa but is very low in β-carotene, the consortium is working to develop sorghum that offers high levels of β-carotene even after months of storage (Che et al., 2016; Magomere et al., 2016), while also increasing the availability of iron and zinc in the grain.", "Agribusiness has more to offer, especially in terms of sharing modern seed technology and other agricultural innovations with developing countries and ensuring the technology gets into the hands of smallholder men and women farmers.", "The investment is already there.", "In 2014, private agricultural companies spent more than $6.4 billion on research and development (USDA-ERS, 2018).", "Historically, both industry and academia have been very careful to protect intellectual property, but that model may be changing as more organizations begin to recognize the competitive advantages of pro-social business practices.", "In an online article titled “The Business of Business is no Longer Just Business,” the Boston Consulting Group, a global management consulting firm, heralded collaborative innovation as the business model of the future, noting, “Companies are part of business ecosystems, which in turn are embedded in local and national economies, which are interwoven with societies” (Reeves and Harnoss, 2017).", "One recent analysis by agricultural economists at the University of Nebraska-Lincoln found that an agribusiness that allowed its GM technology to be used free of charge to fight hunger in developing countries would find itself more than compensated by a corresponding increase in consumer goodwill – and therefore, sales -- in the developed world (Giannakas and Yiannaka, 2018).", "The SPTA and African Biofortified Sorghum efforts follow this path, in which IP has been donated to the project.", "An example of this pro-social mindset with implications for U.S. farmers was research developed to improve crop nitrogen management, undertaken by a number of universities, the USDA, and industry scientists (Kitchen et al., 2017).", "In addition to creating a valuable dataset across a broad geography, the scientists involved in the project also met two other objectives by 1) publishing their work to ensure access to others, and 2) training new scientists.", "Another example of industry data sharing is profiled by Assefa et al. (2017), in which data from multiple proprietary research locations were shared with Kansas State University researchers and supported by the Kansas Corn Commission.", "The analyses included over 124,000 observations from 22 U.S. states and 2 Canadian provinces over 15 years.", "Similar utilization of large industry data sets evaluated planting densities of modern maize hybrids (Assefa et al., 2016) and a long-term study of select contributions to maize yield (Assefa et al., 2018).", "These collaborative efforts provided insight on genetics, agronomic practices, and environment interactions of modern maize hybrids and would not have been possible without the creativity and cooperation of the partners and diversity of scientists involved for the repurposing of valuable data.", "Finding more ways to share agronomic research data and locations amongst scientists may be helpful toward bridging the research gap in the future.", "This ethos of partnering and sharing new technology continues with the Corteva Agriscience Open Innovation platform (OpenInnovation.", "Corteva.com, 2018), through granting access to germplasm, transformation technologies, CRISPR-Cas gene editing, molecular markers, and phenotyping capabilities.", "The vision is to maximize agricultural productivity and sustainability through collaboration and rapid deployment of new technologies through partnering organizations.", "Other examples of sharing IP and materials include official collaborations with the Donald Danforth Plant Science Center and ICRISAT, in which visiting scientists of diverse backgrounds and points-of-view trade materials, technology, and ideas on how to address the intractable problems of important food security crops.", "These collaborations can also advance equity in access to the potential benefits of modern technologies and create greater options for farmers.", "One often-repeated criticism of genetically-modified (GM) crops (and by extension, of multinational agriculture companies) is that they increase the profits of farmers in developed countries while leaving smallholder farmers in developing countries behind.", "The reality, however, is far different.", "In 2016, 185.1 million hectares globally were planted with GM crops; more than half of those hectares (54%) were in developing countries (ISAAA, 2016).", "Out of the more than 18 million farmers who have planted GM crops globally, 90% are smallholder and/or poor farmers (ISAAA, 2016), and smallholder farmers benefit from GM technology far more than those with numerous options and technologies already available (Klümper and Qaim, 2014).", "The public-private partnership model can be an especially powerful tool for overcoming societal barriers that perpetuate low productivity and poverty for smallholder women farmers in developing countries and many projects are now developed with an emphasis on gender equity (ADB, 2014; ADBG, 2009; IFC, 2012).", "In Ghana, a 20-year-old public-private project to restore rubber production has increased the participation of women in cash crop farming, with women representing 30% of the farmers involved (Kassie et al., 2014).", "In Tanzania, a study looking at the impact of adoption of improved maize varieties as part of the Sustainable Intensification of Maize-Legume Cropping Systems in Eastern and Southern Africa (SIMLESA) program found that households headed by women especially benefitted from access to improved maize varieties, with female adopters reporting per capita food consumption of 334 kg annually compared to 217 kg annually for female non-adopters (Kassie et al., 2014).", "More still needs to be done, though, to ensure access to modern agricultural technology by smallholder farmers.", "Greater focus is needed on productivity and quality improvements for crops that feed millions yet have traditionally received limited attention, like millet, cassava, banana, teff, and many legume crops.", "With the advent of genome sequencing, improved plant transformation, and gene editing, molecular biologists and plant breeders have a unique opportunity to make rapid advances in crop improvements and develop greater productivity and sustainability in these crops than would be possible with conventional plant breeding alone (Haque et al., 2018).", "Examples of partnering cited previously in this paper (Assefa et al., 2017; Che et al., 2016; Corteva Agriscience, 2018; Fox et al., 2017; Loussaert et al., 2017; Magomere et al., 2016) offer ideas for creativity towards cutting through intellectual property issues for select crops and technologies, by either donating IP outright, or by creating specific freedom-to-operate opportunities for partnering organizations.", "Greater sharing of plant genetic resources is needed to ensure this valuable resource is not sequestered away and unavailable.", "The U.S. National Plant Germplasm System distributed improved maize germplasm, much of which has been made available by private seed companies, to over 7500 requesters in 80 countries from 1988 to 2015, a valuable resource to plant breeders, molecular biologists, and more importantly to farmers and consumers around the world (Kurtz et al., 2016).", "Greater distribution of germplasm from other crops and regions would likewise contribute to more rapid improvement in food security.", "As with intellectual property challenges, a greater sense of creativity for sharing germplasm is needed to move productivity forward.", "Greater resilience, perseverance, and patience from the donor community will be needed to see research through to the end, in order to deliver valuable products to farmers and consumers.", "Industry can play an important role here since delivering products is the life-blood of maintaining a viable business.", "And finally, science-based, objective, and harmonized regulatory policy is needed to expedite improved seed and cutting edge technology to farmers and consumers.", "Since the advent of transgenic crops, over 1200 approvals have been granted by regulatory authorities in over 28 countries, and all with the same message that the transgenic crop is as safe as the non-transgenic wild type (CropLife International, 2018).", "As Klümper and Qaim (2014) state, smallholder farmers in emerging economies benefit far more from new technology than large-scale farmers in developed economies.", "New technology must be judged on an objective, scientific basis to allow a more equal participation by all regions, from all scientists, for the benefit of all farmers and consumers.", "In summary, agricultural research and innovation have led to steady progress in the battle against food insecurity over the past 25 years.", "Maintaining that progress, however, will be difficult.", "The most recent Future Development report by the Brookings Institution found the estimated number of malnourished people globally rose in 2017 for the first time in many years, and in developing countries low levels of crop productivity bear the brunt of the blame (Kharas and McArthur, 2017).", "This is especially concerning given that this increase in malnourishment is occurring despite low commodity prices globally (FAO, 2017b) and in many countries a far too large share of disposable income is spent on food (The Economist Intelligence Unit, 2018).", "Corteva Agriscience and the legacy companies of DuPont, Dow Agrisciences, and Pioneer Hi-Bred have historically been willing partners and now stand to be a driving force behind scientific and technological efforts to end food insecurity and deliver greater sustainability.", "With the right partnerships, the same science-based innovations that drive revenue for industry can help advance important food crops in developing economies and improve the lives of smallholder men and women farmers." ]	[ "Smallholder farmers are often hindered by lack of land, capital, and improved seed varieties.", "Agricultural innovation increases productivity for all farmers.", "Agriculture advances spark health, gender equality, education, and community development.", "Public funding of basic agricultural research has fallen behind and is needed more than ever.", "Collaboratively sharing new technologies speeds gains in agricultural productivity." ]
Multi-phase SPH model for simulation of erosion and scouring by means of the shields and Drucker–Prager criteria.	A two-phase numerical model using Smoothed Particle Hydrodynamics (SPH) is developed to model the scouring of two-phase liquid-sediments flows with large deformation. The rheology of sediment scouring due to flows with slow kinematics and high shear forces presents a challenge in terms of spurious numerical fluctuations. This paper bridges the gap between the non-Newtonian and Newtonian flows by proposing a model that combines the yielding, shear and suspension layer mechanics which are needed to predict accurately the local erosion phenomena. A critical bed-mobility condition based on the Shields criterion is imposed to the particles located at the sediment surface. Thus, the onset of the erosion process is independent on the pressure field and eliminates the numerical problem of pressure dependant erosion at the interface. This is combined with the Drucker–Prager yield criterion to predict the onset of yielding of the sediment surface and a concentration suspension model. The multi-phase model has been implemented in the open-source DualSPHysics code accelerated with a graphics processing unit (GPU). The multi-phase model has been compared with 2-D reference numerical models and new experimental data for scour with convergent results. Numerical results for a dry-bed dam break over an erodible bed shows improved agreement with experimental scour and water surface profiles compared to well-known SPH multi-phase models.	[ "ENVI" ]	[]	[ "2312" ]	[ "Sediment scouring and fluvial suspension due to liquid flows are common phenomena in many river flows.", "However, the construction of reservoirs interrupts the natural equilibrium of basins and leads to sedimentation.", "Siltation of artificial lakes affects water supply systems by reducing the storage capacity, and eventually affects other reservoir functions such as irrigation supply, energy production, navigation and flood control (Schleiss et al., 2016).", "More than 80% of the world's total storage capacity of artificial reservoirs belongs to hydropower dams (ICOLD, 2012), in which sedimentation may also cause damages to turbines by abrasion or occlusion, and loss of power production.", "The prediction of erosion rates is a key issue for siltation management and long-term operation plans of reservoirs, dams and other hydraulic structures.", "Flows involving sedimentation are characterised by a shear layer of soil particles under large relative motions at the interface between the sediment and the fluid.", "Numerical modelling enables prediction of the changes in the sediment balance upstream and downstream of a hydraulic structure, permitting different scenarios to be investigated quickly with relatively low cost, as well as supporting field solutions.", "However, the presence of two phases, the combination of interfacial and free-surface flows, in addition to particle entrainment of the sediment by the fluid phase are the main challenges of traditional mesh-based methods to simulate erosion and sedimentation processes (Liu, 2003).", "In recent years, the alternative option of meshless methods has emerged (Gingold and Monaghan, 1977; Koshizuka and Oka, 1996; Oñate and Idelsohn, 1998).", "Of all the meshless methods now available, smoothed particle hydrodynamics (SPH) has unique advantages when modelling problems involving large deformation of interfacial and free surface flows or large sediment motions and mixing that occur during soil erosion.", "SPH is a Lagrangian method that was initially developed to study astrophysical problems (Gingold and Monaghan, 1977; Lucy, 1977) and has been extended to a wide range of engineering applications.", "Geotechnical and geoenvironmental applications include flood and river dynamics (Prakash et al., 2014; Vacondio et al., 2012; Williams et al., 2016), soil mechanics (Bui et al., 2011; Bui 2008; Chen and Qiu, 2014; Reyes et al., 2013), simulation of landslides (Manenti et al., 2016; Pastor et al., 2009; Ran et al., 2015; Tan and Chen, 2017; Xenakis et al., 2017) seepage problems (Bui and Fukagawa, 2013; Maeda et al., 2006) and sediment transport (Falappi et al., 2007; Fourtakas and Rogers, 2016; Manenti et al., 2012; Shi et al., 2017; Ulrich et al., 2013).", "The simulation of sediment scour using SPH has developed steadily over the last decade.", "Falappi et al., (2007) simulated sediment scour in a reservoir flushing experiment using a pseudo-Newtonian approach based on Mohr–Coulomb parameters.", "Guandalini et al. (2012) used the Shields’ erosion criterion to predict scouring and in subsequent work Manenti et al. (2012) compared the Mohr–Coulomb pseudo-Newtonian approach with the Shields’ erosion criterion with experimental data.", "The results of Manenti et al. (2012) showed that the eroded volume and the profile evolution were better reproduced by the Shields’ criterion.", "However, the methodology treats the non-eroded sediment particles at subsurface layers as a fixed boundary and prevents the Shields’ approach from being able to simulate the rheology of the material at subsurface layers.", "Ulrich et al., (2013) also used a yield criterion based on Mohr–Coulomb parameters to simulate ship-induced scouring near ports.", "The model combines a linear-elastic approach with a pseudo-Newtonian model for the un-yielded and yielded material, respectively.", "The model was supplemented by a suspension layer based on the Chezy-relation to account for the viscosity transition between the sediment and the water.", "Nevertheless, in order to avoid singularities on the viscosity value the pseudo-Newtonian treatment requires the definition of a maximum value for viscosity which may be case dependent.", "Fourtakas and Rogers (2016), used the Drucker–Prager yield criterion which is also based on Mohr–Coulomb parameters, to simulate the rheology of sediment induced by rapid flows.", "The model uses a pseudo-Newtonian approach based on the Bingham-type Herschel–Bulkley–Papanastasiou (HBP) model that allows simulating the rheology of the un-yielded and the yielded material without needing to define a maximum value for viscosity.", "The Vand equation (Vand, 1948) is used to account for sediment suspension at the surface layer.", "The model showed good results for problems involving impact of rapid flows in the bed of sediment.", "However, numerical tests have shown spurious pressure fields in the sediment at areas of large deformation as, for example, near the wave fronts.", "At these large shear regions, deformation occurs as the kinematics of the liquid phase is sufficient to yield the sediment phase which exhibits slow kinematics and high shear forces.", "This discontinuity at the interface is known to cause the erroneous pressure due to the shear forces discontinuity which requires a second gradient summation for calculating the shear rates (Fatehi and Manzari, 2011).", "These slow kinematics exhibited by the sediment phase under high shear rate have been addressed in SPH more generally using diffusion-based techniques including the XSPH approach of Monaghan (1992) with an additional smoothing (Manenti et al., 2012; Ulrich et al., 2013) or by using the particle shifting algorithm of Lind et al. (2012), as employed in the work of Fourtakas and Rogers (2016).", "These techniques improve the particle distribution and avoid particle clustering which allows for a better sampled kernel.", "Nevertheless the issue is not entirely eliminated.", "In the recent work of Amicarelli et al. (2017), a model for erosional dam break floods is presented.", "The model simulates the sediment bed as a mixture type material using a mixture viscosity and a constitutive model based on Coulomb parameters for fully saturated soil.", "The aforementioned models use the so called weakly compressible SPH (WCSPH) schemes where pressure is related to density through an equation of state.", "Alternatively, truly incompressible SPH (ISPH) formulations that couple the governing equations have also been applied to sediment transport problems (Ran et al., 2015).", "Comparisons of WCSPH and ISPH for free-surface flow have shown that improved velocity and pressure fields are obtained with the ISPH method (Lee et al., 2010; Lee et al., 2008; Xu et al., 2009).", "Incompressible SPH formulations can be an alternative to improve the pressure fields which are essential to better reproduce the dynamics and erosion patterns of the sediment phase.", "In the work of Ran et al., (2015) an ISPH scheme and an interface erosion criterion which is based on the fluid and grain properties (similar to Shields’ criterion), is used to simulate erosion and entrainment of the sediment grain by the water.", "Non-eroded particles are treated as fixed boundaries, like in the model presented by Manenti et al. (2012).", "Satisfactory results with respect to experimental profiles were obtained by Ran et al., (2015).", "However, WCSPH formulations present some advantages over ISPH schemes.", "In terms of computational efficiency, ISPH generally produces reduced pressure fluctuations in the pressure field and larger time steps are possible.", "WCSPH schemes demand smaller times steps to keep density variations within acceptable levels, but on the other hand the formulation avoids solving the Poisson equation which is computationally expensive and numerical solutions are obtained by explicit algorithms.", "In this work, a WCSPH scheme is used to simulate erosion and scouring of a sediment bed.", "A critical bed-mobility condition based on the Shields criterion is imposed to the particles located at the sediment surface.", "Thus, the onset of the erosion process is independent on the pressure field and eliminates the problem of pressure dependant erosion at the interface.", "The Mohr–Coulomb parameters refer to the mechanical properties of the soil.", "Thus, the yield criteria based on Mohr–Coulomb parameters are suitable to simulate the rheology of the subsurface domain of sediment.", "However, erosion at the fluid-sediment interface is dominated by the flux characteristics and physical properties of the sediment (e.g. particle diameter and specific weight).", "The onset of the motion at the surface layer of a bed of sediment may be accurately modelled by the Shield´s parameter, as shown by Manenti et al. (2012) and Ghaitanellis et al., (2017) .", "This paper is intended to bridge the gap between these mechanical and physical approaches by proposing a model that aims to predict erosion rates at the bed surface and scour profiles due to the effect of the water on the sediment.", "The two-phase WCSPH model of Fourtakas and Rogers (2016), applicable to rapidly varying flows, is complemented by the Shields’ theory to deal with the suspension of sediment under low velocity flow.", "These two criteria have been combined in order to maximise the applicability of the model to both the entrainment of soil by water, as occurs in fluvial environments, and the impact of rapidly varying flows in a bed of sediment.", "The computational demands make the problem ideal for SPH development on graphics processing units (GPUs) to enable simulations with a large number of particles required for real engineering problems.", "The open source DualSPHysics solver (Crespo et al., 2015) that is accelerated by GPUs and extended by Fourtakas and Rogers (2016) for sediments has been used to implement the computational algorithm.", "The paper is structured as follows: in Section 2, the computational model is presented.", "A detailed description of the equations for the water and sediment phases, including the erosion criteria and the suspension treatment is given.", "Numerical results are presented in Section 3.", "First, the implementation of the Shields’ criterion is compared to numerical and experimental results.", "Then, a flume experiment over an erodible bed of non-cohesive sediment is presented and the total amount of bed material transported by the wave is compared with the experimental results.", "Finally, the combined Drucker–Prager and Shields’ criteria approach is tested against experimental results of a dam break over erodible beds.", "Conclusions are presented in Section 4.", "The physical domain of the simulation may be represented by five regions, as shown in Fig. 1.", "Region (1) represents the un-yielded sediment, which remains almost static due to its large apparent viscosities; region (2) consists of sediment with an apparent viscosity small enough to allow particle movement (according to a non-Newtonian flow model see Section 2.4.4.)", "; region (3) represents the sediment that reached the critical Shields’ parameter and simulates bed load transport; region (4) is composed of particles suspended in the fluid with a variable viscosity; and region (5) represents only water particles.", "The description of how these regions are a treated using SPH is presented in the following sections.", "In this Section we briefly describe the general governing equations and the SPH modelling technique for the fluid and sediment.", "Vectors and tensors are defined by reference to Cartesian coordinates using Greek superscripts, α, β and γ to denote Cartesian tensor coordinate directions and Latin subscripts, i and j, to identify particle locations.", "Einstein's summation is employed over repeated Greek superscripts.", "In this work, Eqs. (1) to (5) are used for all the phases.", "However, the viscous part of the stress tensor in Eq. (3) is modelled, depending on the phase (water or sediment) via a constitutive model and will be discussed in Sections 2.4 and 2.5.", "This model has been validated for a range of free-surface flows as described in Fourtakas and Rogers (2016)", "The advantage of the HBP model is that it provides information on the pre-yield and post-yield region and thereby avoids the need of setting a maximum threshold for the viscosity as required in other pseudo-Newtonian approaches (Manenti et al., 2012; Ulrich et al., 2013).", "To calculate u* from (20), it is necessary to know a priori the region (laminar or turbulent) where the water particle is located.", "Also, the dependency of z0 on u* by Eq. (21), implies that (20) must be solved iteratively for u.", "An iterative procedure suggested by Manenti et al. (2012) was implemented in DualSPHysics as, starting from the hypothesis that the smooth-flow condition occurs (i.e. ksu/v < 5) as given in Eq. (21) and that the water particle is located inside the viscous sub-layer (i.e. z ≤ δ) until convergence of u.", "Once u is obtained, it is possible to calculate τb and τbcr,0 from (18) and (16), respectively.", "Eq. (31) means that the critical shear stress increases with pressure.", "Therefore, the apparent viscosity, calculated by Eq. (15) will increase with depth, reducing the strain rate at points located at lower layers.", "In contrast to the Shields’ erosion criterion, the Drucker–Prager yield criterion enables computation of the stress tensor for all points at the sediment domain and update of the position and the velocity.", "Thus, while the critical Shield's parameter provides information about the onset of erosion at the bed surface, the critical value of the shear stress defines the yielded or un-yielded state of particles over the entire sediment domain.", "This makes the approach ideal to reproduce the effect of the impact of rapid flows over subsurface layers of a bed of sediments, as occurs for example at dam spillways.", "In order to simulate the dynamics of the subsurface sediment region and to avoid the noisy pressure fields at the sediment surface the Drucker–Prager criterion describing the yield characteristics of the sediment phase was combined with the Shields’ criterion to model the fluvial suspension of the sediment at the interface.", "Regions (1)–(3) in Fig. 1 are modelled as non-Newtonian fluids, with an apparent viscosity computed by the HBP model.", "The apparent viscosity of regions (1) and (2) is obtained by substituting the yield stress (τc) in Eq. (15) by the critical shear stress (τy) computed from the Drucker–Prager criterion.", "The apparent viscosity of region (3) is obtained by replacing the yield stress (τc) in Eq. (15) by the critical bed stress (τbcr) computed from the Shields’ criterion.", "Thus, the 3 regions are well defined.", "Region (1) is un-yielded with a large yield stress, region (2) which is yielded with an apparent viscosity derived from the HBP model and region (3) which is governed by the Shields’ criterion which is pressure independent.", "The transition between regions (1) and (2) is not explicitly defined, but emerges naturally because of the increase in stress with depth using a pressure dependent yield criterion (see Eq. (31)).", "Particles with higher critical shear stress (τy) exhibit larger values of apparent viscosity and remain static.", "The transition between regions (2) and (3) is defined by using the procedure to identify sediment particles located near the bed surface (see Section 2.5.1).", "In addition, in order for a particle to be located at region (3) the condition of Eq. (25) should be assessed.", "If Eq. (25) is valid, then the particle is considered to be at region (3) and the critical bed stress (τbcr) computed by the Shields’ criterion replace the yield stress (τc) in the HBP model to calculate the apparent viscosity (μHBP) through Eq. (15), otherwise the particle is considered to remain at region (1) or (2) and the critical shear stress (τy) of the Drucker–Prager model is used to calculate μHBP by making (τc = τy) in Eq. (15).", "The flowchart of Fig. 2 shows the procedure for calculating the apparent viscosity of particles at different regions.", "Finally, the suspended sediment is identified by the concentration parameter (cv) defined by Eq. (32) and the viscosity is computed from Eq. (33).", "With this approach, the mechanical properties of the bed material are considered by means of the cohesion and friction angle, while the effect of the physical properties are introduced by the particle diameter and the density within the Shields’ erosion criterion.", "Thus, the model is able to reproduce the entrainment of the sediment by the fluid phase which is dominated by the physical properties of the sediment and the hydraulic characteristics of the flow, as well as the behaviour of the subsurface layers of sediment determined by the mechanical properties of the material.", "The computational model herein presented has been implemented in the DualSPHysics code (Crespo et al., 2015), an SPH C++/CUDA open-source solver.", "The previous implementation of Fourtakas and Rogers (2016) was used as the starting point to implement the Shields criterion and combine it with the Druker-Prager yield criterion.", "The predictor-corrector algorithm described by (Monaghan, 1989), bound by the Courant–Friedrichs–Lewy (CFL) condition of force, viscosity and speed of sound is employed for the time step integration.", "Wall boundaries are modelled using the dynamic boundary condition (DBC) of Crespo et al., (2007) implemented in DualSPHysics.", "This section presents numerical results.", "First, the implementation of the Shields’ criterion is validated using a 2-D flushing experiment and comparing the final profile with the experimental result and with numerical results of Manenti et al. (2012).", "Then, a flume experiment carried out by the authors with non-cohesive sediment bed under fluvial conditions is presented.", "This experiment was intended to evaluate the influence of an empirical parameter of the Shields’ formulation (the equivalent grain roughness, ks) on the rate of erosion predicted by the Shields’ criterion.", "Thus, the flume experiment is simulated using only the Shields’ criterion at the surface, treating the non-eroded particles as a fixed boundary.", "Finally, the combined approached presented in Section 2.5.4 is used to simulate a 2-D dam-break problem over erodible bed.", "The results are qualitatively compared to results obtained by the Shield’ and Drucker–Prager criterion separately.", "The flushing experiment of Falappi et al., (2007) represents the scour of a sediment bed due to constant water discharge from a tank, in a similar manner to what happens when a water reservoir gets flushed.", "This is an ideal test case to assess the effectiveness of the Shields criterion in applications where erosion occurs at the bed surface only in the absence of sediment yielding.", "Therefore, the Drucker–Prager criterion is not necessary which deems this test case suitable for validation of the Shields criterion.", "This experiment has been widely used as a benchmark case for erosion models in SPH (Falappi et al., 2007; Manenti et al., 2012; Ulrich and Rung, 2010).", "The case is presented as numerical validation of the implementation of the Shields criterion in DualSPHysics.", "The geometry and dimensions of the experiment are shown in Fig. 3.", "The bulk density of the sediment is ρs = 1750kg/m3, the mean diameter d50 and the kinematic viscosity of the sediment are taken to be 0.1 mm and 0.75 m2/s, respectively.", "A constant water discharge of 0.0079 m3/s was imposed by setting the horizontal particle velocity to 0.658 m/s at the outlet.", "The parameters of the HBP model were set to n = 1 and m = 0 in order to reproduce the Newtonian behaviour of the eroded sediment employed by Manenti et al. (2012).", "In order to avoid excessive creeping of the sediment bed, non-eroded particles are treated as a fixed boundary.", "This is the main disadvantage of the use of the Shields’ criterion at the surface and justifies the need to use other complementary yield criteria to model the rheology of the subsurface sediment domain.", "In order to provide a fair comparison the parameters for the numerical model and the material properties were set in accordance with Manenti et al. (2012).", "To assess the effect of resolution, three different particle spacing were tested: dp = 0.01 m, dp = 0.005 m, and dp = 0.003 m giving simulations with 20,249, 79,480 and 314,871 particles respectively.", "Fig. 4 shows the bed profiles obtained with DualSPHysics at t = 48 s for each resolution.", "The profiles are plotted in Fig. 5 together with the experimental profiles and the one found numerically by Manenti et al. (2012).", "It is observed that the profile computed with DualSPHysics is in close agreement with the experimental one at the upper part, although a small divergence can be seen at the base of the slope for the coarser resolutions.", "Although a direct comparison with Manenti et al. (2012) might indicate that the proposed method is less accurate, no convergence study was presented by Manenti et al. so the effect of particle size is unknown in that work.", "However, in this paper, with particle refinement it is demonstrated that the DualSPHysics result is in increasingly closer agreement with the experimental result indicating convergence.", "To simulate steady flow over a mobile bed, numerical simulations were compared with a flume experiment carried out at the facilities of the Lutheran University of Brazil (ULBRA), campus Palmas, TO.", "An experimental flume mark HM 160, produced by GUNT Hamburg was adapted in order to place a 5.5 cm bed of non-cohesive sediment.", "The flume has a closed water circuit and a trap of sediment was added downstream.", "The cross section of the channel is 86 × 300 mm and the side walls are made of tempered glass, which allows clear observation of the experiments.", "The schematic arrangement of the experiment is shown in Fig. 6.", "The water entered the flume by the inlet reservoir.", "The gate was completely open at the beginning of the experiment.", "The equipment provided a steady flow with a fixed discharge over a mobile bed.", "The bed was built by sand pluviation technique (Chaney and Mulilis, 1978).", "The bed material was a sand of 0.4 mm of mean diameter with a density of 2710 kg/m3.", "The grain size distribution curve is shown in Fig. 7.", "During the experiment the water discharge was kept to a constant value of 2 m3/h and water level measurements were taken at the positions identified as P1, P2 and P3 in Fig. 6.", "The time elapsed from the instant that the water entered the channel and the instant the wave front reached the end of the channel was 8 s.", "The experiment was run for 10 s after the wave front reached the end of the flume.", "Thus, the experiment was run for a total time of t = 18 s.", "The test was performed three times in order to check for repeatability.", "In the numerical simulation, an upstream reservoir filled with water particles was created.", "The reservoir had an orifice of 2 cm height at the entrance of the flume (x = 0).", "A constant water discharge was imposed by setting the horizontal particle velocity to 0.22 m/s at the water inlet (x = 0), which corresponds to the exit of the orifice of the tank, as shown in Fig. 8.", "The imposed velocity was calibrated, considering the width of the flume, in order to obtain the mas flow rate of 2 m3/h.", "The HBP parameters were set to n = 1 and m = 0 in order to mimic a Newtonian behaviour of the eroded material.", "A spatial convergence study in terms of the position of the wave front was conducted using the following initial particle spacings of dp = 0.016 m, 0.008 m, 0.004 m and 0.002 m with the experimental results as the reference solution.", "Fig. 9a shows the position of the wave front at t = 8 s for all particle resolutions.", "Evidently the finer particle resolutions of dp = 0.004 m and 0.002 m reproduced the experimental result (dashed line) as shown in Fig. 9b, which shows a spatial convergence of the model concerning the viscous formulation and its ability to capture the physics of the flow.", "Hence, a minimum particle spacing of dp = 0.004 m is needed.", "Thus, the numerical case with a particle spacing of dp = 0.004 m, leading to 24,909 water particles and 7,188 sediment particles has been used for the same physical time as with the experimental setup of 18 s (10 s after the wave front reach the end of the flume).", "Note that in this approach, particles that do not reach the critical Shields’ parameter remain fixed to the domain.", "For that reason, this approach is appropriate for problems in which only the surface layer of sediment is affected by the flux, as in fluvial channels or when flushing sediment from artificial reservoirs.", "In order to show the ability of the combined approach presented in 2.5.4 to simulate the scouring and erosion caused by the impact of the water on a bed of sediment, the Louvain erosional dam-break of (Fraccarollo and Capart, 2002) is simulated.", "The experiment has been use as a benchmark for various researchers (Fourtakas and Rogers, 2016; Ulrich et al., 2013) and will be used herein to compare the behaviour of the computational model using the erosion criteria separately and to compare the combined approach to other SPH simulations.", "The experiment consists of the collapse of a water column over a loose sediment bed in a prismatic channel.", "The bed was built with cylindrical PVC pellets with an equivalent spherical diameter of 3.5 mm and saturated density of 1270 kg/m3.", "In order to calculate the value of the saturated density, a volume fraction of 0.5 was assumed, following the suggestions of Fraccarollo and Capart (2002).", "Fig. 11 shows the initial set up of the experiment.", "The 2-D computational case was configured using a particle spacing of 0.002 m in order to compare with the same case reported in Fourtakas and Rogers (2016), leading to 25,000 water particles and 49,450 sediment particles.", "Three cases were run using different models for the sediment phase, i.e.:", "Drucker–Prager yield criterion and HBP rheological model for the eroded sediment", "Shields’ erosion criterion and Newtonian model for the eroded sediment.", "Drucker–Prager yield criterion combined with Shields erosion criterion and HBP rheological model", "The mechanical properties of the soil for the Drucker–Prager criterion (case A) were set to ϕ = 310 and c = 0 Pa in order to reproduce a cohesion less material as expected from PVC pellets, since the values are not given by Fraccarollo and Capart (2002).", "Fig. 12 shows the three cases at the same instant in time where the particles belonging to each phase are coloured blue for water and red for sediment in order to show the effect on the deformation of the cases A, B and C.", "The equivalent grain roughness (ks) for the Shields criterion (case B) was set to 5 times the value of the mean diameter, which is expected to represent a transitional flow condition.", "For this case, sediment particles are fixed to the domain unless they reach the erosion criterion (see Fig. 12a), otherwise the impact of the water column over the soil produces excessive deformation of the sediment mass even if high viscosities are used (see Fig. 12b).", "For the example shown in Fig. 12b an upper limit of μmax = 2500 Pa.s following the suggestions of Ulrich et al. (2013) and Ulrich and Rung (2012).", "However, Manenti et al. (2012) set the upper limit to viscosity to several orders of magnitude higher than this, which suggests that the maximum value of the apparent viscosity is case dependent.", "The combination of the criteria (case C) eliminates the need of fixing the particles to the domain position, because the shear stress is computed at each sediment particle as a function of the mechanical soil properties, avoiding the excessive creeping resulting from a fluid model (see Fig. 12c).", "The parameters of the HBP model used in cases A and B were set to n = 1.2 and m = 100 in order to represent a non-Newtonian shear thinning material and to avoid the numerical instabilities for zero shear strain rate.", "Figs. 13–16 show the snapshots of the erosional 2-D dam break experiments at times 0,25, 0.50, 0.75 and 1.00 s.", "The experimental snapshots were presented by Fraccarollo and Capart (2002) showing the estimated interfaces between the sediment bed, the eroded material and the water.", "Numerical experiments show three sub-domains.", "Blue particles represent the water phase, red particles are the un-yielded sediment and green particles denote the eroded material.", "Two features may be analysed from the erosion patterns of Figs. 13–16: the bed profile formed by the motionless particles and the transport layer formed by the moving sediment particles, represented by red and green particles in the numerical snapshots respectively.", "From the (a) snapshots in Figs. 13–15 it may be observed that the bed profiles generated by the Drucker–Prager criterion are similar to the experimental profiles, reproducing the curve formed by the impact of the water in the bed of sediment at position x ≈ 0.05 m. However, the model is not able to correctly simulate the transport layer.", "This effect is better reproduced by the Shields’ criterion, represented by the (b) snapshots where green particles show a well-defined layer of eroded sediment.", "The combined approach presented in the (c) snapshots keeps the positive features of each criterion, i.e. it captures the effect of the impact as well as the entrainment of the sediment particles by the fluid phase, showing satisfactory qualitative agreement between the numerical model and experimental observations.", "The plots of Figs. 17 and 18 confirm this behaviour for t = 0.25 s and t = 1.00 s.", "The Root Mean Square (RMS) error was used to quantify the deviation of the SPH interface location from the experimental interfaces.", "Accordingly, the acronyms RMSE(Zw), RMSE(Zs) and RMSE(Zb) in Fig. 17 and Fig. 18 represent the RMS error of the free surface (Zw), the interface between the water and the moving grains (Zs), and the interface between moving and immobile grains at the bed (Zb).", "The transport layer is defined by the region between the Zs and Zb interfaces.", "The current model shows closer agreement to the experimental data both for the bed surface (Zb interface) and for the transport layer (region between Zb and Zs interfaces).", "Comparing the profiles obtained by the Shields criterion with the profiles obtained by the combined approach here presented at t = 0.25 s, the current model demonstrate greater capability of capturing the yielding effect caused by the impact of the wave at the initial states (see the shape of the wave front in Fig. 17b and 17c).", "The yielding near the dam toe is also well captured by the Drucker–Prager model, as observed in Fig. 17a.", "For t = 1.00 s, the Drucker–Prager yield criterion shows a tiny transport layer near the wave front, while the Shields criterion and the combined approach show reasonable agreement with the experimental data.", "The current model shows the smallest RMS error for the interface between the water and the moving grains (Zs) and for the interface between moving and immobile grains (Zb).", "This means that the scouring profile is satisfactory predicted by the current model.", "The current model that combines the Drucker–Prager and the Shields’ criteria shows the smallest RMS error for the interface between the water and the moving grains (Zs) and for the interface between moving and immobile grains (Zb).", "This allows to conclude that the scouring profile is satisfactory predicted by the current model.", "In this paper, a novel approached that combines two erosion criteria with a non-Newtonian rheological model to simulate surface erosion is presented.", "The Drucker–Prager yield criterion was complemented by the Shields’ erosion criterion in order to model the relevant features of the erosive process by considering the mechanical and hydraulic properties of the sediment bed.", "The implementation of the Shields’ criterion into the DualSPHysics code has been validated using experimental and numerical results.", "The analysis of the equivalent grain roughness parameter showed that it has a strong influence on the on the prediction of the amount of eroded material.", "Significantly different behaviour was observed when the erosion criteria were used separately to simulate a 2-D dam break experiment.", "The Drucker–Prager yield criterion was able to reproduce the effect of the impact of the water in the bed at the dam toe, while the Shields’ criterion showed better agreement on simulating the transport layer formed by the entrainment of the sediment particles by water at final stages.", "Satisfactory results are observed when both criteria are combined keeping the positive features of each criterion, i.e. the shape of the scouring profile and transport layer.", "The combined approach here proposed is able to simulate the yielding of the sediment layer by considering the mechanical properties of the material.", "Thus, the rheology of the domain of sediment under the impact of rapidly varying flows is satisfactory predicted.", "Also, the entrainment of soil by water is accurately simulated by including the physical properties of the sediment and the hydraulic characteristics of the flow through the Shields criterion." ]	[ "A novel multi-phase SPH sediment-water model that combines the yielding, shear and suspension layer mechanics is presented.", "A critical bed-mobility condition based on the Shields criterion is imposed to the particles located at the sediment surface.", "The multi-phase model was implemented in the open-source DualSPHysics code accelerated with graphics processing unit (GPU).", "2-D multi-phase cases are presented for validation.", "Erosion profiles are closely reproduced." ]
Lithological controls on the deformation mechanisms operating within carbonate-hosted faults during the seismic cycle	A significant proportion of moderate-large earthquakes, plus aftershocks, nucleate within and propagate through upper-crustal carbonate-dominated sequences, where the effects of lithological variations on fault behaviour are poorly understood. The Gubbio fault is an active (1984, Ms. = 5.2) normal fault in Italy, hosted in Mesozoic-Cenozoic limestones and interbedded marls. Fault core domains derived from limestone at the studied outcrop are characterised by fractures/hydrofractures and breccias and host a number of localised (<1.5. mm wide) principal slip zones (PSZs). The majority of displacement of up to 230. m is concentrated in these PSZs, which comprise cataclasites, gouges, and calcite veins. Degassing bubbles, 'quenched' calcite, and the transformation of smectite to illite, are also observed within PSZs, implying frictional heating and seismic slip. In contrast, marl-rich domains exhibit distributed shear planes bounding a continuous and pervasive foliation, defined by phyllosilicate-rich pressure-solution seams. Microstructures in the seams include folds/kinks of phyllosilicates and pressure shadows around clasts, consistent with aseismic fault creep. A model is proposed for the behaviour of lithologically complex carbonate-hosted faults during the seismic cycle, whereby limestone-dominated fault core domains behave in a predominantly seismic manner, whereas phyllosilicate-rich domains behave in a predominantly aseismic manner. © 2013 The Authors.	[ "EART" ]	[ "Carbonate fault", "Deformation mechanisms", "Fault zone heterogeneity", "Microstructure", "Phyllosilicate", "Seismic behaviour" ]	[ "1907" ]	[ "The large-scale architecture of upper-crustal faults at <5 km depth comprises either a single high-strain fault core surrounded by a damage zone (e.g. Chester et al., 1993), or multiple high-strain cores, which bound lenses of damaged material (e.g. Faulkner et al., 2003).", "The differentiation between fault core and damage zone is generally based on the presence and spatial distribution of deformation products (Chester and Logan, 1986; Chester et al., 1993; Shipton et al., 2006).", "The fault core, which ranges from a few metres up to a few tens of metres wide, consists of cataclastically deformed fault rocks and typically contains one or more principal slip surfaces (PSSs) (Shipton et al., 2006; Faulkner et al., 2010).", "Damage zones, which are up to a few hundred metres in width, consist of fractured protolith rocks and smaller displacement subsidiary slip surfaces (Faulkner et al., 2010).", "The fault core is where most of the displacement is accommodated, and the deformation processes occurring here are the focus of the present paper.", "Fault core architectures vary widely between faults and appear to be controlled, in part, by the composition of the protolith.", "Upper-crustal faults derived from carbonates (e.g. Agosta and Aydin, 2006; Micarelli et al., 2006; De Paola et al., 2008; Bastesen and Braathen, 2010; Molli et al., 2010; Smith et al., 2011a; Fondriest et al., 2012) and crystalline rocks (e.g. Chester and Chester, 1998; Wibberley and Shimamoto, 2003; Walker et al., 2013) tend to exhibit narrow fault cores that are less than a few metres in width, comprising cohesive and incohesive random-fabric fault rocks such as breccias, cataclasites and gouges (Sibson, 1977).", "The majority of the displacement within these fault cores is localised along discrete PSSs and within their associated principal slip zones (PSZs).", "Numerous field studies of major seismogenic faults suggest that slip during individual earthquake events is localised along these PSZs, which are typically no more than a few cm thick (see Sibson, 2003, for a review).", "Notable examples include: the PSZ associated with the 1999 Mw 7.6 Chi Chi thrust earthquake in Taiwan, which has been estimated to be just 1 mm thick (Kuo et al., 2013); the Nojima fault, responsible for the 1995 M 7.2 Kobe earthquake in Japan, which contains several gouge and pseudotachylyte layers, each less than a few mm thick (Otsuki et al., 2003); and the PSZ responsible for the 2008 Ms 8.0 Wenchuan earthquake in China, which comprises a ∼1 cm thick layer of fault gouge (Li et al., 2013).", "At the microscale, PSZs are often observed to contain sub-zones, which range from a few hundred microns to a few millimetres in width, composed of variably developed cataclasites and gouges and frequently displaying Riedel shear geometries (e.g. Power and Tullis, 1989; Otsuki et al., 2003; De Paola et al., 2008; Smith et al., 2011a; Fondriest et al., 2012).", "In contrast, upper-crustal fault zones rich in phyllosilicates typically display much wider fault cores, often adhering to the multiple fault cores model.", "For example, the exhumed Carboneras fault in SE Spain has a fault core up to 1 km wide (Faulkner et al., 2003), which comprises numerous high-strain gouge zones of a few metres in thickness (Faulkner et al., 2003).", "Similarly, the SAFOD (San Andreas Fault Observatory at Depth) borehole core and field studies of the Median Tectonic Line in Japan have revealed several phyllosilicate-rich fault core strands, each >1 m wide (Zoback et al., 2010; Holdsworth et al., 2011; Jefferies et al., 2006).", "Rather than displacement being localised within PSZs, it is uniformly distributed within each gouge band; and rather than random-fabric fault rocks being the dominant deformation products, phyllosilicate-rich fault cores typically display a continuous, highly foliated fabric, though Riedel shear geometries are still conspicuous features (e.g. Rutter et al., 1986, 2012; Faulkner et al., 2003).", "Consequently, in lithologically heterogeneous, upper-crustal fault zones, where crystalline/carbonate and phyllosilicate-rich protoliths are interlayered, we might expect to see a complex fault zone architecture with separate domains of localised and distributed deformation.", "This geometry has been documented along ancient, exhumed examples of presently inactive major strike-slip faults (e.g. the Carboneras fault zone, Faulkner et al., 2003), low-angle normal faults (e.g. the Zuccale fault in Central Italy, Collettini and Holdsworth, 2004; Smith et al., 2011b) and in accretionary complexes (e.g. the Chrystalls Beach Complex mélange, New Zealand, Fagereng and Sibson, 2010).", "Attention now is turning to active fault zones, in an attempt to understand how lithological heterogeneities within multi-layered sequences cut by a fault may affect not only the fault zone architecture, but also the seismic behaviour of the fault (e.g. Nemser and Cowan, 2009; Chiaraluce, 2012; Gratier et al., 2013; Tesei et al., 2013).", "Quartzo-feldspathic and carbonate rocks typically have sliding friction coefficients in the Byerlee range of 0.6–0.85 (Byerlee, 1978) and experimentally exhibit slip-weakening and velocity-weakening behaviour (Logan et al., 1992; Beeler et al., 1996; Marone et al., 1990; Gu and Wong, 1994; Verberne et al., 2010; Collettini et al., 2011), which is necessary for earthquake nucleation and unstable stick-slip behaviour (Dieterich and Kilgore, 1994; Marone, 1998; Scholz, 1998).", "These lithologies also display dynamic-weakening behaviour during high-velocity rotary shear experiments (see Di Toro et al., 2011, for a review), with the coefficient of friction reducing to sub-Byerlee values (<0.2) at seismic slip velocities, facilitating earthquake propagation.", "In contrast, many phyllosilicates (e.g. talc, smectites) are weak, particularly when wet, (sliding friction ≪0.3, e.g. Behnsen and Faulkner, 2012) and most types exhibit velocity-strengthening behaviour (e.g. Saffer et al., 2001; Saffer and Marone, 2003; Moore and Lockner, 2004, 2011; Ikari et al., 2007, 2009, 2011; Morrow et al., 2007; Tembe et al., 2010; Behnsen and Faulkner, 2012; Sone et al., 2012; Tesei et al., 2012).", "Velocity-strengthening behaviour does not favour earthquake nucleation and rock units displaying this behaviour are expected to act as barriers to earthquake propagation due to a positive stress drop (Scholz, 1998).", "Thus, upper-crustal fault rocks rich in weak phyllosilicate minerals are thought to deform predominantly aseismically by fault creep.", "For example, the creeping behaviour of faults such as the San Andreas is attributed to the presence of smectitic phyllosilicates in fault gouges (e.g. Carpenter et al., 2011; Lockner et al., 2011; Holdsworth et al., 2011).", "Over the course of the seismic cycle, a fault may experience a broad spectrum of slip rates.", "These range from mm/yr, during the interseismic period, to mm/day-week during the pre-seismic (earthquake nucleation) and post-seismic (afterslip) periods and, then, to slip rates of m/s during earthquake propagation.", "It seems reasonable to hypothesise that lithological heterogeneities within the fault core will strongly influence which parts of a fault zone deform seismically or aseismically during the different seismic intervals.", "To further investigate this proposal, we document here the deformational and microstructural characteristics of the Gubbio normal fault (1984, Ms = 5.2) in the northern Apennines of Italy (Fig. 1a).", "This upper-crustal, seismically active fault deforms a succession of alternating limestone and cm-scale marl beds.", "Outcrop to microscale deformation features within the Gubbio fault zone have previously been studied by Bussolotto et al. (2007), who characterised the spatial and temporal relationships of structures in the fault zone, together with a determination of the P/T conditions and fluid behaviour during deformation.", "Here, we use a combination of microstructural (optical microscopy and field emission scanning electron microscopy, SEM) and mineralogical analyses (energy-dispersive X-ray spectroscopy, EDX, and X-ray diffraction, XRD) to study the dominant deformation mechanisms active within the fault core, and to assess the likely influence of lithology on deformation style.", "We use these findings to propose a conceptual model for the long- (inter- and post-seismic period) and short-term (coseismic) frictional behaviour of the fault zone, which can then be tested by future experimental work.", "The northern Apennines of Italy have undergone NE–SW shortening since the middle Miocene, resulting in the development of a NE-verging fold and thrust belt (Barchi et al., 1998b).", "In the Gubbio area, this deformation is represented by the NW–SE striking Gubbio anticline (De Paola et al., 2006 and references therein).", "An upper Pliocene–Quaternary late-orogenic extensional regime is superimposed upon the folds and thrusts, forming a series of extensional basins bounded by NNW–SSE trending normal faults (Barchi et al., 1998a; Boncio and Lavecchia, 2000) (Fig. 1a).", "This extensional regime currently dominates the tectonics of the northern Apennines, although it has a relatively slow separation rate of 2–3 mm/yr (D'Agostino et al., 2009).", "The Gubbio fault is an active segment of a 150 km long fault system known as the Umbria Fault System (Collettini et al., 2003).", "The surface trace is 22 km long, striking NW–SE (∼N130°) (Figs. 1a and 2a).", "Seismic reflection data suggest that it has a listric profile at depth, with average dips of ∼60–70° to the SW at the surface, decreasing to 10–15° at ∼6 km, where it reactivates a pre-existing thrust fault (Mirabella et al., 2004) (Fig. 1a).", "At the surface, the fault juxtaposes Jurassic-Oligocene carbonates, belonging to the Umbria–Marche succession, in the footwall against Quaternary fluvio-lacustrine deposits, of the Gubbio Basin, in the hangingwall (Collettini et al., 2003; Bussolotto et al., 2007) (Fig. 1b).", "A maximum displacement of 3.2 km at the centre of the Gubbio Basin was estimated by Collettini et al. (2003), which accumulated during multiple tectonic phases.", "Historical and instrumental records of moderate-large earthquakes occurring on the Gubbio fault are limited, since no permanent station coverage exists (Collettini et al., 2003; Pucci et al., 2003).", "However in 1984, the Gubbio area experienced an Ms 5.2 (Haessler et al., 1988) earthquake, located ∼10 km south of the town of Gubbio at 7 km depth (ISC, 2001), and this event was attributed to movement on the Gubbio fault (e.g. Collettini et al., 2003).", "A temporary seismic network installed three days after the main event recorded ∼300 aftershocks (M < 3.5), arranged in two linear clusters trending sub-parallel to the strike of the Gubbio fault: a northern cluster (10 km long and 3 km wide in map view), where seismicity was limited to depths of 1–4 km, and a denser southern cluster (14 km long and 5 km wide), where seismicity was limited to depths of 4–6 km (Collettini et al., 2003).", "The aftershocks were located in the hangingwall of the Gubbio fault, rather than on the fault plane itself, and thus were interpreted to represent stress readjustments within the fault hangingwall (Pucci et al., 2003).", "Microseismicity has been recorded in the Gubbio area during a survey conducted from October 2000–May 2001 (Piccinini et al., 2003), which allowed Chiaraluce et al. (2007) to clearly image the subsurface geometry of the northern portion of the Gubbio fault.", "The Umbria fault system has hosted two other major normal-faulting events during recent decades: the Norcia earthquake in 1979 (Mw = 5.8) (Deschamps et al., 1984), and the Colfiorito seismic sequence from 1997 to 1998 (Mw < 6.0) (Amato et al., 1998).", "In addition, the 2009 L'Aquila (Mw = 6.1) (Chiarabba et al., 2009) earthquake was located ∼130 km SE of Gubbio on the Paganica fault, also part of the Apennine fault system.", "Real-time slip analyses using LiDAR-based methods showed that the latter earthquake was succeeded by slow, afterslip deformation in the months following the earthquake (Wilkinson et al., 2010; D'Agostino et al., 2012).", "All of these seismic events nucleated in and propagated through the Mesozoic–Cenozoic carbonatic multilayer bedrock sequence.", "The outcrop that forms the focus of this present study is at Cava Filippi (43°23′21.95″N, 12°29′51.83″E), located ∼1.5 km to the N of the village of Mocaiana, which lies near to the NW termination of the Gubbio fault and where the displacement on the fault is ∼1.5 km (Mirabella et al., 2004).", "This outcrop is part of the footwall of the Gubbio fault.", "The fault rocks at Cava Filippi formed at depths of less than 2.5–3 km within a confined fluid system (Bussolotto et al., 2007).", "Throughout this paper, certain terminologies and abbreviations are used, which we now define.", "Firstly, the fault core contains a number of slip zones, which are zones up to a few 10s of cm wide, containing variably developed brittle fault rocks (breccias, cataclasites; see below).", "These slip zones each contain a principal slip zone (PSZ), which at our study site is never greater than 1.5 mm thick.", "PSZs typically consist of ultracataclasite or gouge.", "Each PSZ is associated with a discrete principal slip surface (PSS), which sharply cuts the PSZ along one of its margins.", "The majority of displacement within the fault core is thought to be accommodated collectively along the PSSs and within the associated PSZs (e.g. Sibson, 2003; Smith et al., 2011a).", "Secondly, we follow the classifications of Woodcock and Mort (2008) for brittle fault rocks that are defined primarily by grain size.", "A cohesive fault rock with ≥30% large clasts (≥2 mm in diameter) is classified as a breccia.", "Anything with <30% large clasts is a cataclasite, and different classes of cataclasite are defined according to the ratio of small clasts (0.1–2 mm in diameter) to cement/matrix (clasts < 0.1 mm in diameter).", "A fault gouge is defined as a fine-grained fault rock that is incohesive (Woodcock and Mort, 2008).", "The breccias are further subdivided based on how well the clasts fit together.", "In crackle breccias, the clasts are separated by thin seams of cement/matrix and have little evidence for rotation of clasts.", "In mosaic breccias, clasts display greater separations and rotations, but adjacent clasts can fit back to their original configuration.", "In chaotic breccias, clast separation and rotation are so large as to preclude reassembly by visual inspection.", "In addition to these random-fabric fault rocks, we also describe a number of foliated brittle fault rocks, where the fabrics are a result of pressure-solution processes dominating over brittle processes.", "Here we refer to the protolith as the “relatively undeformed” host rock, where brittle deformation is at a background level.", "The hangingwall rocks, which are not exposed in the study area, are Quaternary-age lacustrine and fluvial deposits of the Gubbio Basin, which range from clay-rich lignites through to sandstones and conglomerates (GE.MI.NA, 1963).", "Protolith rocks in the footwall of the Gubbio fault dip gently to the NE (Fig. 2c).", "The rocks deformed within the fault core of the Gubbio fault belong to the Scaglia Rossa Formation.", "The Scaglia Rossa Formation is part of a Jurassic to Oligocene carbonate succession in the Umbria–Marche Apennines (Collettini et al., 2003).", "It consists of Turonian to Danian pale grey–red, micritic limestones with regular marl interbeds up to several cm thick (Bortolotti et al., 1970; Trabucho-Alexandre et al., 2011).", "The two deformed members in our study area are a “micritic limestone” member (Fig. 3a), which consists predominantly of beds (up to 20 cm thick) of micritic limestone (∼5 wt.", "% clay; marl interbeds are infrequent), and the “marly limestone” member (Fig. 3b), which is generally more thinly bedded (beds average 5–10 cm thick) and contains frequent marl-rich horizons of up to 2 cm thick.", "Thus, portions of the fault core derived from the marly limestone have a more significant phyllosilicate content (up to ∼20 wt.", "%) compared to those portions derived from the micritic limestone.", "We refer to the damage zone as the portion of the fault zone in which the protolith has been variably fractured during fault-related deformation.", "Several fracture sets are present, some of which emanate from the earlier compressional phase of deformation that resulted in the formation of the Gubbio thrust and anticline (De Paola et al., 2006).", "Here, we describe only those features that are clearly attributed to the current extensional phase of deformation.", "The damage zone is ∼220 m wide, corresponding to a region measured from the damage zone-fault core boundary, where fracture/vein density values peak at ∼15 fractures counted per metre on a 1-D transect (Sagi, 2012), to the protolith, where fracture/vein density decays to background values (<2 fractures/m, Sagi, 2012).", "The fractures are predominantly pure dilational features (Sagi, 2012) and are often infilled by calcite; calcite mineralisation locally forms up to 75% of the rock volume.", "A few shear-extensional fractures are present with offsets up to 5 mm (Sagi, 2012), as well as some dip-slip faults with displacements of up to 5 m (Sagi, 2012).", "All fractures, veins and faults strike sub-parallel to the main Gubbio fault trace, with both synthetic and antithetic dips, often displaying an Andersonian geometry (Fig. 2d).", "Within 2 m of the fault core-damage zone boundary, bedding starts to become highly rotated with beds being dragged into the fault core to dip at steep angles of up to 75° to the SW (Fig. 2c).", "Stylolites are also noteable features within the damage zone.", "Typically, they are either sub-parallel or sub-perpendicular to bedding, and thus are likely a combined product of diagenesis and the prior compressional deformation phase.", "The stylolites in the damage zone have a high-amplitude, sutured morphology, using the classification scheme of Logan and Semeniuk (1976), with very thin seams of insoluble residue (<100 μm thick) comprising <5% of the total rock volume.", "We refer to the fault core at Cava Filippi as the part of the fault zone where the majority of the normal displacement and associated fault rocks are concentrated (Fig. 3c and d).", "This outcrop was selected for its excellent exposures in an active quarry, orthogonal to the main PSSs.", "The damage zone-fault core boundary is marked by a PSS (F1) and thus is very sharp.", "The fault core has a width across-strike of ∼15 m, and is bounded to the SW by a major escarpment that corresponds to the unexposed trace of the Gubbio fault.", "Following the practice of Bussolotto et al. (2007), we subdivide the fault core in the quarry into four structural domains (FD1–FD4), delimited by four main slip zones, which are bounded by the PSSs, F1–F4 (Fig. 3c and d).", "FD1–FD3 are derived from the micritic limestone member of the Scaglia Rossa Formation, whereas FD4 is derived from the marly member, creating a marked lithological heterogeneity within the fault core.", "Bussolotto et al. (2007) used biostratigraphic controls to estimate the individual fault and intradomain displacements, which are illustrated in Fig. 3c and described in Section 3.4.1.", "These estimates suggest that each PSS has several tens of metres of displacement, whereas the displacement within the domains (i.e. within FD1, FD2 and FD3) between the faults is negligible.", "The exception is domain FD4, which, along with F4, is estimated to have accommodated up to 90 m of displacement.", "In total, the cumulative displacement at the Cava Filippi outcrop is estimated to be 150–230 m, which equates to ∼12% of the total estimated throw of the Gubbio fault (Bussolotto et al., 2007), the remainder of the throw being accommodated by the adjacent main Gubbio fault scarp.", "FD1 is characterised by highly rotated bedding planes, which are dipping very steeply compared to the shallowly dipping beds in the adjacent damage zone, at angles of up to 75° towards the SW (Figs. 2c and 3d).", "Some bedding surfaces are striated, in an orientation consistent with slickenlines observed on the PSSs (Fig. 4a), suggesting that they have been locally reactivated as shear planes.", "Moving towards F2, the limestones are increasingly fractured so that bedding is almost completely destroyed leading to the development of breccias (Fig. 3d).", "A few relict bedding features remain in FD2.", "FD2 is characterised by intense fracture and hydrofracture abundance, resulting in heavy brecciation of the rocks (Fig. 4b).", "The exception is a patch of red, marl-rich material, with dimensions of ∼1 × 2.5 m (Fig. 3c), which displays characteristics similar to those described for FD4 (Section 3.4.1.3).", "We measured fracture densities across the fault core along a 1-D transect.", "Fracture density in domains FD1–FD2 is ∼51 fractures/m, three times greater than observed in the flanking damage zone.", "Fractures and veins throughout FD1–FD2 are present in two dominant sets, as in the damage zone.", "Both strike NW–SE (sub-parallel to the principal slip surfaces), with one set dipping SW whereas the other dips antithetically to the NE (Fig. 2d and f).", "The veins have a curviplanar geometry, are up to 2 cm wide, and are filled with calcite (Fig. 4a).", "Several subsidiary slip surfaces occur in FD2 (Fig. 3d), and constraining the displacements for each was not possible, but the lack of any well-developed slip zones and associated gouges or cataclasites suggests that displacements are small, probably less than a few mm.", "The breccias in FD1–FD2 range in composition from 30 to 70% limestone clasts, set in a crystalline calcite cement, and range from mosaic to chaotic breccia types (Fig. 4c).", "Limestone clasts are very angular and randomly oriented, ranging from <1 mm up to 10 cm in diameter.", "Clasts are commonly transected by several very fine calcite veins, which are typically <1 mm thick.", "This geometry creates a crackle breccia texture in certain clasts within the host mosaic/chaotic breccias (Fig. 4c).", "Individual crystals within the calcite cement may be visible with the naked eye and are up to 5 mm in diameter.", "At the mesoscale, it is difficult to differentiate between what may be clasts of calcite and the calcite cement.", "Intermittent clay-rich pressure-solution seams are also present, constituting ∼5% of the rock volume (Fig. 4c).", "Where bedding features are still present, the solution seams are sub-parallel and sub-perpendicular to the bedding, consistent with the geometries observed in the protolith.", "However, where bedding surfaces have been reactivated as shear planes, solution seams are at angles of ∼40° to the plane.", "Domain FD3 is characterised by coarsely foliated breccias (Fig. 4d).", "They range from mosaic- to chaotic-type and the foliation is defined by anastomosing pressure-solution seams (Fig. 4e), which dip at angles ranging from ∼20 to 40° to the SW, thus creating an angle of 30–50° to the dip of the PSSs (Fig. 4d).", "Calcite mineralisation in veins and breccia cements represents 25–85% of the rock volume and limestone clasts range from 10 to 65%.", "Again, clasts are angular and display a wide range of sizes from <1 mm up to 5 cm.", "The solution seams have a spacing of ∼5 cm and locally comprise up to 10% of the bulk rock volume.", "Solution seams within FD1–FD3 (Fig. 4c and e) typically have lower amplitude to wavelength ratio compared to those in the damage zone, and hence have a smoother, less sutured morphology.", "They are also noticeably thicker, being up to 3 mm wide, and occasionally contain visible fragments of calcite and limestone (<1 mm in size).", "Fracture density in FD3 is ∼0.3 fractures/m, slightly lower than in domains FD1–FD2.", "F1–F4 are discrete shear planes, and >10 m in length based on available exposure, which cut sharply through the outcrop (Fig. 5a).", "F1 (displacement = 3 ± 27 m, Bussolotto et al., 2007) lacks a well-developed slip zone, but a PSZ is present, consisting of a clay-rich gouge, up to 2 mm thick and containing sub-mm clasts (∼25%) of calcite and limestone, coats the slip surface.", "F2 (displacement = 130 ± 30 m, Bussolotto et al., 2007) also lacks a well-defined slip zone, although in hand specimen, a zone up to 1 cm thick of chaotic breccia is observed immediately underlying the PSS (Fig. 5b).", "This brecciated zone comprises clasts of chert (20%), limestone (15%) and calcite (10%), averaging 1 mm in size, set in a calcite cement (55%).", "Again, the PSZ consists of a clay-rich gouge layer up to a few mm thick, which coats the PSS.", "F3 (displacement ≤ 50 m, Bussolotto et al., 2007) has a slip zone up to 35 cm wide, which has a foliated S–C fabric (Fig. 5c).", "The C-planes are defined by shear planes running sub-parallel to F3, and the S-planes are defined by phyllosilicate-rich pressure-solution seams that constitute up to 10% of the rock volume.", "They form at angles of up to 50° from the dip of F3 and have average spacings of ∼1.5 cm (Fig. 5d).", "Within the lithons bounded by the foliation, the rock is composed of angular clasts of limestone (15%) and calcite (30%) formed from fragmented calcite veins and/or cement, typically <5 mm in size, set in a calcite cement (55%).", "Given that 30% of the clasts are fragmented veins or cement, then this rock can be classified as a chaotic breccia.", "F3 has a PSZ up to 1 mm wide, defined by a layer of calcite veining (up to 0.5 mm wide) (Fig. 5d), plus a ∼0.5 mm thick gouge layer.", "F4 (displacement of 60 ± 30 m along with FD4) also has a slip zone up to 50 cm wide, associated with an S–C foliated chaotic breccia (Fig. 5e).", "Here, angular limestone and calcite clasts comprise up to 30% each and the calcite cement comprises ∼40% by volume.", "Clasts range from <1 mm to 3.5 cm diameter, averaging ∼5 mm in size.", "Some clasts can still be fitted back together in a jigsaw geometry.", "The phyllosilicate-rich pressure-solution seams are oriented at angles of up to 30° from the dip of F4 (Fig. 5e), and are often very closely spaced (average spacing ∼5 mm).", "This high density means that the clay seams locally form up to 15% of the rock volume.", "In addition, the least-weathered parts of the fault exhibit a PSZ up to 1.5 mm wide composed of ultracataclasite (Fig. 5f).", "The PSSs of F1–F4 are often heavily mineralised, displaying calcite slickenfibres in addition to slickenlines that both indicate predominantly dip-slip fault movement (Figs. 3b and 5a).", "In FD4, which is derived from the marly limestone, the deformation style is very different.", "Compared to other parts of the fault core, fractures are not abundant and shear localisation along through-going faults is rare.", "Instead, the deformation is represented by a continuous and pervasive S–C fabric (Fig. 6a).", "The C-planes have an average strike direction that is sub-parallel to the domain-bounding PSSs (Figs. 2e, f, 6a and b), but they dip at a shallower angle, which is explained by the fact that they appear to have reactivated the pre-existing bedding planes that rotated during deformation.", "The C-planes are defined by an anastomosing network of clay-rich pressure-solution seams (Fig. 6d).", "Individual seams are up to a few mm thick.", "Many C-planes have calcite slickenfibres precipitated on their surfaces (Fig. 6c), which display kinematics consistent with that of the main fault zone, i.e. dip-slip extensional.", "The S-planes are also defined by seams of pressure-solution, which are generally <1 mm thick (Fig. 6d).", "They are inferred to have formed perpendicular to the direction of the local maximum principal stress induced by shearing along the C-planes (Fig. 6b).", "A less well-defined C′-type shear band cleavage locally overprints the primary S–C fabric (Fig. 6a and b).", "The spacing of the C-foliation ranges from <1 mm up to 2 cm, so that in some places, the clay-rich seams comprise up to 20% of the rock volume.", "In contrast to the pressure-solution seams in domains FD1–FD3 and in the damage zone, the seams in FD4 have a very smooth, anastomosing morphology (Fig. 6d).", "This geometry is consistent with the relatively phyllosilicate-rich nature of the marls in FD4, since higher clay content promotes the pressure-solution process (e.g. Rutter, 1976) and favours the development of non-sutured morphologies (Logan and Semeniuk, 1976).", "Calcite mineralisation is present but much more sparse than in FD1–FD3.", "Calcite veins only constitute up to 20% of the rock volume and they tend to be much narrower than those in FD1–FD3, typically <5 mm wide.", "They are formed predominantly perpendicular to the C-planes (Figs. 6d and 10a).", "The veins generally have a tabular morphology, although irregular forms are also present (e.g. Fig. 6d).", "The exception to the predominantly distributed deformation is a localised shear zone that cuts through the centre of FD4 (Fig. 6a).", "This shear zone, ∼25 cm wide, is bounded by two slip surfaces.", "However, the foliated nature of the rocks within this shear zone does not differ significantly to those seen throughout FD4.", "Optical microscope analysis shows that the limestone clasts in the breccias have reduced sizes of <100 μm and are always very angular shapes.", "They are extensively fractured and transected by fine calcite veins ranging from 10 μm up to a few mm wide (e.g. Fig. 7a).", "Consistent with the observations made at outcrop scale, these veins form conjugate sets.", "The calcite veins typically have a blocky texture, and occasionally an elongate blocky texture (Fig. 7a).", "The size of the crystals depends on the width of the vein, but they may be up to 500 μm in diameter in the centre of the widest veins, generally with smaller crystals at the vein boundaries (Fig. 7a).", "Veins typically have a discontinuous and/or branching geometry (Fig. 7a).", "The nature of the calcite crystals within the breccia cement contrasts significantly compared to the vein crystals.", "Calcite crystals in the cement are mostly anhedral with random orientations (Fig. 7b).", "They are also highly inequant, ranging from <20 μm up to 5 mm in size.", "In addition, deformation twins are common within the calcite cement, particularly in the largest crystals, and they may be >1 μm thick and can be curved and taper out within crystals (Fig. 7b).", "Although several twins correspond to type I deformation twins of Burkhard's (1993) classification, which is consistent with deformation at temperatures < 200 °C, ∼50% correspond to type II–type III deformation twins.", "Typically type II–type III deformation twins are associated with temperatures of deformation in the range 150–250 °C, which is high considering the 2.5–3 km of exhumation for the Gubbio fault.", "Further, Bussolotto et al. (2007), used palaeothermometric techniques to calculate temperatures of formation of the fault rocks at Cava Filippi of <50 °C.", "Thus, we infer that the high-strain rates experienced within the fault core enhanced deformation twin morphology (e.g. Rybacki et al., 2013).", "Pressure-solution seams average 50 μm to 1 mm wide and are filled with an extremely fine-grained crystal aggregate.", "EDX analysis with the SEM shows that the seams comprise up to 80% phyllosilicate with the remainder being very fine-grained calcite crystals.", "Calcite crystals are quite equant, ranging from ∼25 to 50 μm in diameter, and are sub-rounded (Fig. 7c).", "Bearded overgrowths (Fig. 7d) occur in some solution seams in domain FD3, suggesting that pressure-solution creep is likely an active deformation mechanism within this domain.", "The solution seams typically cross-cut all other structures (i.e., clasts, cement, veins) in the breccias, indicating that they continued to develop after most other structures.", "PSSs appear as very sharp, straight boundaries that truncate all features within the host breccias (Fig. 8a).", "As described in Section 3.1, the PSS bounds a PSZ.", "The PSZs observed in this study range from ∼0.5 to 1.5 mm thick and are one of two types: those defined by zones of ultracataclasite and those defined by PSS-parallel calcite veins.", "F4 is an example of an ultracataclasite-dominated PSZ (Fig. 8a) with four well-defined sub-zones.", "Sub-zone 1, which ranges from 515 to 915 μm wide, consists of ultracataclasite.", "The ultracataclasite has a random fabric and contains sub-rounded clasts of limestone (10%) and calcite (50%) set in a matrix (40%) of ultrafine-grained calcite and clay (Fig. 8c).", "Clasts range from 20 to 600 μm in size.", "Anything less than 20 μm in size is attributed to the matrix.", "Sub-zones 2, 3 and 4, which have widths of up to 430 μm, 240 μm and 220 μm, respectively, consist of discontinuous, blocky calcite veins that contain crystals ranging from ∼40 to 800 μm in size.", "Each sub-zone is bounded by a slip surface.", "These surfaces are highlighted by the presence of an orange–brown material (Fig. 8d), which at high magnification, and with the SEM, is seen to consist of sub-rounded calcite crystals, <20 μm in size, dispersed within phyllosilicate-rich material.", "The calcite crystals within this material do not appear to have been particularly fractured or abraded, and many of them have concave boundaries.", "SEM investigation of the uppermost slip surface corresponding to the striated F4 plane observed in the field reveals a more complex structure.", "It shows the presence of an ultrafine-grained, phyllosilicate-rich gouge lining the slip surface (Fig. 9a), and this gouge displays further sub-zones, which we refer to here as “shear localisation zones” (SLZs) 1 and 2.", "SLZ 1, which is up to 15 μm thick, has a phyllosilicate content of ∼50%, in which the phyllosilicates are randomly oriented, and contains sub-angular calcite clasts up to 20 μm in diameter.", "SLZ 2, which is <5 μm thick, is much finer-grained, containing nano-sized clasts of calcite, which have a smooth and asymmetric shape (Fig. 9a).", "These clasts are surrounded by finely foliated phyllosilicates, which comprise ∼80% of SLZ 2.", "Also within SLZ 2, several shear surfaces are preserved (Fig. 9a).", "In some specimens from F4, we observed sub-spherical pores within the SLZs that resemble bubbles, which are surrounded by elongate calcite crystals and a small amount (<20%) of phyllosilicate (Fig. 9c).", "Other parts of the SLZs preserve “dog-bone” or “H”-shaped calcite crystals (Fig. 9d).", "SEM analyses have also been performed parallel to the slip surface of F4, i.e. looking down onto the striated fault surface.", "Here, we observed tightly-packed calcite grains with dimensions ranging from 50 to 150 nm, which sometimes exhibit polygonal boundaries and triple-junction contacts (Fig. 9e and f).", "F3 exhibits an example of a calcite vein-dominated PSZ (Fig. 8b).", "The PSZ contains a number of calcite veins, which are characterised by transitions along their length over distances of <300 μm from a blocky to a fibrous texture (Fig. 8b and d).", "The areas of blocky calcite are characterised by crystals with an average size of 100 μm that display a crack-seal texture where fluid inclusions are aligned sub-perpendicular to the slip direction (Fig. 8d).", "The fibrous veins are characterised by much smaller, euhedral crystals, on average ∼15 μm in size with the fibrous texture being created by micron-scale stylolitic surfaces lying sub-parallel to the PSS (Fig. 8e).", "Similar to F4, the slip surfaces that bound the veins within the PSZ are highlighted by the presence of orange–brown phyllosilicate and have a jagged, stylolitic morphology (Fig. 8d).", "The pressure-solution origin of both the C- and S-planes is evident under the microscope, due to the truncation of micro-fossils (Fig. 10b).", "The pressure-solution seams that define the C-planes have an anastomosing morphology, creating interconnected, phyllosilicate-rich networks (Fig. 10b).", "Solution seam spacings range from μm to cm scale (Fig. 10a and b), thus they are much more pervasive than initial field observations suggested, as are the S-planes.", "The C-planes link up with the S-planes, thus bounding lithons of relatively undeformed limestone.", "The solution seams that define the S- and C-planes may be up to 100 μm thick.", "EDX analysis shows that they are filled with ∼50% phyllosilicate and 40% calcite with the remaining 10% comprising quartz and diagenetic apatite (Fig. 10d).", "This material is moderately foliated due to the alignment of phyllosilicate grains (Fig. 10c and d).", "The clasts within the solution seams have relatively smooth, concave boundaries, typical of dissolution (Fig. 10c), and show little evidence of internal deformation that contrasts with the clasts in the phyllosilicate-rich SLZs of F1–F4, which are highly fractured and have sharp, angular boundaries (Fig. 9b).", "Microstructures within the solution seams are inferred to indicate two coeval deformation mechanisms.", "Firstly, asymmetric delamination features and micro-folds (Fig. 10c) imply foliation-parallel sliding along (001) clay lamellae.", "And secondly, pressure shadows around clasts (Fig. 10d) indicate the operation of pressure-solution creep.", "In all cases, the sense of shear interpreted from the asymmetry of these microstructures is consistent with that of the fault zone.", "Calcite veins display similar characteristics to those in FD1–FD3.", "They have a drusy and blocky texture, ranging from <10 μm up to a few mm wide (Fig. 10a).", "Their consistent orientation orthogonal to the prevailing C-plane foliation is characteristic of deformation by pressure-solution.", "The veins are also offset by the pressure-solution seams by distances of up to a few cm (Fig. 10a).", "This geometry may be due to dissolution, or alternatively, some of the larger offsets may be caused by frictional sliding or creep along the solution seams.", "XRD analyses were performed on the clay fraction present within various parts of the fault zone to constrain what clays are present and to assess what influence clay mineral content may have on the behaviour of the different fault core domains.", "A total of six samples were analysed: two samples representing the protolith (one from the micritic limestone protolith and one from the marly protolith), two samples representing pressure-solution seams (one from domain FD3 and one from domain FD4), and two samples representing PSZs (one from F3 and one from F4).", "Sample details and results are summarised in Table 1 and representative XRD traces are shown in Fig. 11.", "In the protolith, the clays present are illite, mixed-layered illite-smectite and a very small amount (<1%) of kaolinite.", "These same clays are present in the pressure-solution seams from both domains FD3 and FD4, although the solution seams show a relative enrichment of kaolinite (up to 25%).", "However, a significant difference in clay content is observed within the PSZs of F3 and F4.", "Whilst illite and kaolinite are still present, smectite forms only 2% of the clay fraction within the PSZ of F4 and is completely absent within the PSZ of F3.", "These results are consistent with the chemical compositions obtained by in-situ EDX analyses of the clay minerals.", "Three dominant styles of deformation are recognised within the core of the Gubbio fault.", "Domains FD1–FD3, derived from the micritic protolith, are characterised by pervasive fractures, veins and chaotic breccias.", "Other features include intermittent pressure-solution seams and subsidiary slip surfaces.", "The style of deformation is thus discontinuous and distributed.", "The main deformation mechanisms inferred from microstructural observations are fracturing and hydrofracturing, with minor amounts of diffusive mass transfer and local frictional sliding along minor faults.", "The slip zones of F1–F4, also derived from the micritic limestone protolith, are up to 50 cm wide, containing chaotic breccias, which can also have a coarsely foliated S–C fabric.", "They each contain a PSS and an associated PSZ.", "PSZs are up to 1.5 mm wide, containing variable amounts of ultracataclasite and gouge, together with slip surface-parallel calcite veins.", "Even narrower SLZs (<5 μm wide) are observed within the PSZs.", "Thus, the deformation is discontinuous and localised, with the main inferred deformation mechanisms being cataclasis and frictional sliding.", "The foliated S–C fabrics within the slip zones and the development of phyllosilicate-rich stylolitic surfaces along some PSSs (e.g. that of F3 described in Section 3.4.2.2) indicate that diffusive mass transfer processes also play a role within the slip zones F1–F4.", "In domain FD4, derived from the marly protolith, the style of deformation is very different to that observed in the rest of the fault core.", "The main deformation product is a pervasive S–C foliation, where the foliation is defined by abundant pressure-solution seams.", "The style of deformation is thus continuous and distributed, with the inferred dominant deformation mechanism being pressure-solution.", "Our findings illustrate that marked contrasts in both the deformation styles and dominant deformation mechanisms occur across the fault core, and we correlate these contrasts to changes in lithology across the core.", "Also, the displacement distribution estimated by Bussolotto et al. (2007) appears to be influenced by lithology because for FD1–FD3 dominated by the micritic limestones, displacement is focused on domain boundaries (i.e. along the PSSs F1–F4), whereas for FD4, significant displacement is accommodated within the domain.", "Thus, with respect to the behaviour of the Gubbio fault during the seismic cycle, we now focus our discussion on those parts of the fault core where most of the displacements appear to be concentrated, i.e. F1–F4 and FD4.", "Although the total displacement accrued at the Cava Filippi outcrop only amounts to ∼12% of the total displacement of the Gubbio fault, the structures here are considered analogues for what may be encountered either along-strike or down-dip of the main Gubbio fault scarp as it traverses lithological heterogeneities in the host rock.", "To better understand the possible behaviour of the Gubbio fault during the seismic cycle, we compare our field, microstructural and mineralogical observations with previous studies of active faults and with experimental studies for deformation of rock types similar to those at Gubbio.", "The displacements accommodated along F1–F4 are concentrated mostly along the PSSs themselves and also within the PSZs.", "The presence of fractured, sub-rounded clasts and ultrafine-grained gouge material suggests that cataclastic deformation occurred within the PSZs.", "The presence of SLZs < 20 μm wide, which themselves contain further shear surfaces and an increasing proportion of phyllosilicates, suggests that shear becomes progressively localised into narrower zones during slip evolution.", "Similar styles of shear localisation are observed in a number of experimental studies and associated with a transition from stable sliding (velocity-strengthening) to unstable, stick-slip (velocity-weakening) behaviour (e.g. Engelder et al., 1975; Moore et al., 1988; Logan et al., 1992; Beeler et al., 1996; Collettini et al., 2011; Ikari et al., 2011).", "These experiments were performed on a wide range of crustal rock types (quartz, calcite, quartzo-feldspathic and phyllosilicate gouges) and under a broad spectrum of experimental conditions.", "On this evidence, it seems possible that the PSSs F1–F4 have behaved in a velocity- and/or slip-weakening manner.", "This interpretation would be consistent with the fact that they are derived from the micritic limestone protolith, since carbonate rocks do experimentally exhibit both velocity- and slip-weakening behaviour under certain conditions.", "For example, Verberne et al. (2010) recorded a change from velocity-strengthening to velocity-weakening in limestone gouge at temperatures between 100 and 150 °C; Collettini et al. (2011) observed an evolution from velocity-strengthening to velocity-weakening with increasing sliding velocity; and Logan et al. (1992) observed this transition at confining pressures > 50 MPa.", "In FD4, displacement is distributed throughout the domain with movement accommodated along pressure-solution seams in the S–C fabric.", "The common occurrence of slickenfibres on C-plane surfaces suggests that the shearing within this domain is fluid-assisted.", "The microstructures observed within the pressure-solution seams, such as folds and kinks of the phyllosilicates and pressure shadows around clasts, are indicative of bulk ductile flow by diffusive mass transfer.", "They are very similar to those reported by Holdsworth et al. (2011) and Hadizadeh et al. (2012) in the phyllosilicate-rich, actively creeping zone of the San Andreas fault, i.e. they are more typical of creeping deformation than seismic slip.", "Interpreting the structures in FD4 to be of aseismic origin would be consistent with their phyllosilicate-rich composition due to the marly protolith, because phyllosilicates have been shown experimentally to be almost exclusively velocity-strengthening (e.g. Morrow et al., 2007; Tembe et al., 2010; Ikari et al., 2011 and references therein).", "The proportion of phyllosilicates within the solution seams is >50%, which experimental evidence suggests is enough for the frictional behaviour to be controlled by the weak phyllosilicates rather than by the stronger carbonate phases (e.g. Tesei et al., 2012), due to the formation of an interconnected clay network (e.g. Schleicher et al., 2010; Tembe et al., 2010).", "Although phyllosilicates comprise <20% of the rock volume in FD4, the fact that they are concentrated along the solution seams means that shear strain can preferentially be focused along these pre-existing zones of weakness (Collettini et al., 2009; Behnsen and Faulkner, 2012), so that ultimately, the strength and frictional behaviour of FD4 is likely dominated by the weak phyllosilicate-rich seams.", "Thus, we hypothesise that FD4 deforms predominantly in a velocity-strengthening, aseismic manner, with the potential to act as a barrier to rupture propagation (e.g. Boatwright and Cocco, 1996; Kaneko et al., 2010).", "Consequently, earthquake ruptures might tend to bypass FD4, and concentrate displacement along the PSSs F1–F4, which lie within the velocity-weakening, micritic limestone part of the fault core.", "As well as having the potential to exhibit velocity-weakening behaviour, carbonate rocks are also widely shown to demonstrate strong dynamic-weakening behaviour at seismic slip rates (>1 m/s), when the frictional strength of carbonate faults reduces dramatically from Byerlee values (μ = 0.6–0.85) to values as low as 0.1 (Han et al., 2007, 2010, 2011; De Paola et al., 2011a; 2011b).", "The weakening mechanisms are thought to be thermally activated due to the frictional heat generated along highly localised slip surfaces during rapid seismic slip (Di Toro et al., 2011).", "Temperatures attained within micron-scale width slip zones at seismic slip rates may be on the order of several hundreds of degrees Celsius and that would be sufficient to trigger physical and chemical modifications to the fault rocks and slip-zone materials.", "For example, in carbonates, these conditions can trigger decarbonation reactions, releasing CO2 gas into the slip zone and producing a thin layer of sub-rounded and spherical nanoparticles of lime (CaO) within the slip zone.", "These nanoparticles may be able to undergo rolling along the slip surface, leading to fault lubrication (Han et al., 2010, 2011; De Paola et al., 2011a).", "As described in Section 3.4.2, within one of the SLZs present within the PSZ of F4, we observed bubble-like features set within a matrix of elongate calcite crystals and phyllosilicate, together with some unusual “dog-bone” or “H”-shaped calcite crystals.", "These microstructures are strikingly similar to those observed by Collettini et al. (2013) within the PSZ of the Spoleto thrust in central Italy.", "Collettini et al. (2013) performed transmission electron microscope analyses on the matrix material within the PSZ and found the presence of amorphous silicate.", "The fault rocks at Spoleto are derived from the Scaglia Rossa Formation, so they are formed from the same protolith as the fault rocks at Gubbio and also formed at similar depths of 2–3 km.", "Collettini et al. (2013) suggested that the observed microstructures are produced by thermally-triggered reactions in the PSZ as a result of rapid heating during earthquake slip.", "They propose that dehydroxylation of clays has resulted in amorphisation, and that the skeletal habit of calcite is a result of disequilibrium crystallisation under rapid cooling conditions (e.g. Faure et al., 2003) following carbonate dissociation.", "The fact that we can make a direct comparison between the microstructures observed along F4 and those described above of Collettini et al. (2013) leads to the conclusion that F4 has slipped coseismically.", "The absence of smectite in the PSZs of F1–F4, as revealed by XRD analyses, may also be due to frictional heating associated with seismic slip, since smectite becomes unstable at temperatures between 120 and 150 °C and collapses to an illite-type structure (e.g. Pytte and Reynolds, 1989).", "The exact mechanism of this smectite-illite transformation has not been well constrained, but it is often associated with the loss of interlayer water (due to dehydration) (Colten-Bradley, 1987).", "We have also observed nanoparticles coating the slip surfaces of F1–F4 (Section 3.4.2.2; Fig. 9e and f), providing further evidence that the thermal dissociation of carbonate may have occurred and that dynamic weakening was operative along these faults.", "In fact, their tightly-packed geometries and polygonal boundaries resemble those produced experimentally by Smith et al. (2013), who suggested that they result from dynamic recrystallisation due to intense frictional heating.", "Thus, there are several lines of evidence to suggest that seismic slip has occurred along the PSSs F1–F4, but we have been unable to identify any seismic signatures within domain FD4, further supporting our hypothesis that FD4 behaves predominantly aseismically.", "While it would be useful if our hypothesis fully described the deformation behaviour of the fault core, evidence suggests that the simple seismic vs. aseismic differentiation between the PSSs F1–F4 and domain FD4 is unlikely.", "Firstly, carbonate rocks do not behave exclusively in a velocity-weakening manner (Logan et al., 1992; Verberne et al., 2010; Collettini et al., 2011) and shear localisation is not always associated with stick-slip behaviour (e.g. Logan et al., 1979; Marone et al., 1990; Rathbun and Marone, 2010).", "Similarly, phyllosilicates do not behave exclusively in a velocity-strengthening manner (e.g. Saffer and Marone, 2003; Ikari et al., 2007; Niemeijer et al., 2012).", "Frictional behaviour is dependent on a number of factors, including the ambient conditions (normal stress, temperature and pore fluid pressure), the imposed rupture velocity, pre-existing structures within the slip zone, and the composition and distribution of mineral phases within the slip zone.", "Secondly, Kaneko et al. (2010) showed that under certain conditions, velocity-strengthening fault patches can host earthquake ruptures, for example, if the earthquake is of large-enough magnitude to overcome the negative stress drop encountered within the velocity-strengthening material, or because of high pre-stress in the velocity-strengthening patch as a consequence of it not being ruptured during previous seismic events.", "This would explain the occurrence of a localised zone of shearing in the centre of FD4.", "Indeed, once transected, seismic ruptures may exploit this localised zone in FD4 by reactivation.", "A complex interplay between seismic and aseismic behaviour is also evident from our microstructural observations.", "For example, we have observed that many slip surfaces associated with F1–F4 have stylolitic morphologies, highlighted by the presence of phyllosilicate (Section 3.4.2; Fig. 8e and d).", "We have also observed SLZs that are enriched in phyllosilicates (up to 80%; Fig. 9a).", "The concentration of phyllosilicates within PSZs is likely to be a result of pressure-solution processes operating along the PSSs during the interseismic period.", "And thus at certain times during the seismic cycle, diffusive mass transfer processes dominate over cataclasis and frictional sliding within the PSZs.", "In fact, grain-size reduction by cataclasis will promote diffusive mass transfer processes and may lead to a transition from frictional to frictional-viscous behaviour (Bos and Spiers, 2001), therefore promoting fault creep.", "Conversely, the dissolution of the asperities along a fault surface may reduce their strength, causing them to break successively and trigger fracturing of the fault plane (Gratier and Gamond, 1990).", "Finally, the variations in calcite vein textures observed within the PSZ of F3 (Section 3.4.2.2; Fig. 8b and d) further point to variations in the seismic history of the fault.", "As proposed by Gratier and Gamond (1990), a crack-seal texture (Ramsay, 1980) may reflect sliding occurring at a very slow rate, during which successive microcracks are healed.", "The presence of small euhedral crystals, on the other hand, may reflect precipitation of calcite into larger fluid-filled cavities created during more rapid displacement events (Gratier and Gamond, 1990), whilst the PSS-parallel stylolites are indicative of fault healing by pressure-solution welding (e.g. Yasuhara et al., 2005).", "Despite these complexities, we propose a simplified conceptual model, which summarises possible scenarios for the mechanical behaviour of the Gubbio fault (Fig. 12).", "During an earthquake, the micritic limestone-dominated portions of the fault core accommodate the coseismic displacements along the localised PSSs F1–F4.", "This localisation is accompanied by fracturing and/or hydrofracturing within domains FD1–FD3.", "At these times, domain FD4, which does not accommodate either of these modes of deformation, remains loaded, and this residual stress is then relaxed during the post-seismic period by slow, creeping afterslip deformation concentrated in the marly sections of the fault core.", "Alternatively, it may be that domain FD4 and certain parts of domains FD1–FD3, i.e. the pressure-solution seams, and certain portions of the PSZs of F1–F4, creep continuously during the interseismic period, whilst the other parts of the fault core remain locked.", "This behaviour will increase the stresses in the locked parts of the fault core until they fail seismogenically.", "In addition, Sagi (2012) found that the marl-rich units of the Scaglia Rossa Formation act as fault seals, inhibiting fluid flow.", "This behaviour is consistent with the observation that calcite veining in FD4 occurs to a much lesser extent than in domains FD1–FD3.", "It is possible that the fluids involved in the pressure-solution processes in FD4, transport material away from FD4 to be deposited in the veins within FD1–FD3.", "Either way, the preferential transport of fluids away from FD4 and towards FD1–FD3 will generate a fluid overpressure and result in the hydrofracturing and hydraulic brecciation that we observe in domains FD1–FD3.", "This fluid overpressure can also trigger seismic slip along the PSSs F1–F4, which would be consistent with the fact that some PSZs are characterised by calcite veining (e.g. Fig. 5d) and chaotic breccias (e.g. Fig. 5b).", "A similar mixed-mode fault slip behaviour has been proposed, and supported experimentally, by Collettini et al. (2011) for the Zuccale fault zone, which similarly consists of domains of competent carbonate material surrounded by foliated, phyllosilicate-rich horizons (Collettini and Holdsworth, 2004; Collettini et al., 2011).", "Although there is no recorded evidence of afterslip deformation occurring at Gubbio specifically, one of the best-documented examples of afterslip deformation comes from L'Aquila, ∼130 km south of Gubbio, after the 2009 Mw 6.1 earthquake, where post-seismic deformation may have amounted to as much as 50% of the coseismic slip (Wilkinson et al., 2010; D'Agostino et al., 2012).", "Evidence of interseismic creep in the Apennines is also limited, but D'Agostino et al. (2009) identified, through the study of GPS velocity fields, a significant deficit in seismic release in the Umbria–Marche Apennines, which makes aseismic activity a real possibility.", "The one place where interseismic creep is documented in the northern Apennines is along the Alto-Tiberina low-angle normal fault, of which the Gubbio fault is an antithetic splay (Chiaraluce et al., 2007; Hreinsdóttir and Bennett, 2009).", "These observations show that the lithologies through which the Gubbio fault penetrates are able to accommodate aseismic creep.", "The driving forces and mechanisms by which afterslip and interseismic creep occur are not yet fully understood, but a number of recent studies show a coincidence between the operation of pressure-solution creep and the aseismic sliding of active faults (Gratier et al., 2011, 2013; Holdsworth et al., 2011; Hadizadeh et al., 2012).", "Other factors besides lithology can control the distribution of seismicity within a fault zone such as geometric complexities, which can induce different strain rates within a fault zone (e.g. Tesei et al., 2013), and the location of the mainshock rupture patch, since post-seismic strains will preferentially occur in the areas surrounding the location of the mainshock (e.g. Johnson et al., 2006; Ozawa et al., 2011; D'Agostino et al., 2012).", "But still, playing down the importance of lithology is hard in these cases.", "Geometric complexities within fault zones are often a result of lithological heterogeneities within the sequences through which a fault traverses (e.g. Bonson et al., 2007; Nemser and Cowan, 2009; Tesei et al., 2013).", "Also, lithology can control the location of earthquake rupture patches, since, as discussed in Section 5.2, earthquakes will only nucleate in materials that are velocity-weakening.", "The majority of displacement at the Cava Filippi outcrop is accommodated along the PSSs F1–F4 and in domain FD4.", "Along F1–F4, the dominant deformation mechanisms are cataclasis and frictional sliding.", "The resulting microstructures and localised deformation features are similar to those observed in both natural and experimental examples of major earthquake-hosting faults.", "We therefore propose that earthquake nucleation and propagation are favoured along F1–F4, which are hosted in micritic limestones.", "Domain FD4 is characterised by continuous, distributed deformation with the dominant deformation mechanism being pressure-solution.", "The structures observed in FD4 are comparable to those observed in natural and experimental examples of creeping faults.", "Thus, we propose that domain FD4 behaves in an aseismic manner and we attribute this behaviour to its phyllosilicate-rich host rock.", "The behaviour of the Gubbio fault over the course of the seismic cycle is therefore likely to be complex, with a lithologically-controlled interplay between localised earthquake nucleation and propagation, and distributed interseismic and post-seismic deformation.", "Combined field, microstructural, geodetic and experimental studies of faults in varying lithologies, and under a range of conditions, will help us to better constrain the role of lithology in controlling fault behaviour, so that lithological variations along or across a fault may be considered when assessing the past seismicity and future seismic hazard of a fault." ]	[ "Field and microstructural study of active fault in interbedded limestones/marls.", "Limestones deform predominantly by fracturing, cataclasis and frictional sliding.", "Marls deform predominantly by pressure-solution.", "Textural and mineralogical evidence for frictional heating in carbonate slip zones.", "Lithological heterogeneities may control distribution of seismic and aseismic slip." ]
Monocytes Latently Infected with Human Cytomegalovirus Evade Neutrophil Killing	Molecular Mechanism of Behavior; Immunology; Immune Response; Virology	[ "MULT" ]	[ "Immune Response", "Immunology", "Molecular Mechanism of Behavior", "Virology" ]	[ "1000" ]	[ "Human cytomegalovirus (HCMV) is a human herpesvirus that can cause severe disease in immune-suppressed, immune-compromised, and immune-naive individuals.", "For instance, it is the leading viral cause of birth defects in the developed world (Terrazzini and Kern, 2014).", "There is no effective vaccine against HCMV, and routinely used antivirals suffer from a number of drawbacks including poor bioavailability, toxic side effects, and the risk of emergence of drug resistance (Benzi et al., 2012; Hantz et al., 2010; Komatsu et al., 2014; Scott et al., 2007).", "One other aspect of HCMV biology also makes antiviral targeting difficult, and this is the ability of the virus to establish a latent infection in certain cell types in vivo.", "During latent infection, the maintenance of viral genome is underpinned by a latency-associated transcription program but in the absence of virus production.", "This likely helps the virus to avoid immune detection and clearance by host immune responses (Poole et al., 2014a, 2014b; Poole and Sinclair, 2015; Sinclair and Poole, 2014; Wills et al., 2015).", "However, it is now clear that a substantial level of HCMV disease in immunocompromised subjects results from reactivation of virus from these latently infected cells (Sissons and Wills, 2015).", "One site of HCMV latency, in vivo, is in the myeloid cells such as CD14+ monocytes and their CD34+ progenitors.", "As these cells differentiate into dendritic cells (DCs), or macrophages, viral lytic gene expression reactivates leading to viral DNA replication and de novo production of infectious virions.", "Consequently, HCMV lifelong persistence likely results from constant reactivation of the virus from latency, but, in the immune-competent individuals, these reactivation events are kept sub-clinical by normal host immune responses (Poole et al., 2014a, 2014b; Poole and Sinclair, 2015; Sinclair and Poole, 2014; Wills et al., 2015).", "Understanding latent carriage is clearly important for a full understanding of how this persistent human pathogen interacts with its host, and, lately, substantial progress has been made in identifying the effects of latent infection on the latently infected cell.", "For instance, although the transcription program of key lytic genes is heavily repressed during HCMV latency, a number of viral genes are known to be expressed in latently infected myeloid cells (Cheng et al., 2017; Dupont and Reeves, 2016; Shnayder et al., 2018) and the effects of some of these on latently infected cells have been reported (Humby and O'Connor, 2015; Keyes et al., 2013; Lau et al., 2016b; Poole et al., 2014a, 2014b; Weekes et al., 2013).", "This has uncovered a number of ways by which latency-associated viral gene expression manipulates the cell to optimize carriage and reactivation of latent viral genomes (Mason et al., 2012; Poole and Sinclair, 2015).", "Importantly, such studies have also led to proof of principals for chemotherapeutic (Krishna et al., 2017b; Weekes et al., 2013) and immunotherapeutic strategies to target the latent reservoir (Krishna et al., 2016) in vitro, although whether these will have clinical benefits is still under investigation.", "Besides the known changes in the latently infected cell, which can modulate, e.g., cell survival (Krishna et al., 2016; Mason et al., 2012; Poole et al., 2008, 2015; Poole and Sinclair, 2015; Krishna et al., 2017a; Lau et al., 2016b), it is also clear that other latency-associated changes in cellular gene expression can manipulate the cellular microenvironment to also help latently infected cells evade T cell surveillance (Lau et al., 2016b; Mason et al., 2012, 2013).", "However, whereas there is a wealth of data regarding the effector functions of natural killer cells and neutrophils during lytic infection with HCMV (Bennett et al., 2010; Falk et al., 2002; Heo et al., 2015; Magri et al., 2011; Stern-Ginossar et al., 2008; Wills et al., 2005; Yamin et al., 2016), much less is known about whether these immune cells can detect and target latently infected cells and, if so, how latent infection combats such antiviral functions.", "Neutrophils comprise about 50% of all leukocytes.", "These cells are rapidly recruited to sites of infection or inflammation by chemotaxis.", "Here, they shape the immune landscape through interactions with macrophages, DCs, and cells of the adaptive immune response by direct cell-cell contact or via soluble mediators (Mocsai, 2013; Moraes et al., 2006; Nani et al., 2015; Peters and Sacks, 2009; Ribeiro-Gomes and Sacks, 2012; Williams and Chambers, 2014).", "Once recruited, neutrophils become fully activated, characterized by their ability to release granule proteins, their acquisition of phagocytic capabilities, the production of reactive oxygen species (ROS), and their ability to produce neutrophil extracellular traps (NETs), all of which enhance the cells' effector capacity (Brinkmann et al., 2004; Kruger et al., 2015).", "In addition, neutrophils are able to mediate antigen-dependent cell cytotoxicity (ADCC) via Fc receptors on their cell surface, which allows recognition of pathogen-infected cells and target them for killing (Sionov et al., 2015; Sips et al., 2016; Yu et al., 2016).", "Recently, neutrophil-mediated ADCC killing has been the subject of intense research with clinical trials in place for using this function of neutrophils to target cancerous cells (Challacombe et al., 2006; Di Carlo et al., 2001a, 2001b; Matlung et al., 2018; Rajasekaran et al., 2015; Treffers et al., 2018).", "There is also an increasing body of evidence to suggest that neutrophils target virally infected cells (Fujisawa, 2008; Jegaskanda et al., 2013; Worley et al., 2018).", "Neutrophils have been shown to play antiviral roles via ROS for a number of viruses (Drescher and Bai, 2013), and herpesviruses, including HCMV, have been shown to induce ROS in phagocytic cells during lytic infection (Gonzalez-Dosal et al., 2011; Speir et al., 1996).", "Similarly, a number of viruses have also been implicated to be targeted by NETs (Saitoh et al., 2012) as well as ADCC (Sionov et al., 2015; Sips et al., 2016; Yu et al., 2016).", "Indeed, it is known that cells lytically infected with HCMV both induce interleukin (IL)-8 (Costa et al., 2013) and express a virally encoded CXCL1 homolog, UL146 (Luttichau, 2010), which both attracts neutrophils and then uses these attracted immune cells as a “Trojan horse” to help disseminate infectious virions (Pocock et al., 2017).", "Although it is clear that such a strategy could aid the dissemination of virus from lytically infected cells, this will not occur during a latent infection as no virion production occurs during this phase of the virus life cycle (Hargett and Shenk, 2011; Poole et al., 2014a, 2014b) In addition, previous reports have not identified UL146 as being expressed during latency (Goodrum et al., 2002; Shnayder et al., 2018; Slobedman and Cheung, 2008).", "Consequently, we have analyzed whether neutrophils can detect latently infected cells, whether any such response is antiviral, and, if so, how latently infected cells avoid this response.", "Our studies show that latently infected cells can, indeed, be recognized and killed by neutrophils at high Effector to Target (E:T) ratios and that this appears to be via ADCC, which is dependent on the presence of antibodies to the viral US28 protein in HCMV-seropositive serum.", "However, we also show that latently infected cells inhibit recruitment of neutrophils by decreasing the expression of S100A8 and S100A9, two secreted cellular proteins that are known to mediate neutrophil recruitment.", "Preventing this latency-associated downregulation of S100A8/A9 resulted in increased recruitment of neutrophils to latently infected cells and their subsequent killing by ADCC.", "Taken together, our work shows that myeloid cells latently infected with HCMV can be targeted and killed by neutrophils through ADCC in a US28 antibody-dependent manner.", "However, a latency-associated decrease in the expression and secretion of cellular S100A8/A9 prevents recruitment of neutrophils to latently infected monocytes, thereby helping to avoid neutrophil-mediated targeting and elimination of the latent HCMV reservoir.", "Previous analyses have shown that host T cell responses can recognize and target myeloid cells latently infected with HCMV.", "However, changes in the latency-associated secretome inhibit these T cell effector functions (Lau et al., 2016b; Mason et al., 2012, 2013).", "Whether other immune cells can also recognize and target latently infected cells is not known.", "For instance, it is becoming increasingly clear that neutrophils also target virally infected cells (Fujisawa, 2008; Jegaskanda et al., 2013; Worley et al., 2018) and, in the case of HCMV, neutrophils appear to be recruited to lytically infected cells and used to disseminate infectious virions (Pocock et al., 2017).", "However, it is not known if neutrophils recognize latently infected cells and, even if they did, it would be difficult to see how this could be pro-viral, as is seen during lytic infection, because no virion production occurs during latent infection (Hargett and Shenk, 2011; Poole et al., 2014a, 2014b).", "Consequently, we asked if latently infected cells are recognized by neutrophils and what effect this has on the latently infected cell.", "To address this, we generated a recombinant HCMV carrying an mCherry expression cassette under the control of the cellular GATA-2 promoter (TB40E-GATA2-mCherry, detailed in the Methods section), which allowed detection of latently infected cells based on mCherry expression (Figure S1A).", "This virus showed no growth defects during lytic infection (Figure S1B), established latency normally (Figure S1C), produced infectious virus only after reactivation by differentiation (Figure S1D), and resulted in the detection of mCherry expressing latently infected cells for a much longer time frame compared with a recombinant SV40-GFP-HCMV (Figure S1A) in which GFP expression is known to wane after 3–4 days (Krishna et al., 2017b).", "We then used this recombinant virus to analyze whether neutrophils were capable of targeting latently infected cells and what effect this would have on the cells.", "To this end, monocytes were latently infected with TB40E-GATA2-mCherry virus and the monocyte population, both latently infected (red) and bystander uninfected monocytes, were co-stained with calcein dye.", "The monocytes were then co-cultured with purified neutrophils at high E:T ratios; we used higher than physiological E:T ratios of neutrophils to monocytes (with physiological ratios in peripheral blood being approximately 1:5 monocytes:neutrophils) to overcome any potential inhibitors of neutrophil function that might have been produced by the latently infected monocytes.", "Figure 1A shows that addition of neutrophils to experimentally latently infected monocytes from an HCMV-seronegative donor had little effect on the number of experimentally latently infected cells in the population compared with uninfected bystander cells at any E:T ratio in the presence (serum, green line) or absence (media, blue line) of serum from the same seronegative donor.", "However, we reasoned that, besides ROS- and NET-mediated killing, an important mechanism by which neutrophils are known to mediate cell killing of virally infected cells is by ADCC, an antibody-dependent mechanism (Sionov et al., 2015; Sips et al., 2016; Yu et al., 2016).", "Consequently, we repeated the analysis using monocytes and serum from an HCMV-seropositive donor (Figure 1B).", "Figure 1B shows that the number of experimentally latently infected monocytes (that are mCherry positive) from an HCMV-seropositive donor are profoundly reduced, particularly at high E:T ratios of neutrophil:monocytes, when cells were co-cultured in serum from the same HCMV-seropositive donor (serum, green line).", "No such decrease in latently infected monocytes was observed if the analysis was carried out in the presence of media only (media, blue line).", "These data were consistent with the view that neutrophils are able to target and kill latently infected cells but only in the presence of serum from an HCMV-seropositive donor suggesting that this killing may be mediated via ADCC.", "Clearly, addition of neutrophils to latently infected cells in the presence of HCMV serum from a seropositive donor resulted in decreases in the number of monocytes expressing mCherry after latent infection with TB40E-GATA2-mCherry virus.", "Although this was likely due to death of the latently infected cells, we wanted to rule out that this decrease was simply due to, e.g., neutrophil-mediated silencing of mCherry expression in these latently infected cells rather than their neutrophil-mediated killing.", "To do this, we employed live-cell imaging using monocytes latently infected with TB40E-GATA2-mCherry virus and co-stained with calcein such that uninfected bystander monocytes would stain blue/green (calcein positive) and mCherry-positive latently infected cells would stain yellow/white.", "These cultures were co-cultured for 6 h with neutrophils (unstained) at an E:T ratio of 1:20 (this E:T ratio was chosen as it gave good statistically significant neutrophil-mediated reduction of latently infected cells in Figure 1A).", "Figure 2A shows that when experimentally latently infected cells (mCherry) were co-cultured with neutrophils (colorless), in the presence of serum from the HCMV-seropositive donor, uninfected monocytes (green) maintained their cellular integrity as expected at all time points shown.", "In contrast, calcein-positive latently infected cells (yellow/white) in the same field of view (circled in the 6-h panel) clearly blebbed and died.", "This killing of experimentally latently infected cells, easily detectable by 6 h post co-culture, only occurred in the presence of HCMV-seropositive donor serum (Figure 2B, lower panel), but not in the presence of serum from a seronegative donor (Figure 2B, upper panel).", "Interestingly, killing of experimentally latently infected cells by neutrophils in the presence of HCMV-seropositive donor serum only appeared to occur upon close contact between neutrophils and the latently infected monocyte (Figure 2C, black circles 3-h and the 6-h panels).", "In addition, Figure 2C shows that similarly close contact between uninfected monocytes (blue/green) in the same population and neutrophils (colourless) showed no such killing (a monocyte in contact with a neutrophil is circled red in the 3-h panel, and this contact is lost as shown in the 6-h panel, where the monocyte alone is circled in red).", "Quantification of these data from multiple fields of view in Figure 2D shows that, of the latently infected (mCherry) and calcein-stained monocytes (yellow/white) that had contacted a neutrophil only 6% survived.", "However, of the uninfected monocytes in the same population (green) that contacted a neutrophil 84% survived.", "None of the latently infected monocytes (mCherry) and those co-stained with calcein (yellow/white) became green over time, suggesting that the decrease in the number of latently infected monocytes was due to cell death and not silencing of the mCherry cassette (Figure 2D).", "Taken together, these observations suggest that, in the presence of HCMV-seropositive donor serum, neutrophils can target and kill latently infected monocytes and that this requires cell-to-cell contact between the neutrophil and the latently infected cell.", "Our data, so far, had shown that the ability of neutrophils to target and kill experimentally latently infected monocytes required serum from an HCMV-seropositive donor; this suggested that the killing involved ADCC.", "To examine this in more detail, we first tested whether blocking all Fc receptors on the neutrophils had any impact on their ability to kill latently infected monocytes.", "Figure 3A shows that co-culture of latently infected monocytes with neutrophils in control media (with no HCMV-seropositive serum) showed no latently infected cell killing, as expected (media, blue line).", "Importantly, co-culture of latently infected monocytes with neutrophils in the presence of autologous HCMV-seropositive serum, now also showed no killing if the neutrophils were pretreated with media containing an Fc receptor block before incubation with the latently infected CD14+ monocytes (Fc block, red line) when compared with the media-only control (blue line).", "As serum from an HCMV-seropositive donor and not a seronegative donor mediated killing of latently infected monocytes by neutrophils, it seemed likely that this effect was due to an HCMV-specific antibody and one that recognized antigen on the surface of latently infected cells.", "Although it is now becoming clear that the complexity of HCMV latency-associated gene expression is higher than first thought (Goodrum et al., 2002; Rossetto et al., 2013; Shnayder et al., 2018; Slobedman and Cheung, 2008), one well established latency-associated viral gene product, which is likely to be expressed on the surface of latently infected cells, is US28 (Avdic et al., 2016; Krishna et al., 2017b).", "Indeed, although the levels of US28 expression by latently infected CD14+ were variable, US28 was detectable on non-permeabilized cells (Figure S2).", "This confirmed cell surface expression of US28 and argued that US28-specific antibodies in HCMV-seropositive donor serum might recognize US28 on the surface of latently infected cells.", "To test whether recognition of this viral antigen was involved in the observed neutrophil-mediated killing of latently infected cells, we tested whether blocking of US28-specific antibodies in the serum from HCMV-seropositive donors could prevent neutrophil-mediated killing of latently infected monocytes.", "To do this, we removed the Fc portion from the US28-specific rabbit antibody (to prevent any potential contamination of the rabbit Fc regions of the antibodies interfering in the assay), and the resulting F(ab')2fragment of the US28 antibody was then used to pretreat latently infected monocytes before repeating the killing assay in the presence of serum from a seropositive donor.", "Figure 3B shows that when this F(ab')2 fragment specific to US28 was used, neutrophil-mediated killing of latently infected cells in the presence of serum from a seropositive individual was inhibited.", "In contrast, an F(ab')2 fragment from the relative isotype control had no such effect.", "Similarly, Figure 3C shows that if an F(ab')2 fragment specific to viral IL-10 (another well-established latency-associated viral gene product) was used to pretreat the latently infected monocytes, there was also no inhibition of neutrophil-mediated killing.", "Finally, Figure 3D shows microscopy of four representative cells from the experiment shown in Figure 3B and confirms that pretreatment of latently infected monocytes with US28-specific F(ab')2 fragment, but not the relevant isotype-matched control, prevented their killing despite being physically contacted by neutrophils.", "Taken together, these data show that neutrophils can detect and kill HCMV latently infected CD14+ monocytes, at least in part, mediated by US28-specific antibody.", "Thus far, our results had shown that neutrophils could kill latently infected cells but only at high E:T ratios.", "This suggested to us that latently infected cells may be suppressing neutrophil targeting/killing, which could be overcome by using high levels of neutrophils in the assay.", "To investigate this further, we decided to assess changes in total cellular protein expression during latent infection using an unbiased proteomic screen of latently infected CD14+ monocytes in an attempt to identify latency-associated changes in monocyte gene expression, which might be involved in such neutrophil evasion.", "Most previous studies, analyzing global changes in cellular gene expression during HCMV latency, have been carried out against a background of bystander uninfected cells (Mason et al., 2012; Poole et al., 2011, 2014a, 2015; Poole and Sinclair, 2015; Rossetto et al., 2013; Slobedman and Cheung, 2008), which can confound the identification of true latency-associated changes and their effects on cellular gene expression.", "To analyze global changes in cellular proteins accompanying HCMV latent infection of CD14+ monocytes, we initially enriched for the latent HCMV population by infecting monocytes with a clinical isolate of HCMV, TB40E, which expresses GFP from an SV40-GFP expression cassette, and isolated latently infected GFP-positive cells 72 h following infection (Lau et al., 2016a).", "GFP-positive and GFP-negative populations were cultured for a further 3 days (6 days latency, in total) before harvesting for proteome analysis.", "We initially confirmed that the GFP-positive population was latently infected by RT-qPCR for UL138 transcription in the absence of detectable lytic immediate early (IE) gene expression (Figure 4A).", "As further proof of latent infection, we also inoculated indicator fibroblasts with supernatants from latently infected cells.", "These fibroblasts showed no evidence of the presence of infectious virus.", "In contrast, supernatants from latently infected monocytes, which had been reactivated by differentiation into DCs, showed clear evidence of infectious virus production (Figure 4B).", "As expected, we observed no viral gene expression or virion production from the GFP-negative population (Figures 4A and 4B).", "Once we had confirmed that our target cells were latent, they were lysed and proteins analyzed by tandem mass tagging (TMT) and mass spectrophotometry proteomics (Figure 4C).", "We compared GFP-positive and GFP-negative cells (Figure 4C), and as a control, we also compared mock infected cells with cells treated with UV-inactivated virus (Figure S3), which allowed the identification of specific changes in cellular proteins resulting from latency-associated viral gene expression rather than just interactions with virions.", "Total changes in cellular proteins from 2 × 106 cells per condition were quantified by Mascot and Proteome Discoverer.", "This analysis identified the robust induction and repression of a number of cellular proteins in latently infected cells.", "The graph in Figure 4C represents 1,993 proteins detected (the full list of targets is shown in Table S1).", "This analysis resulted in the identification of a number of cellular proteins that changed as a result of latent infection of monocytes and included S100A8 and A9 (detected with 14 and 11 unique peptides, respectively), which were two of the most robustly downregulated proteins during latent infection (Figure 4C).", "No such downregulation of S100A8 and S100A9 was observed by infection with UV-inactivated virus when compared with mock infected cells (Figure S3).", "Consistent with our proteomic analyses, western blot analysis of GFP-positive latently infected monocytes confirmed the downregulation of S100A8 and S100A9 (Figure 4D).", "As S100A8 and S100A9 are secreted as heterodimers (Edgeworth et al., 1991), we tested supernatants of these latently infected cells for secreted S100A8/A9 by ELISA (Figure 4E).", "Consistent with downregulation of both proteins in latently infected cells, a significant decrease in secreted S100A8/A9 was observed in supernatants from latently infected CD14+ monocytes.", "We also observed identical results if monocytes were latently infected with TB40EmCherry-GATA2 virus (Figures S1E and S1F).", "Together, these results show that S100A8/A9 is downregulated during latent infection with HCMV both intra- and extracellularly.", "Intriguingly, S100A8/A9 is a known neutrophil chemoattractant (Gomes et al., 2013; Newton and Hogg, 1998; Ryckman et al., 2003).", "Consequently, we reasoned that downregulation of these proteins during latency could be part of a mechanism to help the latently infected cell avoid neutrophil-mediated killing.", "We first tested whether S100A8/A9 could act as a neutrophil chemoattractant in our system using transwell assays.", "These assays showed that recombinant S100A8/A9 did attract neutrophils and that this was decreased in a dose-dependent manner upon addition of neutralizing antibody to S100A8/A9 in the assays (Figure 5A).", "On the basis that S100A8/A9 is routinely secreted at high levels by myeloid cells, but that its secretion is decreased in latently infected monocytes, we next tested whether supernatants from latently infected CD14+ monocytes had reduced ability to recruit neutrophils compared with uninfected monocytes.", "To do this, CD14+ monocytes were latently infected with TB40E-SV40-GFP virus for 3 days and then GFP+ cells were sorted by fluorescence-activated cell sorting.", "These GFP+ cells were then cultured for a further 3 days (6 days latency) before supernatants were harvested and used to assess their ability to attract neutrophils.", "Figure 5B shows that, as expected, supernatants from control monocytes were capable of recruiting neutrophils, whereas this recruitment was severely impaired if supernatants from latently infected monocytes were used and this impairment could be partially rescued by the addition of recombinant S100A8/A9.", "In essence, the ability of monocytes to recruit neutrophils through the S100A8/A9 axis was severely compromised if these cells were latently infected with HCMV.", "Finally, we asked if addition of S100A8/A9 to latently infected monocyte cultures (to replete the downregulated S100A8/A9) would overcome the low level of neutrophil recruitment to latently infected cells we had previously seen.", "To do this we latently infected monocytes (with TB40E-GATA2-mCherry) that had also been pulsed with calcein.", "These were then added to the lower wells of a transwell plate, and these cells were supplemented with recombinant S100A8/A9.", "We then added neutrophils to the top of the transwell plate (at an E:T ratio of neutrophils that was able to elicit killing of latently infected monocytes as shown in Figures 1 and 3).", "Figure 5C shows that when recombinant S100A8/A9 was added to the latently infected cells to overcome latency-associated S100A8/A9 downregulation, neutrophils now migrated to the latently infected cells, resulting in their killing.", "These results show that the latency-associated reduction in neutrophil migration to, and killing of, latently infected monocytes can be overcome by the addition of recombinant S100A8/A9 to the latently infected cultures.", "Taken together, these data show that neutrophils are able to target and kill HCMV latently infected monocytes via ADCC.", "However, in culture, efficient killing only occurs at high E:T ratios as high numbers of neutrophils are required to overcome the latency-associated downregulation of the neutrophil chemoattractants S100A8/A9, which prevent neutrophil recruitment to the latently infected cell (at low E:T ratios), which results in them being less efficiently targeted.", "It is becoming increasingly clear that latent infection of myeloid cells with HCMV is far from silent or quiescent but results in profound changes in the latently infected cell to optimize viral carriage and reactivation.", "It is also clear that these changes in the cell act, in part, as pro-survival signals (Poole et al., 2011, 2015; Poole and Sinclair, 2015; Slobedman and Cheung, 2008) as well as to prevent surveillance and targeting of the latently infected cell by host immune responses, particularly CD4+ and CD8+ T cells by regulating the latency-associated cell secretome of latently infected CD34+ cells (Mason et al., 2012).", "Exactly where in the host such changes in secreted cell proteins would be most effective is unclear, and there is no tractable animal model for HCMV, so in vivo studies are difficult.", "However, although we do not rule out that such latency-associated changes during latent infection could affect, e.g., CD4+ and CD8+ T cell effector functions in the periphery, we think it likely that such latency-associated changes could aid T cell evasion in, e.g., the microenvironment around latently infected cells in tissues such as bone marrow.", "By the same argument, we feel that latently infected CD14+ cells may also create a microenvironment in sites of latency, and we, therefore, favor the view that this likely occurs in the bone marrow or other tissue sites of latency.", "The routine secretion of S100A8/A9 by monocytes (Ryckman et al., 2003 and Figure 4E) suggests that neutrophils may well be routinely chemoattracted to monocytes.", "Our view is that, because of this, neutrophils may well be constantly sampling potential targets but these would only be routinely killed if they were expressing recognizable signals for neutrophil-mediated killing.", "This would be consistent with neutrophils playing a role in routine surveillance and removal of cancerous (Challacombe et al., 2006; Di Carlo et al., 2001a, 2001b; Matlung et al., 2018; Rajasekaran et al., 2015; Treffers et al., 2018) or virally infected cells (Sionov et al., 2015; Sips et al., 2016; Yu et al., 2016) during normal surveillance.", "However, downregulation of S100A8/A9 from monocytes during HCMV latency could help to reduce this neutrophil surveillance and decrease the likelihood of their killing.", "The ability of a pathogen to limit its visibility to multiple branches of the innate immune system is one immune evasion strategy often employed by pathogens and, in particular, those pathogens that establish latent or persistent infections, and this also includes avoidance of neutrophil killing.", "ADCC-mediated killing of virally infected cells by neutrophils has been reported for a number of viruses (Ackerman et al., 2016; Ashkenazi and Kohl, 1990; Bradford et al., 1992; Chai et al., 2017; Ihara et al., 1986; Siebens et al., 1979; Smalls-Mantey et al., 2013; Veillette et al., 2015).", "However, except for vaccinia virus, which is known to express a protein that interferes with this (Al-Mohanna et al., 2001), little has been reported on the strategies by which other viruses evade neutrophil killing.", "Our results now show that HCMV employs a strategy during latent infection to prevent targeting and killing of latently infected cells by neutrophils.", "This neutrophil targeting is combated by the ability of latent infection to suppress the secretion of the neutrophil chemoattractants S100A8/A9, thereby preventing neutrophil attraction, which otherwise would result in high levels of neutrophil recruitment to, and killing of, latently infected cells.", "The ability of latently infected monocytes to target neutrophils is likely to have far-reaching implications.", "Neutrophils are rapidly recruited to sites of infection or inflammation by chemotaxis where they shape the immune landscape through direct and indirect interactions with macrophages, DCs, and cells of the adaptive immune response.", "Although it is clear that neutrophils play a central role in the control of HCMV lytic replication (Heo et al., 2015; Yamin et al., 2016), what role, if any, neutrophils play in the context of a latent infection has so far been unclear.", "We now show that, at high density, neutrophils are able to detect and kill latently infected cells via ADCC by targeting US28 expression on the latently infected cell, as this killing was blocked by anti-Fc receptor antibody as well as by US28-specific antibodies.", "The ability of HCMV to target S100A8/A9 during latent infection may have additional roles beside evasion of neutrophil targeting.", "For example, it is known that S100A9 prevents DC differentiation (Cheng et al., 2008), and, therefore, any latency-associated reduction of S100A8/A9 in monocytes might modulate their responses to differentiation signals, ensuring efficient reactivation only when conditions are optimal.", "It is now clear that latent infection of CD14+ monocytes results in major changes in cellular gene expression at the protein level and that this includes changes in cellular proteins that interfere with neutrophil-mediated killing of latently infected cells by decreasing their expression of the neutrophil-chemoattractant S100A8/A9.", "Therapies to target virus-infected cells with neutrophils are currently ongoing for HIV-1 and influenza infection (Chai et al., 2017; Stephenson et al., 2016; Wines et al., 2017).", "Knowing that monocytes, which carry latent HCMV, can be targets of host neutrophils, but that this is combated by latency-associated changes in the cell, opens up the possibility that this viral immune evasion mechanism during latency could, itself, be exploited by strategies to help target the latent reservoir.", "This work shows that avoidance of neutrophil recruitment is an immune evasion strategy during experimental latency of HCMV.", "However, as there is no tractable animal model for HCMV latent infection the findings cannot, as yet, be verified in vivo.", "All human samples were obtained under ethical approval and after approval of protocols from the Cambridgeshire 2 Research Ethics Committee (REC reference 97/092) conducted in accordance with the Delcaration of Helsinki.", "Informed written consent was obtained from all the volunteers included in this study before providing blood samples, and all experiments were carried out in accordance with the approved guidelines.", "All methods can be found in the accompanying Transparent Methods supplemental file." ]	[ "Neutrophils can target HCMV latently infected monocytes for ADCC-mediated killing", "Killing of latently infected cells by neutrophils requires high E:T ratios", "Latent infection reduces secretion of the neutrophil chemoattractants S100A8/A9", "Decreased S100A8/A9 secretion prevents neutrophil targeting of latently infected cells" ]
Selective formation of copper nanoparticles from acid mine drainage using nanoscale zerovalent iron particles	Nanoscale zerovalent iron (nZVI) has been investigated for the selective formation of Cu nanoparticles from acid mine drainage (AMD) taken from a legacy mine site in the UK. Batch experiments were conducted containing unbuffered (pH 2.67 at t = 0) and pH buffered (pH < 3.1) AMD which were exposed to nZVI at 0.1–2.0 g/L. Results demonstrate that nZVI is selective for Cu, Cd and Al removal (>99.9% removal of all metals within 1 h when nZVI ≥ 1.0 g/L) from unbuffered AMD despite the coexistent of numerous other metals in the AMD, namely: Na, Ca, Mg, K, Mn and Zn. An acidic pH buffer enabled similarly high Cu removal but maximum removal of only <1.5% and <0.5% Cd and Al respectively. HRTEM-EDS confirmed the formation of discrete spherical nanoparticles comprised of up to 68% wt. Cu, with a relatively narrow size distribution (typically 20–100 nm diameter). XPS confirmed such nanoparticles as containing Cu° with the Cu removal mechanism therefore likely via cementation with Fe°. Overall the results demonstrate nZVI as effective for the one-pot and selective formation of Cu°-bearing nanoparticles from acidic wastewater, with the technique therefore potentially highly useful for the selective upcycling of dissolved Cu in wastewater into high value nanomaterials.	[ "ENVI" ]	[ "Acid mine drainage", "Aluminium", "Cementation", "Copper", "Nanoscale zerovalent iron particles" ]	[ "2304", "2305", "2307", "2310", "2311" ]	[ "Acid mine drainage (AMD) is an acidic solution that typically contains elevated concentrations of heavy metals and is caused by the microbial oxidation of pyrite and other sulphidic minerals, as a consequence of the mining and processing of metal ores and/or coals [1,2].", "Sulphidic minerals are extremely common and consequently many effluents derived from abandoned mines and mine wastes therefore exhibit a net acidity [1].", "If left untreated AMD can contaminate ground and surface water and thereby damage ecosystems and potentially impact human health.", "As such the generation of AMD is often cited as one of the most prominent environmental issues currently facing the mining industry [1].", "For example, in the UK alone it is estimated that as many as six percent of all surface water bodies are currently adversely affected by AMD derived from legacy metalliferous mines [3].", "Similarly in the USA it is estimated that as many as 19,300 km of rivers and more than 180,000 acres of lakes and reservoirs have been damaged by AMD [4].", "Consequently, the development of cost-effective remediation solutions for AMD has been the subject of intense research for several decades.", "Despite such interest, conventional AMD remediation technologies remain relatively expensive and/or require significant construction space (e.g. passive treatment), which poses a major challenge [1].", "AMD often contains a wide range of metals and metalloids which while being toxic to the environment would also be beneficial to recover (e.g. Cu, Ni, Zn).", "Whilst it is possible to recover metals from dilute aqueous effluents using existing active treatment technologies e.g. ion exchange [1], the absence of widespread commercial practices for the recovery of metals from AMD indicates that the economic value of the metals rarely offsets the operating costs of recovery.", "As such a fundamental barrier exists where either the cost of AMD treatment needs to significantly decrease or the economically valuable metals present in such waste needs to be valorised into higher-value products, i.e. products which are worth significantly more than their raw metal value.", "With regard to the latter, one potential solution is to develop methodologies to directly synthesise high-value nanomaterials from AMD.", "Such nanomaterials could then be directly utilised as high value materials and/or reagents in a range of different processes, and as such provide significantly greater economic return than their equivalent bulk scale metal.", "In recent years, nanoscale zero-valent iron (nZVI) has gained much attention for its use in a wide range of water treatment applications due to its unique properties, including high surface area to volume ratio and an ability to be injected into the subsurface as a colloidal suspension [5–8].", "To date nZVI has been demonstrated as highly effective for the removal and/or degradation of a wide range of metals and metalloids (e.g. [7,9–17]), inorganic anions (e.g. [18]) and organic contaminants (e.g. [19–21]) from solution.", "Very little is known, however, with regard to the physicochemical properties of metals and metalloids when sorbed and/or chemically reduced by nZVI, and in particular whether such precipitates/particles could be recovered for economic gain.", "In particular almost all studies to date have investigated the use of nZVI for the sorption and/or enmeshment (i.e. the immobilisation) of metal and metalloid species.", "In contrast the use of nZVI for the in situ synthesis and recovery of functional (nano)materials from wastewater or contaminated land is a new research arena.", "Preliminary evidence has already been presented on the formation of iron-bearing nanomaterials from AMD [22], however, to the best of our knowledge no studies have yet investigated the selective formation of heavy metal (e.g. Cu, Zn, Al) nanomaterials from AMD.", "It is well known that nZVI is highly efficient for the removal of aqueous Cu under a range of different chemical conditions (e.g. [10,23] and [24]), however, the extent at which nZVI might be selective for the removal of aqueous Cu from wastewater (such as AMD) remains unknown.", "Furthermore very little is known with regard to the physico-chemical properties of such recovered Cu, such as whether, and if so under what conditions, discrete Cu-bearing nanoparticles might form due to the cementation reaction between aqueous Cu and Fe°.", "This study has been established in order to investigate these phenomena in order to gain an understanding of the extent at which nZVI could be used for the selective formation of Cu-bearing nanoparticles from AMD which could prove a highly useful mechanism for the valorisation of Cu-bearing AMD, thereby unlocking a new economic incentive for AMD treatment.", "Such novel approaches are urgently required from addressing AMD which is one of the most prominent environmental issues currently facing the mining industry, and regarded by the European Environment Bureau and the US Environmental Protection Agency and as “second only to global warming and stratospheric ozone depletion in terms of global ecological risk” [25].", "The AMD used in this study was collected from Parys Mountain which is a disused open cast Cu-Pb-Zn mine on Anglesey (Wales, UK) [26].", "Samples were collected from the Duffryn Adda adit (GPS location: 53°23′40.96 N, 4°21′01.80 W) and sealed in high-density polyethylene bottles (without headspace) and stored at 4 °C until required (maximum storage time was 7 days).", "The AMD initially contains low concentrations of dissolved oxygen (DO), (e.g. <2 mg/L), however, it quickly equilibrates with the atmosphere when in surface waters to reach oxygen concentrations more typical for that of vadose and/or surface waters (e.g. ∼7–10 mg/L), changing its redox potential and associated heavy metal transport properties in the process.", "Pure nZVI were synthesised following the methodology first described by Glavee et al., [27] and then adapted by Wang and Zhang [28].", "7.65 g of FeSO4·7H2O was dissolved in 50 mL of Milli-Q water (>18.2 MΩ cm) and the pH was adjusted to 6.8 using 4 M NaOH.", "NaOH addition was performed slowly, drop-wise, to avoid the formation of hydroxo-carbonyl complexes.", "The salts were reduced to nZVI by the addition of 3.0 g of NaBH4.", "The nanoparticle product was isolated from the aqueous phase via centrifugation (Hamilton Bell v6500 Vanguard centrifuge, 6500 rpm for 120 s), rinsed with absolute ethanol (Fisher Scientific, 12478730; ratio of 50 mL/g of nZVI) and then centrifuged (Hamilton Bell v6500 Vanguard centrifuge, 6500 rpm for 120 s).", "This step was then repeated three more times.", "The nanoparticles were dried in a vacuum desiccator (approx. 10−2 mbar) for 72 h and then stored in an argon filled (BOC, 99.998%) MBraun glovebox until required.", "Prior to conducting any nZVI-AMD exposure experiments the AMD was removed from the refrigerator and allowed to equilibrate in the ambient laboratory (temperature = 20.0 ± 1.0 °C) for 24 h. Unless specified differently all batch systems comprised 200 mL volume of the AMD in 250 mL clear soda lime glass jars.", "Following nZVI addition the batch systems were immediately sonicated for 120 s using an ultrasonic bath (Grant, XB3).", "Each system was then sealed (using a screw cap) and placed on the benchtop in the open laboratory.", "Periodic sampling of dissolved oxygen (DO), oxidation reduction potential (ORP) and pH was conducted by gently agitating each batch system in order to ensure homogeneity.", "The pH, Eh and DO probes were calibrated prior to each measurement.", "The measured Eh values were converted to Eh (vs. standard hydrogen electrode) by subtracting the difference between the measured Eh of the reference solution (220 ± 5 mV) with the true Eh of the reference solution (Mettler Toledo 220 mV/pH 7 reference solution).", "5 mL aqueous-nZVI suspensions were periodically taken using an auto-pipette.", "The extracted suspensions were centrifuged at 4000 rpm (3077 g) for 240 s after which the supernatant became clear (i.e. all of the nZVI was centrifuged to the bottom of the vial).", "The supernatant was then extracted using a 10 mL syringe and filtered through a cellulose acetate filter 0.2 μm filter.", "The filtered water was prepared for inductively coupled optical emission spectrometry (ICP-OES) by the additional of HNO3 at a concentration of 2% by volume.", "The solid nZVI plug at the base of each centrifuge vial was prepared for X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and high resolution transmission electron microscopy (HRTEM) by an absolute ethanol wash.", "This was conducted by adding 20 mL of ethanol (Fisher Scientific, 12,478,730) and then gently agitating the centrifuge vial in order to suspend the nZVI plug.", "The vial was then centrifuged at 4000 rpm (3077 g) for 240 s in order to separate the solid and aqueous phases.", "The supernatant was then removed and the process was repeated a further two times.", "Each time the supernatant was decanted the nZVI plug at the bottom of the centrifuge vial was maintained in place using an Eclipse 20mm Neodymium Pot Magnet (length 25mm, pull force 28kg).", "Once the ethanol washing process was completed the nZVI plug was pipetted onto a glass optical microscope slide (Agar Scientific, G251P) for XRD and XPS analysis and a Au coated holey carbon film (TAAB, C062/G) for HRTEM analysis.", "Samples were then dried in a vacuum chamber at <1 × 10−2 mbar for a minimum of 2 h prior to analysis.", "In order to test the influence of a constant pH on the metal/metalloid removal behaviour from the AMD onto nZVI pH buffered batch systems were created.", "180 mL of the AMD was first added to two different 250mL clear soda lime glass jars.", "20 mL solutions of 0.05 M or 0.1 M HCl with both containing 1.0 g of potassium hydrogen phthalate (C8H5KO4) was then added to each of the batch systems.", "The solutions were then gently stirred and left for 24 h to equilibrate prior to nZVI addition.", "Sorption-desorption experiments were then conducted following the same methodology outlined above.", "In order to test the reusability of nZVI for the removal of metal/metalloids from the AMD the nZVI were exposed to the AMD (40 mL of AMD and nZVI at 1g/L) inside 50 mL centrifuge vials.", "The batch systems were first sonicated using an ultrasonic bath (Grant, XB3) for 120 s and then left for 30 min.", "The batch systems were then centrifuged at 4000 rpm (3077 g) for 240 s.", "The supernatant was then filtered through a cellulose acetate filter 0.2 μm filter and prepared for ICP-OES analysis following the technique described above.", "The nZVI plug at the bottom of the centrifuge vial was maintained in place using an Eclipse 20mm Neodymium Pot Magnet (length 25mm, pull force 28kg) and the remaining supernatant was decanted.", "40 mL of fresh AMD was then added and the steps listed above were repeated six times.", "All sorption-desorption experiments were conducted at room temperature (measured to be 20.0 °C ±1.0 °C) and ran as duplicate pairs, with the average data used to create the figures/tables displayed herein.", "A Phillips Xpert Pro diffractometer with a CoKa radiation source was used for XRD analysis (generator voltage of 40 keV; tube current of 30 mA).", "XRD spectra were acquired between 2h angles of 10–90°, with a step size of 0.02° 2 and a 2 s dwell time.", "ICP-OES analysis of aqueous samples was performed using a PerkinElmer Optima 2100 DV ICP-OES.", "HRTEM analysis was performed using a JEOL JEM-2100 microscope at 200 kV.", "Energy dispersive spectroscopy (EDS) analysis and mapping was performed using Oxford Instruments X-MaxN analyzer and Aztec software.", "A beryllium sample holder was used in order to prevent any background Cu from being detected.", "Nanoparticle size distribution from HRTEM images was measured using ImageJ software (Java 1.6.0_24) with 50 nanoparticles analysed per sample.", "X-ray photoelectron spectroscopy (XPS) spectra were collected using a Thermo K-Alpha+ spectrometer.", "Spectra were collected at a pass energy of 40 eV for narrow scans and 150 eV for survey scans with a 0.1 and 1 eV step respectively.", "Charge neutralisation was achieved using a combination of low energy electrons and argon ions.", "Spectra were quantified in CasaXPS using Scofield sensitivity factors and an energy dependence of -0.6.", "In order to determine the relative proportions of Fe2+ and Fe3+ in the sample analysis volume, curve fitting of the recorded Fe 2p3/2 photoelectron peaks was performed following the method of Scott et al. [29].", "The Fe 2p3/2 profile was fitted using photoelectron peaks at 706.7, 709.1, 710.6 and 713.4 eV corresponding to Fe°, Fe2+ octahedral; Fe3+ octahedral and Fe3+ tetrahedral.", "These parameters were selected on the basis that the surface oxide was assumed to be a mixture of wüstite and magnetite, as the oxide Fe2+ is in the same coordination with the surrounding oxygen atoms in both forms of oxide.", "Characterisation of the as-formed nZVI was performed using BET surface area analysis, HRTEM, XRD and XPS.", "BET surface area analysis determined that the surface area of the nZVI was 50.9 m2/g.", "HRTEM analysis determined that the nZVI were spherical, generally within a size range of 10–150 nm and an average diameter of 61 nm (Fig. 1).", "Each individual nZVI particle was recorded to contain a discrete outermost layer (density contrast), which is attributed to be the presence of an oxide shell surrounding the Fe° core.", "In addition dark mottles were recorded within the metallic cores which indicates that individual particles are either polycrystalline or comprised isolated metal crystals in an otherwise amorphous matrix.", "Individual nZVI particles were aggregated into chains and rings due to their high surface energy and magnetic properties [30].", "A single diffraction peak at 52.381° 2θ was recorded using XRD and attributed to the (110) lattice reflection of α-Fe° (Fig. 2).", "The peak was relatively broad which indicates that the nZVI are relatively amorphous.", "XPS analysis determined the outer surface of the nZVI is comprised of a mixed valent (Fe2+/Fe3+) oxide overlying a Fe° core.", "Given the mean free path of Fe is equivalent to approximately 6 atomic layers, this detection of Fe° in the XPS analysis volume therefore indicates that the oxide thickness is likely to be <5nm, which corroborates the aforementioned nZVI oxide thickness measurement using HRTEM.", "The results are summarised in Table 1.", "Prior to nZVI addition the pH, Eh and DO of the AMD was measured along with the concentrations of dissolved metals and metalloids using ICP-OES.", "The water was acidic (pH = 2.67) and oxygen-rich (DO and Eh 395 mV and 8.98 mg/L respectively).", "In order to highlight the highly polluted nature of the water, concentrations of notable metal and metalloids present in the AMD are presented along with their World Health Organisation (WHO) recommended drinking water human health guideline concentrations [31] in Table 2, and were used throughout this paper as reference conditions.", "Several elements were recorded as exceeding the guideline concentrations, namely Mn, Ni, Cu and Cd by factors of 44.8, 1.9, 22.7 and 56.7 respectively.", "The concentrations of all metals and metalloids analysed using ICP-OES is displayed in Table S1.", "The addition of the nZVI to all batch systems containing AMD (no pH buffer) resulted in a rapid decrease in Eh and DO and a concurrent increase in pH (Fig. 3).", "Most significant change was recorded for the batch systems containing the largest concentration of nZVI with only relatively minor changes recorded for the batch systems containing nZVI at <0.5 g/L.", "Eh minima were recorded for the AMD exposed to nZVI at concentrations of 0.2, 0.5, 1.0 and 2.0 g/L within the first 1 h of reaction, with 428, -21, −231 and −319 mV respectively recorded.", "In contrast an Eh minimum of 457 mV was recorded after 24 h exposure of the AMD to nZVI at 0.1 g/L.", "The Eh in all systems was 395 mV at t = 0.", "Maxima in pH were recorded within the first four hours of exposure of the AMD to nZVI at concentrations of 0.2, 0.5, 1.0 and 2.0 g/L, with 3.02, 4.52, 6.02 and 6.24 recorded respectively.", "In contrast a pH maximum of 2.82 was not recorded until after 24 h exposure of the AMD to 0.1 g/L nZVI.", "The pH was 2.67 at t = 0 in all systems.", "Such redox and pH changes are attributed to the rapid oxidation of nanoparticulate surfaces during their initial exposure to the AMD, consuming DO and H+ and reducing the Eh of the system.", "Following this initial reaction period gradual reversal to ambient pH, Eh and DO conditions was observed for all systems, which is attributed to the reactive exhaustion of the nZVI (i.e. total transformation of Fe° to Fe3+ (hydr)oxide products).", "Changes in pH, Eh and DO recorded following exposure of the nZVI to the pH buffered AMD is displayed in Fig. S1.", "It can be observed that similar changes in Eh and DO occurred when compared to the unbuffered AMD, however, only a minor increase in pH was recorded, with maxima of 2.30 and 3.09 recorded after 24 h for the batch systems containing buffer reagents 0.1 g/L C8H5KO4 and 0.01M HCl 0.1 g/L and 0.1 g/L C8H5KO4 and 0.005M HCl respectively (pH was 1.91 and 2.42 at t = 0 respectively).", "Figure S2 displays pH, Eh and DO changes for AMD when fresh AMD was added to the nZVI every 30 min.", "Similar changes in pH, Eh and DO were observed for the first two exposures, however, after the third exposure of the nZVI to the AMD a clear return to ambient electrochemical conditions was recorded and attributed to the reactive exhaustion of the nZVI.", "This was corroborated visually; the batch systems eventually became clear due to the total dissolution of the nZVI into the AMD.", "The addition of the nZVI to the AMD (no pH buffer) resulted in significant changes in the aqueous concentration of several different metals and metalloids (Fig. 4 and Table 2).", "The most significant concentration decreases were recorded for Cu, Al and Cd with removal of these metals to below ICP-OES detection limits (after 10X dilution) recorded within 2 h reaction time when using nZVI concentrations of ≥ 1 g/L, followed by retention below WHO specified drinking water guideline concentrations (Table 2) for time periods ≥24 h.", "A decrease in the concentrations of Zn and Ni was also recorded but at a lower magnitude (both in terms of relative quantities removed and the duration of removal).", "The Zn concentration minimum was 14.9 mg/L (nZVI concentration: 2 g/L, sampling time: 48 h), or 78.8% removal.", "However, Zn removal of <50% was recorded for all other sampling times with the exception of the 24 h sampling time (when using a nZVI concentration of 2 g/L), where removal of 72.0% was determined.", "Removal of Ni to below WHO specified drinking water guideline concentrations was recorded throughout the entire reaction period except the 28 d sampling time for the batch system containing nZVI at a concentration of 2 g/L, however, removal of only <50% was typically recorded for all other nZVI concentrations.", "Minimal concentration changes were recorded for major cations: Ca, Mg, K and Mn (Table 2) which demonstrates that nZVI is selective for the removal of certain metals from solution (namely those whose solubility is redox sensitive).", "It has been reported that the removal mechanism of metals and metalloids by nZVI is typically via adsorption/surface complexation for metal ions which have the standard electrode potentials (E°) for reduction to a metallic state that are very close to, or more negative than Fe° (-0.44 V), such as Zn2+.", "Moreover, for metal ions such as Cu2+ which have a E° that is much more positive than Fe°, removal is typically via surface-mediated cementation [7], Eq. (1).", "Fe° + Cu2+ = Fe2+ + Cu° E° = 0.78Metal cations such as Ni2+ which are only slightly more electropositive than Fe° are typically removed from solution via a combination of adsorption and (partial) chemical reduction.", "The E° for the cathodic reduction of aqueous K+, Ca2+, Na+, Mg2+ and Mn2+ to their metallic state is −2.92, −2.84, −2.71, −2.37 and −1.18 V respectively.", "The lack of removal from solution of such ions onto nZVI is therefore likely to be due to their more negative E° (for reduction to a metallic state) than Fe°.", "Significant removal of Al onto nZVI was recorded, however, despite the E0 for the cathodic reduction of aqueous Al3+ to its metallic state being −1.66 V, and therefore considerably lower than −0.44 V. However, within the pH range of approximately 4–8 A l is known to readily precipitate from solution as a hydroxide, namely gibbsite (Al(OH)3) [32].", "Similarly, the E0 for the cathodic reduction of aqueous Zn2+ and Cd2+ to their metallic state are −0.76 and −0.40 V respectively and as such their removal by nZVI recorded herein is likely also to be due to the pH increase, resulting in the co-precipitation (and adsorption) of Zn and Cd with nZVI corrosion products and/or iron (hydr)oxides derived from native iron within the AMD [32].", "In contrast to other metal/metalloids the concentrations of Fe, Na and B were recorded to increase in all batch systems, with a relatively linear correlation with the mass of nZVI added observed, and as such their behaviour is attributed to the dissolution of such metals from the nZVI, with Na and B likely to have been derived from the NaBH4 used in the nZVI synthesis.", "Fig. 5 displays dissolved metal concentration data as a function of time for the pH buffered AMD following exposure to the nZVI at a 1.0 g/L concentration.", "It can be observed that in contrast to the unbuffered AMD system (Fig. 4) no appreciable removal of Al, Zn or Cd was recorded, which is attributed to the increased solubility of such metals at the lower pH [32].", "Fig. 6 displays changes in the concentration of notable dissolved metals as a function of time for the AMD when fresh AMD was added to the nZVI every 30 min.", "Results demonstrate that under such conditions nZVI was least able to retain Al (desorption occurred upon the second exposure to the AMD) followed by Cu, Ni and Cd (desorption of such metals occurred upon the third exposure to the AMD).", "In contrast removal of >20% of dissolved Zn was recorded in all instances, which suggests that the nZVI are suitable to be reused for Zn removal from solution.", "Fe and B concentrations were recorded to initially increase and then decrease in the latter stages of the experiment.", "This is attributed to the dissolution of the nZVI into the acidic AMD which was also observed visually: the nZVI suspensions became progressively clearer due to the progressive and eventually total dissolution of the nZVI into the AMD.", "It can be observed in Table 2 and Fig. 4 that following their initial removal from the aqueous phase significant release of heavy metals (namely Al, Zn, Ni and Cd) was recorded in all systems.", "This behaviour is in agreement with other studies (e.g. [33]) and was coincident with the reactive exhaustion of the nZVI and consequent reversal of the solution chemistry to pre-nZVI addition conditions, resulting in a combination of dissolution and desorption of metals from corrosion products as pH reverted to lower values and Eh increased in response to atmospheric oxygen ingress.", "The re-dissolution of Cu was recorded to commonly occur in the latter stages of the exposure of nZVI to both unbuffered and pH buffered AMD and probably resulted from the oxidative dissolution of Cu° to Cu2+ as Eh climbed above ∼200 mV.", "XPS observations (see Section 3.5.2.)", "provide further evidence of this mechanism.", "This behaviour provides clear evidence that nZVI is only suitable for the recovery of Cu from DO-bearing AMD over relatively short time periods (e.g. <48 h), with the recovery of nZVI (and cemented Cu) necessary within such a time period, unless either favourably low Eh can be maintained or pH can be buffered to remain circumneutral or basic.", "Interesting this phenomena could potentially be harnessed as a new method to concentrate Cu whereby following the selective recovery of Cu from the AMD the nZVI (and sorbed Cu) could then be separated into a smaller volume of AMD (e.g. via centrifugation or coagulation/flocculation) with the Cu then allowed to be release back into the aqueous phase, thus concentrating the Cu for final recovery.", "XRD was used to determine the bulk crystallinity and composition of nZVI solids extracted from all batch systems at periodic time intervals during their exposure to the AMD (Fig. S3).", "A transition from Fe°, with a well-defined peak centred at 52.6° 2θ (lattice reflection: Fe(110)), to a broad (and low intensity) peak centred at approximately 25° 2θ was recorded for all systems, indicating the formation of an amorphous iron hydroxide phase.", "In systems containing nZVI at concentrations of 0.1–1.0 g/L Fe° was not detected at the first sampling time of 30 min.", "In contrast Fe° was detected until 24 h for the system containing nZVI at 2.0 g/L.", "In all systems the amorphous iron hydroxide phase remained until the end of the 28 d reaction period, however, goethite (α-FeOOH) emerged after 7 d reaction for the batch system containing nZVI at 2.0 g/L, with greatest intensity peaks centred at 24.6, 38.8, 42.8, 46.9, 48.2 and 62.6° 2θ recorded corresponding to lattice reflections of 110, 130, 111, 210, 140 and 221 respectively.", "Goethite continued to crystallize into the latter stages of the experiment to become the sole crystalline corrosion product after 28 d. However, the intensity of the goethite peaks remained relatively low.", "This was not unexpected given that the transformation of amorphous iron hydroxides to goethite can be efficiently retarded by the presence of co-precipitated Al [34].", "XRD spectra recorded for nZVI extracted from the pH buffered AMD is displayed in Fig. S4.", "It can be observed that, similar to the unbuffered system, a transition from Fe°, with a well-defined peak centred at 52.6° 2θ (lattice reflection: Fe(110)), to a broad (and low intensity) peak centred at approximately 25° 2θ was recorded and attributed to the formation of amorphous iron hydroxide.", "XPS was used to characterise the surface chemistry of nZVI following exposure to the unbuffered and pH buffered AMD including any sorbed or cemented species (Table 3 and Fig. 7).", "Fe2p3/2 photoelectron peaks recorded for unreacted nZVI confirmed the outermost surface of the nanomaterial to be a mixed valence oxide, with a Fe2p3/2 peak centred at 710.9 eV (Section 3.1).", "Fe° was also detected, which as discussed in Section 3.1.,", "indicates that the oxide thickness of the as-formed nZVI is likely to be <5nm.", "Subsequent XPS analysis of nZVI extracted from both unbuffered and pH buffered AMD at periodic time intervals determined a slight shift in the location of the Fe2p3/2 peak to between approximately 711.1 and 711.5 eV for nZVI extracted after time periods ≤1 h (Fig. S5).", "This was then maintained at approximately ± 0.2 eV for the entire reaction period.", "In addition Fe° was not detected in any samples.", "This provides clear evidence that the entire mass of Fe° located in the outer ∼5 nm of each nZVI was oxidised (to Fe2+) within 30 m of exposure to the AMD (i.e. the oxidation was rapid and did not leave behind any regions of unoxidised Fe°).", "O1s spectra recorded for the unreacted nZVI (Fig. S5) confirmed that the outmost surface was a mixture of metal oxide (binding energy 529–531 eV) with a small component of surface hydroxyl (binding energy 530-532 eV) and H2O (binding energy (531–533 eV), which agrees with previous studies (e.g. [8]).", "Following exposure of the nZVI to the AMD a decrease in the proportion of metal oxide photoelectron peaks relative to hydroxyl and H2O was recorded and attributed to the oxidation of nZVI surfaces and consummate sorption of H2O and formation of metal hydroxide phases.", "Al2p spectra (only detected for the nZVI following exposure to the unbuffered AMD) were centred at binding energies of ranging from 74.4 to 74.9 eV (mean = 74.7 eV) with no shoulder peak(s) recorded at 72.7 ± 0.2 eV.", "XPS spectra for Al oxides and hydroxides are known to overlap each other [35] and as such differentiation between such phases was not possible, however, this result indicates that Al° was not present (Al° is typically recorded at a binding energy of 72.7 ± 0.1 eV) [35].", "No clear change in the Al2p photoelectron peak shape or binding energy was recorded as a function of time for the samples indicating that no change in the composition of sorbed or precipitated Al occurred.", "Cu2p3/2 spectra were asymmetric, with curve fitting yielding two or three photoelectron peaks centred at binding energy ranges of 932.8–933.3 eV (mean = 933.0 eV) and 935.2-935.9 eV (mean = 935.5 eV) along with two shake-up satellites centred within the range of 940.5-940.9 eV (mean = 940.8 eV) and 943.4-943.9 eV (mean = 943.6 eV), Fig. 6.", "Cu° and Cu+ are typically cited as exhibiting XPS Cu2p3/2 spectra centred at binding energies of 932.6 and 932.4, however, an additional shake-up satellite centred at approximately 945.0 eV is also typically indicative of the latter specie (in the form of Cu2O) [36].", "Tetrahedral and octahedral Cu2+ is typically cited as exhibiting a photoelectron peak at approximately 933.6 and 934.0 eV respectively along with shake-up satellites centred at approximately 940.8 and 943.6 eV [37].", "It is therefore likely that the Cu was likely present as a mixture of Cu° and octahedral Cu2+, with the former species likely formed via cementation with Fe° from the nZVI (Eq. (1)).", "The presence of octahedral (rather than tetrahedral) Cu2+ indicates that it was perhaps incorporated into the nZVI structure via substitution upon octahedral sites, such as spinel (e.g. magnetite) [37].", "A clear decrease in the proportion of Cu° relative to Cu2+ was recorded as a function of time for the nZVI when exposed to the AMD (Fig. 6).", "This coincides with the significant release of Cu which was recorded using ICP-OES (Fig. 4), which therefore indicates that such Cu release is likely to be related to Cu° reoxidation.", "Table 3 displays the surface concentration of different elements (at.", "%) determined using XPS analysis of the nZVI extracted at periodic time intervals following exposure to the AMD.", "Following the addition of the nZVI to the AMD an increase in the proportion of O and a commensurate decrease in the proportion of Fe was recorded and attributed to the build-up of nZVI corrosion phases at the surface of the nanomaterial.", "Appreciable surface concentrations of Cu was also recorded for nZVI extracted from both unbuffered and pH buffered AMD, however, in contrast to the nZVI extracted from the unbuffered AMD very low (or zero) concentrations of both Al and Zn were detected.", "Cu concentration maxima of 6.60 at.", "% was recorded for pH buffered system and attributed to the greater dissolution of Fe (from the nZVI) in such systems which in turn decreased the ratio of solid Fe to Cu.", "HRTEM-EDS images of the nZVI after exposure to the AMD at a concentration of 1.0 g/L for 4 and 24 h are displayed in Fig. 8 with additional EDS maps displayed in Figures S6 and S7 and metal concentration data displayed in Table 4.", "It can be observed that Fe, O, Al, Cu, Zn, Si and S comprise >99% of all samples analysed with trace quantities of Mg, Ca, Mn and Cl recorded.", "Three types of nanomaterial can be observed: (i) “spherical” nanoparticles which exhibit a core-shell structure; (ii) “spherical” nanoparticles which do not exhibit a core-shell structure; and (iii) “needle” or “rod” shaped nanoparticles.", "The latter two nanomaterials were more prevalent in the samples extracted during the latter periods of the experiment (e.g. >7 d), indicating that they were likely to be either nZVI corrosion products or precipitated native iron (hydr)oxides.", "In the vast majority of instances Cu was detected as being relatively diffuse (i.e. no clearly defined pure Cu nanoparticles were detected without also being co-located with Fe) for the nanoparticles produced by the exposure of the nZVI to the unbuffered AMD water.", "In contrast clearly defined discrete Cu-bearing nanoparticles were recorded for the pH buffered system (Figs. 9 and 10).", "A summary of the physico-chemical properties of such Cu-bearing nanoparticles is displayed in Table 5.", "Similar to the nZVI which were exposed to the unbuffered AMD Fe and O were recorded as comprising a major component (i.e. typically >80 wt.", "%) of the material detected using EDS for the nZVI exposed to pH buffered AMD.", "However, in contrast no Al or Zn was detected and Cu was detected in higher concentrations.", "It addition it is observed that a major proportion of the Cu-bearing nanoparticles detected were located in regions that are distinctly separate to the Fe bearing nanoparticles, indicating that they are discrete nanoparticles and thus able to be separated from each other.", "In some instances, however, the Cu-bearing nanoparticles were recorded as surrounding a larger Fe bearing nanoparticle.", "This indicates that such Cu nanoparticles are likely formed via a surface mediated process (e.g. the Cu was deposited as a nanoparticulate layer upon the surface of each dissolving nZVI particle).", "Almost all conventional methods for AMD treatment (e.g. alkali dosing or passive bioreactors) are not designed with metal recovery in mind.", "As such the wastes produced (e.g. ochreous sludges or metal sulphide-rich organics) can require expensive long-term management associated with their treatment and/or disposal.", "Instead a paradigm shift is required in order to reconsider AMD as a resource rather than simply an environmental liability.", "One option is to develop novel methods that selectively recover key metals/metalloids from AMD and thus provide economic value in order to offset the costs of such remediation activities.", "An intrinsic barrier associated with this, however, is that valuable metals in AMD are often present at relatively low concentrations (e.g. <100 mg/L) and as such their recovery and conversion to bulk/sheet metal is not typically economically viable.", "Instead, if metals could be directly converted into valuable (nano)material products then a clear economic driver could be realised.", "For example, the current raw metal value of Cu is approximately 6800 USD/t [38] compared to either approximately 10,000 USD/t for bulk orders of Cu° nanoparticles of relatively low purity and unconstrained particle size distributions (PSD) [39] to upwards of approximately >50,000 USD/t for nanopowder of well constrained purity and PSD (e.g. [40]).", "Aside from this clear economic incentive the formation of nanomaterials directly from waste streams (such as AMD) could also have a significantly positive influence on the economics and sustainability of the nanomaterial production industries.", "Here we have demonstrated a new method for the one pot selective formation of Cu bearing nanoparticulate products from AMD by simply exposing the AMD to nZVI whilst an acidic pH is maintained.", "AMD is an ideal candidate waste stream for conversion to nanomaterials because it typically contains appreciable concentrations of high value metals (such as Cu) but is also is a globally significant environmental problem.", "The Cu nanoparticle synthesis mechanism has been determined to be via the cementation of aqueous Cu with nanoscale Fe° which comprises the dissolution of Fe from the nZVI which is coupled to the simultaneous chemical reduction of Cu ions into discrete Cu° nanoparticles.", "As displayed in Eq. (1) the reaction is spontaneous and results in the conversion of Cu2+ to Cu° in a 1:1 M ratio with the oxidation of Fe° to Fe2+.", "The synthesised nanoscale Cu0 are then likely to be recoverable using conventional clarification and/or centrifugation processes, which are well proven technologies for nanoparticle recovery (e.g. [41]), with the former typically more cost effective but would require a further step in order to remove the coagulant from the nanomaterial.", "Whilst being expensive centrifugation could also potentially be utilised, however, for the separation of the Cu nanoparticles from any other unwanted nanoparticles (e.g. nZVI corrosion phases) as a function of their relative density.", "In addition, the HRTEM data demonstrates that a significant proportion of the Cu nanoparticles are physically bound (electrostatically) to the nZVI, and as such could be recovered using a magnetic field.", "Other factors to consider includes the requirement for the treatment/recycling of any residual AMD (which will contain elevated aqueous Fe concentrations due to nZVI dissolution) prior to being released back into the environment, however, it is likely that this will be able to be achieved using conventional passive or active AMD treatment processes (e.g. a constructed wetland).", "Where AMD is anoxic, maintenance of anoxia will also be beneficial for Cu nanoparticle recovery and thereby limit the unwanted reaction between nZVI and DO and thus maximise the proportion of nZVI available for selective Cu nanoparticle formation.", "Whilst many studies have determined the performance of various different (nano)sorbents for the removal of metals and metalloids from wastewaters/effluents very few have also investigated the physico-chemical composition of the resultant sorbed/precipitated nanomaterial(s).", "Here nZVI have been investigated for the removal of metals and metalloids from AMD with a particular focus on the synthesis of nanoscale Cu°.", "The following can be concluded:", "AMD treatment required a relatively high concentration of nZVI, with concentrations <0.5 g/L imparting minimal changes to the solution electrochemistry (pH, Eh and DO) and metal/metalloid concentrations.", "Addition of nZVI concentrations ≥ 1 g/L to the AMD resulted in the rapid and near total selective removal of Cu, Al and Cd solution (>99.9% removal within 1 h) through a combination of mechanisms including cementation (for Cu), precipitation and sorption to corrosion products (for Al and Cd).", "In contrast minimal changes in the concentrations of numerous other metal and metalloid ions present in the AMD (namely: Na, Ca, Mg, K, Mn and Zn) were recorded.", "Following near total removal of Al, Cu and Cd from the aqueous phase during the initial stages of the reaction (<48 h) significant re-release was recorded and attributed to the reactive exhaustion of the nZVI and ingress of oxygen allowing recovery of system Eh, DO and pH.", "The selectivity of nZVI for Cu can be further enhanced by the application of an acidic pH buffer.", "This is likely due to the restriction of Zn, Cd sorption onto nZVI corrosion products along with the concurrent prevention of hydrolysis and precipitation of Al as Al(OH)3.", "The outermost surface of the Cu nanoparticles formed were determined using XPS to comprise a mixture of Cu° and Cu2+, with detection of the former species clear evidence that such nanoparticles formed via a cementation (redox) reaction between aqueous Cu and Fe° from the nZVI.", "Overall the results demonstrate that the application of nZVI can be fine-tuned for the highly selective formation of Cu° bearing nanoparticles from acidic wastewater, such as AMD, which might then find application in a wide range of applications including catalysis, optics, electronics and as antifungal/antibacterial agents.", "The method is easily scalable and does not require any electrical input and is therefore a potentially highly useful “green” method for the upcycling of metals from metal-bearing waters and effluents into high value nanomaterials." ]	[]
Comparative measurement of the breakup length of liquid jets in airblast atomisers using optical connectivity, electrical connectivity and shadowgraphy	A comparative examination of the optical connectivity, electrical connectivity and shadowgraphy techniques for the measurement of the break-up length of atomising liquid jets from a co-axial airblast atomiser is presented. The atomiser was operated over air-to-liquid Momentum Ratios between 27 and 335 and Momentum Flux Ratios between 0.67 and 8.27. Shadowgraphy records instantaneous images of the shadow of the atomising liquid jet when it is back-illuminated by a light source. The electrical connectivity uses the continuity of an electrically conducting atomising liquid jet to measure the potential during the presence of an electrical connection between the spray nozzle and a probe further downstream. The optical connectivity visualises the atomising liquid jet, doped with a fluorescing dye, as it is illuminated from within the nozzle using a laser beam. Comparison of the measured breakup lengths with time resolved shadowgraphy, optical connectivity and electrical connectivity, following the proposed novel processing of the time-dependent potential, showed that the mean values are all within ±15% of each other. The advantages and limitations of each technique are discussed.	[ "ENGI", "PHYS" ]	[ "Electrical connectivity", "Liquid atomisation", "Liquid breakup length", "Optical connectivity", "Shadowgraphy" ]	[ "2208", "3105" ]	[ "The atomisation of liquid jets by injection in a gaseous environment is a process, which is utilised in many practical applications, such as automotive and aviation engines, agricultural sprays, dust control, firefighting and nuclear reactor core cooling.", "The atomisation process is complex and several parameters, such as the physical properties and the flow characteristics of both the injected liquid and of the surrounding air and the geometry of the atomiser, contribute to the formation of the final spray.", "However, the importance of all the influencing parameters is not known.", "Yet, there is high demand for precise control of the spray characteristics.", "For example, the injected liquid fuel in a burner must atomise quickly near the nozzle to form small droplets, which disperse well into the surrounding air, so that they evaporate quickly and mix with the air to improve fuel consumption and reduce combustion emissions.", "A lubricating liquid jet on the other hand needs to maintain coherence for some distance beyond the nozzle, so that the lubricant is delivered to the precise location it is required in order to reduce its consumption and avoid contamination of surrounding areas.", "The control of the spray characteristics requires a deeper understanding of the atomisation process, which is broken down in smaller sub-processes that can be independently examined and understood.", "The most fundamental description of the atomisation process follows a two stage approach, namely the primary and the secondary atomisation [1,2].", "Primary atomisation begins immediately at the nozzle exit, where destabilising forces operate on the gas–liquid interface to break up the continuous liquid stream into discrete liquid fragments.", "The liquid jet usually maintains continuity for several liquid jet diameters downstream of the nozzle exit.", "After the continuity of the liquid jet is interrupted, secondary atomisation takes place, which involves further breakup of the liquid fragments into progressively smaller sizes until stable droplets are obtained.", "This complex process involves many spatial and temporal scales.", "One of the key spatial scales is the length of the continuous liquid jet past the nozzle exit, which is known as the “breakup length”.", "The breakup length is critical because it defines the spatial extent of the primary atomisation region.", "The measurement of the breakup length is therefore crucial in spray studies, because it is essential for the physics of atomisation and the performance of atomisers [3–6] and for the evaluation of computational models for atomisation [7].", "A number of techniques have been developed for the measurement of the break-up length.", "The most prominent is photography.", "It is the most widely used method [6,8–11], because it is straightforward to apply, it places only moderate demands on equipment and the photographic images provide detailed information of the continuous liquid geometry.", "In this method, the atomising liquid jet is imaged directly by a camera.", "An intense and fast light source is required to illuminate the liquid jet, so that the exposure time is short compared to the time scales of atomisation in order for the jet image to remain sharp.", "The liquid jet may be illuminated from different directions, but usually back illumination is preferred (Shadowgraphy).", "In this way, the shadow of the jet is recorded against a bright background, delivering high contrast images where the liquid jet contour is well defined (Fig. 1).", "The shadowgraphy technique is extensively presented in [12] where a theoretical analysis on the formation of the shadow of the visualised object is provided and the capabilities of shadowgraphic visualisation are highlighted.", "Shadowgraphy facilitates the visualisation of the whole liquid jet from which many morphological characteristics can be extracted, including the breakup length.", "While this method is easy to implement and gives good results under certain flow conditions, it has the disadvantage that, when atomisation becomes intense and the density of droplets around the liquid jet increases, the optical access to the liquid jet core is reduced.", "In this case, there is poor jet visualisation and, therefore, ambiguity in the estimate of the break-up length and other liquid jet characteristics.", "A different approach for the measurement of the breakup length is the electrical connectivity technique, which does not rely on the visualisation of the continuous liquid jet and, therefore, does not share the same difficulties.", "This technique is based on the conduction of electricity along the length of the continuous liquid jet downstream of the nozzle.", "A potential is applied between the spray nozzle and a probe downstream.", "If there is continuity of the liquid phase between the nozzle and the probe, a closed electrical circuit will ensue.", "The probe can be moved across different positions to determine the continuity of the liquid jet as a function of the downstream distance.", "If the measured potential between the nozzle and the probe is low, then there is electrical connectivity between the two, indicating continuity of the liquid jet core.", "Discontinuity of the liquid jet can be inferred when the measured potential between the nozzle and the probe is high.", "The theoretical basis of the electrical connectivity method was developed in the seminal work of Hiroyasu et al. [13].", "A theoretical analysis of the expected measured potential between the needle probe and the nozzle was performed by Chehroudi et al. [14] and Yule and Salters [15].", "These authors consider the continuous liquid jet as a resistor.", "Therefore, the jet resistance increases with the jet length and decreases with the jet cross sectional area and consequently the measured potential across the nozzle and the probe.", "Various morphologies of the continuous liquid jet core were examined from which its resistance is estimated.", "However, no consensus exists on the expected output signal behaviour among authors, since different assumptions were made on the shape of the jet.", "Additionally, there are differences in the implementation of the technique between different researchers.", "Hiroyasu et al. studied the breakup length of a high-speed liquid jet by measuring an electrical resistance between the nozzle and a fine wire screen detector located in a spray jet [13].", "Chehroudi et al. tried to determine the shape and length of the intact liquid core by applying a voltage between the nozzle unit and fine needles, rods and screens [14].", "Their results indicate that electrical current can be transmitted, not only by continuous liquid cores, but, also, by atomised unconnected liquid fragments, which would represent a serious limitation for the technique.", "Yule and Salters investigated the breakup zone of a transient diesel spray as a function of time and position employing a wire probe [15].", "For this reason a visual inspection of the process is necessary in order to assess how the measured potential should be interpreted.", "A novel technique for the measurement of the break-up length was recently proposed by Charalampous et al. [16,17], which is based on the optical connectivity of a continuous liquid jet and aims at addressing some of the shortcomings of shadowgraphy and electrical connectivity.", "In this approach, the liquid jet is illuminated from within the nozzle by a laser beam.", "The beam propagates along the liquid jet by reflecting at the gas–liquid interface.", "Due to the higher index of refraction of the liquid jet to that of the surrounding gas, when the incident angle of the laser beam rays on the gas–liquid interface, defined from the normal to the interface, is greater than the angle for total internal reflection (about 48° for water), the laser beam is reflected completely back into the liquid stream and propagates downstream for a long distance, in the same way that light propagates along optical fibers.", "This phenomenon was demonstrated as early as the 19th century [18,19].", "The addition of a fluorescent dye, such as Rhodamine WT [20] in the liquid jet, causes some of the intensity of the laser beam to be absorbed and re-emitted at a longer wavelength as fluorescence.", "This causes the volume of the continuous jet to become luminous, which allows the evaluation of the break-up length.", "Beyond the point of liquid discontinuity, the laser beam is diffused and its intensity is significantly reduced.", "A schematic of the process is shown in Fig. 3.", "An extension of the theoretical principle is related to the propagation of the laser beam within a continuous liquid jet and the subsequent fluorescent emission from the continuous liquid jet.", "This has been performed numerically for ideal jet geometries [21].", "It is found that in straight jets, such as those observed in coaxial airblast atomisers considered here, the laser beam can propagate sufficiently to illuminate liquid jets over 10 liquid nozzle diameters.", "This numerical investigation was expanded for instantaneous liquid jet geometries derived from Large Eddy Simulations of liquid jets in an air coflow [22], which were close to observed jet geometries under intense atomisation regime (We = 1040 and MR = 336).", "It was found that the laser beam can illuminate the full length of the liquid jet with some fluorescent intensity decrease close to the tip, where the jets become narrower.", "This technique offers the unique advantage that the continuous liquid jet is visualised without significant interference from the surrounding droplets and, therefore, does not suffer from the same issues as shadowgraphy.", "As such, the breakup length can be resolved in cases where atomisation is intense and shadowgraphy fails.", "These are the cases of significant interest as in most practical applications intense atomisation for the generation of fine droplets is required.", "There is a limitation of the jet length that can be visualised using optical connectivity, as light intensity losses by refraction occur at the jet interface.", "Nevertheless, this issue is mitigated by the fact that optical connectivity is useful for cases where atomisation is intense.", "In such cases, the continuous liquid jet length is short and the visualisation of the full jet length with optical connectivity is successful.", "Other techniques exist for the measurement of the breakup length, such as X-ray absorption [23–25] and ballistic imaging [26–29].", "While these techniques are powerful, they require advanced optical instrumentation, which is not commonly available in an engineering laboratory.", "Additionally, complicated experimental arrangements make these techniques unavailable to most researchers at present, while additional evaluation of their measurement accuracy is required.", "It is the purpose of this paper to provide a comparison between the measurement of the breakup length of a liquid jet by shadowgraphic imaging, electrical connectivity and optical connectivity, which can be implemented in most research laboratories.", "Different processing methods of measured data using the electrical connectivity technique are evaluated and the best approach is proposed.", "The remaining of the paper is structured in the following sections.", "In the next section, the details of the experimental configuration of the tested co-axial airblast atomiser are presented along with the description of the implementation of each of the three considered techniques.", "The evaluation of data processing approaches of the electrical connectivity signals and the description of the results and their interpretation follows in Section 3.", "Section 4 presents a discussion of the results where the merits and weaknesses of each of the tested techniques are exposed.", "The paper ends with a summary of the conclusions.", "The investigation was conducted on the liquid jet injected by the air-blast atomiser of Fig. 4.", "The internal and external diameters of the liquid nozzle were DL = 2.3 mm and 2.95 mm respectively and the internal diameter of the gas nozzle was DG = 14.95 mm.", "The length of the liquid nozzle was over 20DL which ensures that the mean velocity profile of the liquid jet is sufficiently developed, while the gas nozzle had a contraction with an area ratio of about 30.", "The two working fluids were water and air.", "The atomiser was set up vertically and exhausted downwards in order to avoid complexities due to gravitational effects that would cause the liquid jet to deflect from a straight path.", "The flow rate of the water and the air was controlled by and measured using rotameters.", "The resolution of the water rotameter was 0.1 l/min while the resolution of the air rotameter was 50 l/min.", "Corrections were applied to the measured air flow rate to account for compressibility effects due to the increased pressure of the air supply.", "All measurements were obtained at room temperature and pressure.", "The shadowgraphic imaging measurements were performed with a 10-bit Photron Fastcam-APX RS high speed CMOS camera.", "The camera was fitted with a 105 mm lens and the imaged region was resolved within approximately 30 μm per pixel, resulting to a spatial resolution of about 75px over the diameter of the liquid jet at the nozzle exit.", "Therefore, the liquid jet breakup length can be resolved in increments smaller than 5% of DL.", "The camera frame acquisition rate was set to 20,000 frames/s for all flow conditions.", "This frequency is greater than the breakup frequency of the liquid jet core for all tested flow conditions.", "As such, temporally resolved image sequences were recorded for each flow condition.", "The purpose of acquiring temporally resolved image sequences was to examine the possibility of improving the accuracy of the determination of the location of the liquid break-up in cases where the full length of the atomising jet is not clearly visible.", "This corresponds to flow conditions for which the products of atomisation obstruct the direct optical access to the liquid jet.", "If the liquid jet images are uncorrelated, the ambiguity cannot be overcome.", "However, the breaking point of the liquid jet could be traced upstream from a temporal sequence of correlated images.", "The source of illumination was a 100W lamp that was placed behind the liquid jet, so that the shadow of the liquid jet was imaged in the forward scattering direction.", "The measurements with the electrical connectivity technique were performed with a stainless needle probe with 1.1 mm diameter, which crossed the axis of the atomiser.", "The probe was sufficiently robust to withstand the impact of the liquid without deflection as verified by shadowgraphy.", "The needle probe was supported on a robust beam to minimise vibrations.", "The distance between the needle probe and the nozzle was adjusted by a vertical traverse in steps of 1 mm from the nozzle exit.", "This is sufficient to resolve the breakup length in increments of up to 40% of DL.", "The spatial resolution of the electrical connectivity was not as fine as that of shadowgraphy.", "The current setup was appropriate to deliver spatial resolution of the breakup length measurements better than half the liquid jet diameter, which is more than adequate for the purposes of this investigation and a good compromise between spatial resolution and measurement time.", "An electrical circuit was designed (Fig. 2) to measure the electrical potential between the nozzle and the probe.", "A constant voltage of 10 V from a stabilised battery supply was applied between the liquid nozzle of the atomiser and the probe.", "The 10 V electrical potential was selected because it offers a natural scale to compare the measurements and, as shown previously, the results of the technique do not depend on the applied electrical potential in the range between 6 V and 60 V [15].", "The positive pole of the power supply was connected to the needle probe in series with a 1 MΩ resistance to regulate the current in the circuit.", "The negative pole of the power supply was wired straight to the nozzle of the atomiser.", "A low potential between the nozzle and the probe indicates good electrical connectivity and, therefore, continuity of the liquid jet.", "Conversely, a high potential between the two indicates poor electrical connectivity and, therefore, marginal continuity or complete discontinuity of the liquid jet.", "An analogue oscilloscope was used to monitor the potential between the nozzle and the probe, while acquisition of the potential was performed by a 12-bit analogue to digital converter (National Instruments PCI-6023E) capable of sampling at 200 kHz.", "However, the sampling rate of the electrical connectivity was set at 20 kHz, so that the electrical connectivity measurements could be synchronised to the camera of the high speed shadowgraphic measurements for simultaneous measurements.", "The measurements of the breakup length with the optical connectivity technique were reported elsewhere [16].", "However, they are used here for comparison purposes.", "The details of the technique have been described elsewhere, however, we provide here a brief summary of the implementation of the technique.", "A stainless steel light guiding tube with a quartz window was inserted into the tube that delivers the liquid jet to the nozzle.", "The beam from a Nd:YAG laser, operating at 532 nm, was steered into the light guiding tube by a system of mirrors and lenses, so that it entered in a direction parallel to the tube axis to minimise reflections on the internal surfaces of the tube.", "The laser beam exited just before the contraction of the liquid jet nozzle (see Fig. 4), in a direction parallel to the nozzle axis, so that it follows the liquid stream.", "The atomising liquid was doped with Rhodamine WT dye, which is excited by laser light at 532 nm and fluoresces strongly with a peak at 587 nm.", "As a consequence, the liquid jet became luminous along the length of propagation of the beam.", "An Andor Classic iCCD camera was used to record images of the fluorescent liquid jet.", "A long pass optical filter that absorbs the laser wavelength at 532 nm and transmits the longer wavelengths of the fluorescent light was placed in front of the camera lens to suppress scattered light.", "The optical connectivity measurements were not temporally resolved.", "The resulting liquid Reynolds number was 5442, while the Weber number ranged between 80 and 1041, the momentum flow ratio was between 27 and 335 and the momentum flux ratios between 0.67 and 8.27.", "For this range of flow conditions the breakup frequency of the liquid core of this atomiser is below 2000 KHz [9] which is ten times lower than the sampling rate used in the present investigation and the sampling rate used for the high speed measurements is more than adequate to temporally resolve the breakup of the liquid jet.", "A summary of the tested conditions is presented in Table 1.", "The results from each of the considered techniques are presented in this section.", "The methodology for the evaluation of the breakup length for the shadowgraphic imaging measurements and the electrical connectivity is also presented here.", "The methodology for the optical connectivity has been presented at a previous publication [16].", "Example images from each of the flow conditions considered here are shown in Fig. 5.", "Atomisation can be observed to progress from a destabilisation regime of the main jet at low MR to the stripping of droplets from the jet surface at high MR. It is possible to evaluate many aspects of the morphology of the liquid jet, such as the waves on the surface of the jet, the shape of the detached liquid, the flapping of the liquid jet and the breakup length.", "The evaluation of the breakup point from shadowgraphic images is a matter of interpretation.", "This is because it is not always possible to identify clearly the location of the breaking point.", "One issue is the presence of ligaments and lamella that can connect detached masses of liquid with the main liquid jet that is injected from the nozzle.", "This can be observed in all cases in Fig. 5.", "For example in Fig. 5a, the liquid jet breaks up in a succession of liquid fragments.", "While the continuity of the liquid jet is interrupted at about the middle of the image, there is a fine webbing of liquid ligaments that connect the main jet with the liquid fragment directly downstream.", "The same is true for the case of the most intense atomisation of Fig. 5f, where, at the location of the breakup point half way between the nozzle exit and the needle probe, fine ligaments connect the main jet with the detached segment downstream.", "This fine interconnection between successive liquid masses not only causes problems in the interpretation of the breakup length, but also makes the automated treatment of the liquid jet images unreliable, since thresholding of the image intensity will not correctly identify the main jet and lead to longer measured breakup lengths.", "Another difficulty with shadowgraphy is that the detached liquid droplets and ligaments can obscure the observation of the breakup point.", "This type of ambiguity is demonstrated in Fig. 6.", "If the jet is illuminated from behind the page and imaged from the front, the observer should see the gap between the continuous liquid jet and the segment of detached liquid immediately downstream, to identify the breakup location correctly.", "However, if the jet is illuminated from the left and imaged from the right, the optical path between the illumination source and the camera is blocked by a mass of detached liquid immediately to the right of the breakup location and the gap will not be seen.", "Instead, a continuous liquid jet will appear, which, in fact, comprises of three distinct liquid masses and the measured breakup length will be considerably longer.", "For the current example, the measurement error is about 50%.", "The difficulty in the measurement of the breakup length from single images can be alleviated from temporally resolved shadowgraphic images.", "The uncertainty of the breakup location can be reduced by examining the sequence of the time-dependent images frame by frame.", "For example, in the first frame of Fig. 7, the liquid jet appears to be continuous.", "However, after 50 μs, it is revealed that there is a discontinuity upstream of the location of the needle, which becomes greater in the subsequent frames, revealing that the liquid is divided in two distinct segments leading to a measured breakup length considerably shorter than identified from the first image.", "In summary, shadowgraphy can give accurate measurements of the breakup length provided a temporal record exists.", "However, the evaluation of the breakup length requires considerable input from the investigator as a frame by frame analysis is required, which is not fully automated and contains a degree of subjectivity.", "During the application of the electrical connectivity method, the electrical potential is measured between the nozzle and the probe at distinct locations downstream of the nozzle.", "The location of the liquid jet breakup is determined by analysing the measured potential against the distance from the nozzle.", "The separation of the continuous liquid jet and the downstream liquid fragments is not always complete.", "This can be observed in Fig. 8 for two flow conditions with different morphological development of the liquid core.", "In the first case (Fig. 8a), close to the breaking point there is a liquid membrane that connects the main jet to the liquid fragment downstream.", "In the second case (Fig. 8b), the vicinity of the breaking point is dominated by ligaments.", "This has also been observed by [15] for diesel sprays and it was proposed that diesel sprays do not breakup in a single column, but in a number of interconnecting liquid ligaments.", "While there are differences between the diesel sprays of that study and the air-blast atomiser considered here, the connection between the liquid jet and downstream liquid fragments conducting electricity applies to both cases.", "For the jets in Fig. 8, while the physical connectivity between the liquid jet fragments and the main liquid jet is small, the measured electrical potential is significant (4.1 V and 3.9 V respectively, with 10 V indicating complete discontinuity).", "Therefore, an appropriate criterion of the measured potential needs to be defined that indicates the location of the breakup even if there is electrical connectivity between the nozzle and the probe.", "The breakup criterion is also affected by the change of the measured potential with the apparent contact of the liquid jet on the electrical probe.", "This is illustrated in Fig. 9, which shows a sequence of images of the atomising liquid jet along with the measured voltage drop across the jet nozzle and the probe.", "The progressively decreasing area of contact between the liquid jet and the needle probe in frames 312–320 of Fig. 9 shows an increasing trend in the measured voltages between the nozzle and the probe, while in frames 321–322 the increasing contact between the jet and the probe decreases the value of the measured voltage.", "From a low voltage of about 4.5 V in frame 312, the measured potential rises to a peak value of about 6 V in image 320, where the apparent contact between the jet and the probe is very small.", "Since, neither the contact area between the liquid jet and the probe nor the presence of interconnecting ligaments between the main liquid jet and the downstream fragments can be accounted for in the practical application of the technique, the probability function of the measured voltages needs to be considered as a function of the distance from the nozzle.", "The shape of the probability function is presented in Fig. 10, and, as the probe is placed farther from the nozzle, the mean value of the measured voltage increases, the probability peaks at an intermediate location over the range of measured values and the skewness of the histogram shifts from right to left.", "Therefore, various statistical parameters can be considered to identify the appropriate breaking point criterion for the electrical connectivity technique.", "The first considered parameter is the mean value of the measured potential between the nozzle and the probe.", "The mean value of measured voltage is presented in Fig. 11 as a function of the distance from the nozzle exit for all the considered flow conditions.", "In the near nozzle region, the mean measured potential is the same for all cases and its value increases linearly with the distance from the nozzle.", "The linear increase of the electrical resistance with length is consistent with that of a conductor of constant cross section and indicates that the liquid jet cross section is largely intact for the full length of that region.", "A linear baseline of the measured potential of the unbroken liquid jet with distance from the nozzle is thus established and is indicated in Fig. 11 by a dashed line and is compatible with the behaviour of the potential across a straight conductor with the resistivity of water.", "The baseline is then used as a guide for the detection of the length of the continuous liquid jet.", "The results for most flow conditions indicate an initial linear increase of the measured potential with distance from nozzle exit, followed by a sharp increase of the measured potential at a certain distance.", "This sharp increase can be explained by a sharp decrease of the mean cross section of the liquid jet and is indicative of liquid jet discontinuity.", "This axial location is marked in Fig. 11 with a dot for each flow condition and occurs progressively closer to the nozzle exit as the atomisation improves.", "All the dots that indicate the jet breakup fall on the baseline established along the length on the intact jet, which indicates that this method of identifying the breakup location with electrical connectivity is independent of the considered flow regimes.", "Beyond the breakup region, the slope of the mean potential is reduced.", "This is due to the increasing number of instances, where the maximum value of 10V potential is measured.", "The advantage of this method for identifying the breakup length is that the average potential provides a clearly defined criterion that is applicable across many flow regimes.", "The drawback is that the threshold is not fixed and the slope of the voltage with distance needs to be analysed.", "This may pose difficulties when the slope of the potential does not change dramatically with distance from the nozzle.", "The second considered parameter is the standard deviation of the fluctuations of the measured potential, which is indicative of the spread of the measured potential values.", "The evolution of the measured standard deviation with the distance from the nozzle exit is presented in Fig. 12 for all flow conditions.", "The observed general trend is that the standard deviation begins to increase at some location downstream of the nozzle exit and reaches a peak and then decreases again.", "This behaviour can be explained as follows.", "Close to the location of the liquid jet breakup, as evaluated from the plot of average measured potential of Fig. 11, and marked with a black dot on the standard deviation plots in Fig. 12, there is an increase of the fluctuations of the measured voltages due to the formation of a discontinuity of the liquid jet.", "The black dots fall in the range of 0.2–0.4 V, which appears to be the threshold value for breakup when the standard deviation of the fluctuations of the potential is considered as a criterion.", "As the probe moves further downstream, greater fluctuations of the measured voltages are measured, since the interconnecting ligaments between the main liquid jet and the downstream liquid fragments become finer.", "The peak value of the standard deviation identifies the distance where the probe starts to detect complete discontinuity of the liquid jet, meaning that there is no connectivity between the main jet and detached liquid masses even through thin ligaments.", "As the liquid jet becomes completely discontinuous for most of the time further downstream from the nozzle exit, the fluctuations of the measured potential decrease and tend to zero.", "Since the threshold value of the standard deviation is not obvious from the trends of Fig. 12, the standard deviation of the fluctuations of the measured potential is not a good criterion for detection of the liquid breakup.", "The third considered statistical parameter for the interpretation of the measured electrical potential is the skewness of the histogram of the measured potential.", "While the probe is traversed downstream from the nozzle exit, the skewness of the measured samples changes from positive to negative.", "This can be observed in Fig. 10 for flow We = 526 and MR = 171 and is similar for all other tested flow conditions.", "The development of the skewness of the samples of the measured potential is shown in Fig. 13 as a function of the distance from the nozzle exit for all flow conditions.", "Closer to the nozzle, the values of the measured potential are skewed to the right (Fig. 10a).", "This means that, when the breakup of the jet is initiated, it develops fast and there are many samples, where the reduced electrical connectivity of the breaking liquid jet results to high values of measured potential.", "As the probe is traversed downstream, the probabilities of larger and lower electrical connectivity than that of the liquid jet become equal and the skewness becomes zero (Fig. 10b).", "As the probe is traversed further downstream, the tail of the measured samples will be at low potential values as the electrical connectivity of the liquid jet is diminished (Fig. 10c).", "From the above, it can be asserted that when the liquid jet is discontinuous on average, the skewness of the histogram of the measured values of the potential will be zero.", "This break-up criterion is easy to apply, because the evaluation of the breakup location is unambiguous and does not require any interpretation of the signal.", "However, as it will be discussed later the estimated breakup length with this method is considerably longer than what is evaluated when using the other two methods.", "Another approach for the evaluation of the average breakup length of the liquid jet may be attempted by considering the fraction of the samples, where the measured potential falls below a certain threshold.", "In Fig. 14, the Cumulative probability Distribution Function (CDF) of the measured potential being below 3.0 V, 4.0 V, 5.0 V, 6.0 V, 7.0 V 8.0 V, 9.0 V and 9.9 V respectively is presented.", "Very close to the nozzle exit, the measured potential between the nozzle and the probe is always below the threshold values and therefore the CDF is 0.", "As the probe moves further away from the nozzle, the CDF increases, signifying the proportion of time that the measured potential is below the selected threshold.", "Naturally, higher threshold values result to the CDF shifting to greater distances from the nozzle exit.", "It is difficult to estimate an appropriate threshold just from examining Fig. 14.", "For this reason the value of the potential that corresponds to 50% of the cumulative density function is selected.", "This represents the median value of the samples and is presented in Fig. 15 as a function of the distance from the nozzle exit.", "Fig. 15 shows considerably similar behaviour with Fig. 11, which shows that there is a sudden change of the slope of the mean value of the potential with distance from the nozzle exit.", "The location of the change in the slope is almost identical to that of the mean value of Fig. 11.", "This occurs because the distribution of the measured potential is mono-modal and the mean and median values are similar.", "Therefore, there is no additional information that can be gained from the CDF approach.", "In the optical connectivity technique, unlike shadowgraphy, the main liquid jet can be directly imaged without too much interference from the detached fragments of liquid [16].", "This is demonstrated in Fig. 16, where an atomising liquid jet was imaged using shadowgraphy and optical connectivity simultaneously.", "In the shadowgraphic image, all of the liquid phase along the line of sight between the light source and the imaging camera is visualised without distinction between the continuous liquid jet and the surrounding detached liquid.", "In the image of the optical connectivity technique, only the continuous liquid jet is visualised through which the laser beam propagates.", "The interpretation of the shadowgraphic image for the determination of the breakup length is challenging, since both the background and parts of the continuous liquid jet have the same intensity.", "The interpretation must be based on the change of the image intensity along the boundary of the liquid jet in order to decide which bright image regions are within and which are outside the continuous liquid jet.", "Such a procedure is complex and automatic image processing is difficult.", "In the optical connectivity, the liquid phase is clearly seen as bright intensity and the gas phase is clearly seen as dark.", "Therefore, there is negligible uncertainty at the detection of the location of the continuous liquid phase.", "Consequently, the breakup length of the liquid jet can be clearly identified.", "In addition to the breakup length, other useful characteristics of the liquid jet morphology can be identified, such as the wavelength of the interfacial waves and the midline of the liquid jet [31,32].", "The procedure for the processing of the optical connectivity images, a discussion of the advantages and the limitations of the technique for the determination of the breakup length as well as the structure of the liquid jet have been presented elsewhere [16,33] and only a summary is provided below.", "The intensity of the fluorescent light is tracked downstream of the liquid nozzle exit.", "Close to the nozzle exit, the fluorescent intensity remains fairly constant.", "In the vicinity of the breaking point, there is a sharp reduction of the fluorescent intensity of the liquid jet.", "The breaking point is identified by considering a threshold value that is a fraction of the intensity at the nozzle exit.", "This value is in the region of 10–20% of the fluorescent intensity at the nozzle exit.", "Since the measured breakup length does not change by more than 10% when changing the value of the above threshold, the determination of the breaking point of the liquid jet is relatively easy and can be performed through automatic image processing which is not susceptible to large uncertainty.", "The comparison of the measurements of the average liquid jet breakup length, obtained with the electrical connectivity technique, high and low speed imaging with shadowgraphy and optical connectivity are presented in Fig. 17 as a function of the air-to-liquid momentum ratio (MR).", "For all measurements approaches and different processing approaches of each method, with the exemption of the skewness criterion for electrical connectivity, the measurement of the breakup length is within 8–10 mm, which corresponds to about 4DL.", "When considering that the average breakup length varies in the range of 25 mm to 10 mm, depending on flow conditions, the relative uncertainty [34,35] among the different measuring techniques ranges from ±20% to ±50%, with the uncertainty being more significant for the high MR range, where the average breakup length is shorter.", "The interpretation of the electrical connectivity measurements with the skewness criterion results to breakup length measurements, which are longer by about 50% than the other methods in most cases.", "Therefore, this criterion does not provide an accurate evaluation of the breakup length.", "The interpretation of the results of the remaining methods should be considered with respect to the MR of the measured flow conditions, where the merits and shortcoming of each technique can be highlighted.", "For the lower values of the MR range (Fig. 17), the atomisation is less intense.", "In this flow regime, the continuous liquid jet is longer, the shedding of liquid from the surface of the jet is not intense and close to the breakup location there is formation of liquid lamella (Fig. 5a–c).", "For this range of MR, there is good agreement of the measured breakup length among the high and low speed shadowgraphic imaging and the electrical connectivity.", "The high and low speed shadowgraphic measurements agree well, because the morphology of the jets is such that the liquid jet can be visualised adequately.", "The good agreement of the electrical connectivity with shadowgraphy for this range of MR values can be hypothesised to be due to the morphology of the constriction of the liquid jet that develops at the breaking point, which is different than the constriction at higher MR values.", "However, this is a hypothesis and more evidence is required, which can be obtained with high magnification imaging measurements that resolve the morphology of the breaking location better than the resolution used in this study.", "Considering the optical connectivity technique, the measured breakup length is shorter than for the other techniques.", "This is explained by Charalampous et al. [16] and is a result of the optical connectivity technique being based on the propagation of a laser beam within the length of a liquid jet in the same way that light propagates in optical fibers.", "Close to the breaking point the laser beam cannot propagate considerably within the ligaments that connect the continuous core and the downstream liquid jet fragments.", "As a result, the breakup length measured by the optical connectivity technique will be weighted towards the beginning of the breakup point, while the shadowgraphic techniques will measure a somewhat longer breakup length, since they are more likely to detect the breakup point at a later stage.", "For higher values of MR (Fig. 17), the atomisation is more intense (Fig. 5d–f), and the results show that there is good agreement between optical connectivity and high speed shadowgraphic imaging.", "Low speed shadowgraphy gives longer breakup lengths and electrical connectivity gives shorter breakup lengths.", "For low speed shadowgraphy under intense atomisation, due to visual interference of the breakup location from droplets and liquid fragments, it is more difficult to identify the breakup location correctly.", "Therefore, the measured breakup length can appear longer.", "However, when using high speed shadowgraphy, the temporal record of the acquired images can help to overcome the interference from the detached liquid segments by tracking the discontinuity upstream in the liquid jet.", "In the optical connectivity method, since the laser beam is introduced through the nozzle and only the main liquid jet is illuminated, optical interference with the surrounding droplets is avoided.", "Additionally, as the breakup length is naturally short under intense atomisation, the losses of the laser beam intensity along the interface of the continuous liquid jet due to refraction are small and the illumination of the continuous jet is complete.", "The shorter breakup length detected by electrical connectivity might be attributed to the nature of the technique.", "As the probe is physically inserted into the flow, electrical connectivity will be able to detect the narrowing of the continuous jet close to the nozzle.", "However, at high MR, the flow is fast and the narrowing does not have sufficient time to develop to a discontinuity within the probe location indicating a short breakup length.", "Overall, confidence in the measurement of the breakup length for the region of higher MR is higher for the high speed shadowgraphy and the optical connectivity, while electrical connectivity has greater uncertainties.", "Overall, when considering the 3 most reliable measurement methods of the breakup length, namely optical connectivity, high speed shadowgraphy and electrical connectivity interpreted according to the mean value of the measured potential, the maximum measurement error among them is within ±15% of the mean measured value.", "This range is much narrower than the ±50% range for all the measurement techniques and processing approaches considered.", "From an application point of view, the selection of the appropriate technique depends on the implementation of the experiment and the results, which are sought.", "The most common way to measure the breakup length is low speed shadowgraphy.", "It has the advantages that it is non-intrusive, very easy to set up and requires equipment that is available at all laboratories.", "There is no limit on the length of the breakup length that can be measured and, in addition, valuable information can be extracted regarding the overall atomisation process and the behaviour of the detached liquid segments.", "On the downside, shadowgraphy requires optical access to the atomising spray from two opposite sides, one for the illumination and one for the imaging system, which could be a problem when the spray is contained.", "Also, the analysis of the images is susceptible to the subjective interpretation of the researchers, since the location of the breakup point must be determined visually.", "This is a significant issue when atomisation is intense and there is no clear view of the liquid jet and therefore there is ambiguity in the measurements.", "This ambiguity can be overcome by using high speed shadowgraphy.", "The setup remains essentially the same, but the cost of the required imaging system is much higher and may not be available at all laboratories.", "In this case, however, the analysis of the shadowgraphic record requires substantial user effort, as the breakup location needs to be traced upstream which needs to be done manually.", "In general, shadowgraphy is a good tool for the overall investigation of the liquid jet breakup with some limitations depending on the atomisation intensity, which can be overcome with temporally resolved measurements.", "The optical connectivity approach for the measurement of the liquid breakup length is most appropriate when there is optical obstruction of the liquid jet from surrounding detached liquid fragments, since it visualises only the main jet.", "This is particularly important for cases where many droplets surround the liquid jet core.", "The optical connectivity images of the liquid jet present clearly the continuous liquid jet.", "This makes the determination of the breakup length straightforward by tracing the fluorescent intensity along the jet length, which is an advantage over shadowgraphy and can be performed with automatic image processing.", "Additional information can also be extracted for the overall morphology of the liquid jet, since the full image of the continuous jet is obtained [31,32].", "The technique does pose increased demands on equipment compared to shadowgraphy because a laser source is required for illumination.", "However, lasers are available at most laboratories studying atomisation.", "Another difficulty is that the atomiser must be modified, so that the laser beam can be guided through the nozzle.", "In case of complex atomiser designs, this might not be possible.", "Finally, it has been shown that there will be a decrease of the laser beam intensity as it propagates along the liquid jet length due to refraction [16] at the liquid interface.", "Therefore, this technique is recommended for cases where the breakup length is expected to be short, which are related to intense atomisation and, therefore, for most practical applications.", "This is also the case when low speed shadowgraphy becomes problematic.", "Therefore, the two techniques can be complementary.", "Finally, the electrical connectivity technique is an alternative method to optical approaches and is dedicated to the measurement of the breakup length.", "This technique is attractive, since it is easy to set up, has little demand on equipment and produces small datasets.", "Like shadowgraphy, there is no limitation of the maximum breakup length that can be measured.", "The drawbacks of the technique are that the estimated breakup length can be somewhat shorter than what is measured by optical techniques.", "This is due to the interpretation of the measured potential between the nozzle and the probe.", "The requirement for physical access to the near nozzle region may be a problem for some applications, especially in limited spaces and in physically hazardous environments, where the direct contact of the spray with the probe can cause damage or contamination of the surface that would alter the electrical resistance of the probe and interfere with the measurement.", "The time required to characterise a single flow condition is also longer compared to other techniques, since the probe has to be traversed along the jet.", "Finally, a requirement of the technique is that the liquid must be electrically conductive.", "Overall this technique is promising when only the breakup length is required and demands on equipment needs are minimal.", "The advantages and disadvantages of the considered techniques are summarised in Table 2.", "A comparative investigation of the measurement of liquid jet breakup length of a co-axial airblast atomiser was conducted using electrical connectivity, optical connectivity and shadowgraphy for a range of flow conditions with air-to-liquid Momentum Ratios (MR) between 27 and 335 and momentum flux ratios between 0.67 and 8.27.", "The findings are as follows:", "During shadowgraphic measurements, the optical access to a central liquid jet is obstructed by the presence of detached liquid segments along the line of sight between the jet and the camera resulting to bias towards long breakup length measurements.", "High speed shadowgraphy provides the temporal evolution of the liquid jet, which can alleviate some of the uncertainty in the interpretation of the shadowgraphs and produce more reliable breakup length measurements.", "The electrical connectivity technique was affected by the presence of thin liquid ligaments that interconnect the detached liquid fragments and are sufficient to allow the conduction of electricity.", "Different criteria for processing the measured values of potential in order to identify the breakup length were considered, which were assisted from the availability of understanding gained from simultaneous electrical connectivity and shadowgraphic measurements.", "The criteria included the mean value of the measured potential, its standard deviation, its skewness and its median value.", "The methods, based on the mean and the median value of the potential, were appropriately reliable to measure the mean value of the breakup length, but they have to be evaluated at different injector geometries.", "Therefore, if an appropriate criterion to process the electrical connectivity potential measurements is used, the measured breakup length is within the same range of the other techniques.", "The optical connectivity technique can overcome the optical obstructions caused by droplets surrounding the liquid jet, which become more significant with the increase of Momentum Ratio of the flow conditions.", "Overall, it is found that the three methods have merits and should be used complementary depending on the requirements of the investigation and the morphology of the investigated liquid jets.", "If applied correctly, the maximum measurement error between the techniques is within ±15% of the mean value of the breakup length." ]	[ "Comparison of measurement methods for breakup length of liquid jets.", "Shadowgraphy, optical connectivity and electrical connectivity considered.", "Guidelines for implementation given.", "Maximum measurement error between techniques within ±15%." ]
Depression and playfulness in fathers and young infants: A matched design comparison study	Background Depression in fathers in the postnatal period is associated with an increased risk of some adverse child developmental outcomes. One possible mechanism for the familial transmission of risk is through the negative effects of depression on parenting and the parent-child relationship. So far, evidence indicates that depressed fathers tend to be more withdrawn in their early interactions. However, the interaction dimensions studied to date may not be able to detect and accurately classify unique features of father-infant play – including physically stimulating and highly rousing episodes of play. Hence, in this matched design comparison study, we set out to examine, for the first time, links between diagnosed paternal depression in the postnatal period and playfulness in father-infant interactions. Methods Fathers and their infants were assessed when the infants were 3 months old. Paternal depression was diagnosed using a structured psychiatric interview. Currently depressed (n = 19) and non-depressed (n = 19) fathers were individually matched on age and education. Fathers were filmed playing with their children. Four dimensions were coded for paternal playfulness during free-play: physicality, playful excitation, tactile stimulation and active engagement. Results Depressed fathers, compared to non-depressed fathers, engaged in fewer episodes of playful excitation (mean scores: 0.71 vs.2.53, p = 0.005), less gentle touch (mean time: 38.57 vs. 53.37, p = 0.015) and less active engagement (mean scores: 2.29 vs 3.24, p = 0.044). When controlling for infant fretfulness, the findings remained largely unchanged. Limitations The sample size was small and the sample was limited to mostly white, well-educated fathers. Conclusions Playful paternal behaviours as early as 3 months differ between fathers with and without depression. These changes may help in understanding children's risk in relation to paternal psychopathology and could be a target for future family interventions.	[ "MEDI", "PSYC" ]	[]	[ "2738", "3203" ]	[ "A developing body of literature now documents that mood disorders are not limited to mothers in the postnatal period.", "Fathers experience depressive symptoms as well, with prevalence rates of around 5–10% within the first year of a child's life (for reviews see, Cameron et al., 2016; Edward et al., 2014; Paulson and Bazemore, 2010).", "Furthermore, depression in fathers has been linked with multiple indicators of adverse child outcome, including behavioural and emotional problems, impaired cognitive development, and an increased risk of the development of psychopathology (Barker et al., 2017).", "One possible mechanism for the familial transmission of risk is through its negative effects on parenting and the father-child relationship (Kane and Garber, 2009; Nath et al., 2016; Sethna et al., 2015, 2012).", "So far, evidence indicates that paternal depression is associated with less optimal father-infant interactions with a few exceptions (Field et al., 1999; Lundy, 2002).", "For example, in comparison to non-depressed fathers, depressed fathers are less responsive in free-play with their 3-month old infants (Parfitt et al., 2013), tend to engage in fewer activities, such as, outdoor play, singing or reading to their children aged 9-months (Paulson et al., 2006), touch their 4-month old infants less frequently during routine tasks (Zaslow et al., 1985), and are less engaged during free play at 3 months and 12 months (McElwain and Volling, 1999; Sethna et al., 2015).", "In general, while these studies have linked depressive symptoms to more withdrawn paternal behaviours, they also highlight an important limitation.", "That is, the scales employed to code father-infant interactions have usually been developed using mother–infant dyads.", "Hence, while they may be particularly sensitive to the interaction style of mothers; they may not be able to detect and accurately classify unique features of father-infant interactions (McElwain and Volling, 1999; Paquette and Dumont, 2013; Sethna et al., 2015).", "For instance, episodes of sudden vocal and behavioural activity in close proximity to the infant are coded as ‘intrusive’ using a maternal coding scheme (McElwain and Volling, 1999; Sethna et al., 2015).", "However, in the context of father-infant interactions, these playful behaviours (which excite, surprise, and stimulate children) tend to promote attention and positive communication between the dyad.", "Consequently our understanding of early parenting by fathers is incomplete due to the conventional (though changing) maternal focus on parenting behaviours.", "We have previously reported that fathers with depression are less intrusive, or more withdrawn, in their interactions (Sethna et al., 2015).", "Like much previous research, a measure of parent-child interaction that had been developed for mothers was used.", "Hence we were restricted to studying the key interaction dimensions typically examined in relation to mother- infant interactions.", "In this study, we extend previous work with depressed fathers by using a measure of parent-child interaction mainly developed to code paternal playful behaviours.", "Since playfulness comprises a greater proportion of fathers’ interaction with children than it does for mothers (Craig, 2006).", "Thus, the current study examines, for the first time, links between diagnosed paternal depression in the postnatal period and playfulness in father-infant interactions with 3 month old infants.", "Early research on father–infant relations focused primarily on the interaction styles of fathers compared to mothers, and conclusions were made in the 70's that fathers were not simply a replacement for mothers - their interaction styles were qualitatively different (Belsky, 1979; Clarke-Stewart, 1978; Lamb, 1977; Weinraub and Frankel, 1977; Yogman, 1977).", "In subsequent research, father-infant interactions were described as less predictable and highly rousing episodes of behaviours - comprising a rapid increase and decline of emotional excitement.", "In contrast, maternal interactions comprised regulated sequences of social exchange including gaze, vocalizations and affect.", "Moreover, while mothers were more likely to follow their infant's focus of attention, fathers, in contrast, appeared more directive and disruptive (Belsky et al., 1984; Power, 1985; Stevenson et al., 1988; Yarrow et al., 1984).", "More recent reports suggest that mothers and fathers adopt multiple roles in the family system (Tamis-LeMonda, 2004).", "For instance, both parents may function as caregivers, role-models, teachers, breadwinners and spouses - determined by, for instance, work arrangements, family structure, parent's preferences, obligations and health needs.", "As a result of these multiple roles, similarities may exist in the way that both parents interact with their children (Parfitt et al., 2013; Roggman et al., 2004).", "For example, fathers who are the primary care-givers, tend to be more comforting, sensitive and engage in less physical play (Lamb et al., 1982; Lewis et al., 2009).", "In their complimentary influences, fathers are likely to perform roles played by mothers, and vice versa, in response to family circumstances that entail adjustment (Cabrera et al., 2014).", "Nonetheless, differences in the way mothers and fathers interact with their children are also evident - such that, on average, fathers tend to engage in more physical and competitive play routines (Stgeorge and Freeman, 2017).", "Notably, fathers engage more frequently than mothers in vigorous physical play from the first year of their child's life (MacDonald and Park, 1986).", "This increase in the prevalence of more active play in fathers has been linked to socio-emotional development (Paquette and Dumont, 2013), and recent meta-analytic evidence points towards positive links with multiple domains of children's behaviour, including, social competence, emotional development and the ability to self-regulate (Stgeorge and Freeman, 2017).", "Hence, in this investigation, we focus on playfulness that characterises father-infant interactions.", "Based on existing evidence, playfulness is operationalized and measured by the following four interaction domains: (i) physicality (gross motor stimulation) (ii) playful excitation (sudden, unexpected verbal or non-verbal behaviours), (iii) tactile stimulation (touch), (iv) active engagement (stimulation via paternal behaviour, affect, facial expression, and tone of voice).", "Play1* is a significant component of the parent-infant relationship and an important context for infant learning (Teti et al., 1988).", "Physicality in play during the first six months, corresponds to neuromuscular maturation, evident in the improved control of specific motor patterns, and in the subsequent development of socialization skills (Pellegrini and Smith, 1998).", "Father-infant interactions can also encompass episodes of ambiguous, unexpected behaviours (e.g., unpredictable movements within the infant's visual field or a change in pitch and volume of voice) (Dixon et al., 1981; Labrell, 1994).", "Although not empirically tested in infancy, there are suggestions of a positive association between these rousing play episodes and the decoding of emotion (happiness, sadness, anger, fright and neutrality) in pre-school children (Carson et al., 1993).", "We are not aware of specific evidence of the impact of parental depression on physicality in play and sudden excitatory arousal or playfulness.", "It is, however, well recognized that depression impacts on behaviours including linguistic production (rate, volume, and pitch), facial expression, gaze, posture and gestures (Segrin, 2000).", "In view of these findings and those which suggest that depressed fathers tend to be more withdrawn in their early interactions, we predict that depressed fathers will be less physical (Hypothesis 1) and engage in fewer episodes of playful excitation (Hypothesis 2) when interacting with their infants, compared to non-depressed fathers.", "Touch serves a communicative function, signalling emotions of positive (Pelaez-Nogueras et al., 1996) and negative valence (Hertenstein and Campos, 2001), and in the transmission of more specific messages, for example, security in the presence of the caregiver (Tronick, 1985).", "Research on tactile stimulation in father-infant interactions is very limited (Shields and Sparling, 1993; Zaslow et al., 1985).", "For example, Zaslow and colleagues (1985) have shown that in the context of paternal ‘blues’ fathers show diminished contact with their infants aged 4 months.", "While studies from the maternal literature similarly report that depressed compared to non-depressed mothers spend less time touching their infants, they also report that depressed mothers use more negative touch (for e.g. poking, shaking, tickling, pulling or tugging limbs) as opposed to positive touch (gentle stroking, or playfulness) (Ferber, 2004; Herrera et al., 2004; Malphurs et al., 1996b).", "We aim to investigate differences in the quantity and quality of tactile stimulation, i.e. touch in depressed and non-depressed fathers.", "Based on the available evidence, we predict that depressed fathers in comparison to non-depressed fathers will spend less time touching their infant, and are more likely to engage in negative (i.e. vigorous) touch, as opposed to positive (i.e. gentle) touch (Hypothesis 3).", "Active stimulation using a range of modalities during communication helps modulate infant arousal and reinforces behaviours.", "Within this cycle, an infant responds to parental stimulation with increased attention, communication and positive affect (Bridges and Connell, 1991; Bridges et al., 1997; Diener et al., 2002; Yarrow et al., 1984).", "For example, Bridges et al. (1997) have found that, in the presence of an actively communicating father, infants displayed positive affect and were generally more communicative.", "Infant developmental outcomes studied in relation to an early animated environment include, for example, emotional regulation, cognitive competence, and exploration and motivation (Clarke-Stewart, 1978; Diener et al., 2002; Yarrow et al., 1984).", "So far, the impact of depression on paternal use of animation/stimulation has not been specifically addressed.", "We predict that depressed mood will interfere with the father's ability to stimulate the infant vocally, physically, emotionally and psychologically- their interactions will be quiet and contained (Hypothesis 4).", "This cross-sectional matched sample study comprised a total of 38 fathers.", "Matching variables included age and education in line with previous research (Tamis-LeMonda et al., 2004).", "We had 19 fathers with current major depressive disorder in the study, and then identified and individually matched fathers without current depressive disorder and with equivalent or the most similar values of the two covariates.", "This created a new sample of matched participants − 19 fathers with current major depressive disorder and 19 fathers without depression - previously cited (Sethna et al., 2012).", "The matching procedure was based on a random order nearest available matching method (Rosenbaum and Rubin, 1985; Rubin, 1979).", "The first depressed participant was randomly matched with the closest non-depressed participant (N), followed by the second depressed participant being matched with the yet unmatched closet non-depressed participant (N-1), and so on, until all depressed fathers were matched.", "By construction, the currently depressed and non-depressed fathers in the matched sample had identical (or nearly identical) values of age and education.", "Participants were drawn from a larger prospective cohort study examining the impact of paternal depression on child development (Ethical approval: 06/Q1604/63).", "They were recruited from postnatal wards in Oxfordshire and Buckinghamshire UK, and assessed at 3, 12 and 24 months.", "For this study, exposure and outcome data were concurrently assessed at the 3-month time point.", "All fathers recruited into the study met inclusion criteria of being 18 years or older, speaking English, being married or in a stable relationship and residing with their infant.", "Infants were of gestational age >36 weeks, birth weight ≥ 2 kg, and had no major congenital anomalies.", "Seven weeks after the birth of their child all consenting fathers were sent a questionnaire including the Edinburgh Postnatal Depression Scale (EPDS, Cox et al., 1987).", "All the fathers screening positive (10 or above on the EPDS), and a random one-in-four sample of those screening negative (an EPDS score less than 10) were subsequently invited to take part in the study.", "Those agreeing to participate further, were visited at home when their infant was approximately 14 weeks old.", "During this visit fathers were interviewed using the Structured Clinical Interview for DSM-IV (SCID; First et al., 2002) to establish whether they met criteria for major depressive disorder - as previously reported elsewhere (Edmondson et al., 2010; Ramchandani et al., 2011).", "Subsequently two study groups were identified: (i) currently depressed – including fathers diagnosed as experiencing current major depressive disorder using the SCID, (ii) non-depressed – including fathers with neither current nor past diagnosis of major depressive disorder on the SCID or elevated symptoms on the EPDS screening questionnaire.", "Exposure: Diagnosed paternal depression", "Outcome: Four categories of behaviour that characterize father-infant interaction including: (i) Physicality in play, (ii) Playful excitation, (iii) Tactile stimulation and (iv) Active engagement.", "Data for this study were collected three months post birth.", "All assessments were conducted in the families’ homes during times when the infants were typically alert and fed.", "Father – infant dyads were videotaped during two interactive settings, including 3 min of free play, which was the focus of this investigation.", "Fathers were asked to place their infants in a supine position on a floor-mat, and to play with and talk to their infant, as they would normally do, without the use of toys.", "Parents were asked not to allow their children to use pacifiers during the videotaping so that the researchers could hear children's vocalizations.", "Following the videoed observational assessment, all fathers were interviewed using a structured psychiatric interview.", "The main study variables were measured using the following tools:", "The Structured Clinical Interview for DSM-IV Axis 1 Disorders (SCID; First et al., 2002), was used for diagnosing major depressive disorder.", "The SCID has been found to have high reliability (Zanarini et al., 2000) and validity (Basco et al., 2000).", "All interviews were conducted by trained graduate psychologists or psychiatrists.", "Four categories of behaviour that characterize father-infant interaction were coded and detailed below (manual available from the authors upon request).", "They include: (i) Physicality in play, (ii) Playful excitation, (iii) Tactile stimulation, (iv) Active engagement.", "Gross-motor stimulation (movement of infant limbs and body position), coded using a 5 point Likert scale of measurement.", "The scale comprises progressive levels of physicality from gross motor energetic stimulation, whereby the infant is lifted off the mat (5) to low-energy physical stimulation, holding of the infant's arm/legs with minimal movement (1).", "Thus coding is based on progressive levels of physicality, with 5 as the highest level of physicality in play.", "This scale is modified from previous work on fathers by Crawley and Sherrod (1984) with 10–13 month old infants, and by Yogman (1981) with infants in the first six months of life.", "A frequency count of sudden, emotional, unpredicted and repetitive short sequences / and or single occurrence of behaviour.", "This was adapted from previous work in which paternal use of teasing entails ‘unexpected ambiguous behaviours which destabilize the infant and whose expectations are contradicted’ (Labrell, 1994, p. 128).", "In this category of playful behaviour we code for specific instances of unpredictable movements within the infant's visual field, change in pitch and volume of voice quality which stimulate and arouse the infant.", "Examples of arousal behaviours coded include: sudden paternal vocalization, i.e. high pitched speech or non-verbal exclamation, unpredicted physical activity around the infant or distal stimulation by motor movements and high positive arousal through sudden animated facial or vocal expression.", "Higher scores suggest the increased episodes of arousal behaviours and hence increased playful excitation during interactions.", "The quantity of overall touch (i.e. total time in seconds spent in physical contact with the infant during the 3 min interaction), and the quality of touch (i.e. the proportion of time, measured in seconds, spent in a certain type of touch) were both coded.", "Quantity of overall touch comprised a rating of the proportion of the interaction (i.e. the total time in seconds across the entire play episode) during which the father touches the infant either as part of a game; to gain or maintain the infant's attention, as an affectionate gesture or in response to the infant's physical movements.", "In line with previous studies we used proportions to account for any differences in the overall duration of the interaction.", "Higher scores indicate increased time spent in tactile behaviours.", "Quality of touch was based on previous work (Moreno et al., 2006; Polan and Ward, 1994).", "The mean time spent in each of the following touch items was measured: (i) Inactive touch (ii) Light touch (iii) Firm touch (iv) Stimulating touch and (v) Active touch and (vi) Practical touch.", "Individual touch items were also combined into two composites, and subsequently used for the current analyses: Gentle touch -the mean time (in seconds) spent in inactive touch, light touch and practical touch.", "This variable corresponds to previous research (Feldman and Eidelman, 2003; Ferber et al., 2008; Pelaez-Nogueras et al., 1996).", "Vigorous touch -the mean time (in seconds) spent in firm, stimulating and active touch, also comparable to previous studies (Stack and Muir, 1992; Weiss, 1988; Weiss and Wilson, 2006).", "The level of vitality and liveliness within the session, coded through paternal behaviour, affect, facial expression and tone of voice.", "This dimension taps into vocal, emotional, psychological and physical availability of the parent- the parent effectively communicates his awareness of the infant's presence, is receptive to ongoing communication and is available to respond or initiate social interaction empathically and appropriately.", "Rated on a likert scale of measurement from 1 to 5; higher ratings (5) indicate increased stimulation throughout the play session with momentary episodes of calm to regulate arousal, and lower scores (1) indicate minimal use of stimulation.", "Active engagement is a reflection of the effort the father puts into the interaction to create a lively, vigorous environment (higher scores) as opposed to a quiet and contained one (lower scores).", "All videotapes were coded by two researchers blind to paternal depression status.", "Inter-rater reliability between the two coders was established using Intra-class coefficients (ICC) on interval data (Playful excitation: ICC = .97; Touch types: ICC ranged from .74 to .92), and weighted Kappa on ordinal data (active engagement: k = 0.78; physicality in play: k = 0.81).", "Observational measures of infant behaviour were coded using The Global Rating Scales for Mother– Infant Interaction (GRS, Murray et al., 1996).", "The GRS comprise individual infant behaviours rated on five point scales.", "We assessed ‘Infant fretfulness’, which comprised two infant behaviour scales from the GRS: (i) happy–distressed and (ii) non-fretful–fretful.", "Sampling bias in the current study was minimized as the non-depressed fathers were selected from the same community sample which included the depressed fathers.", "They differed mainly on the exposure under consideration.", "Recall bias was minimized as participants completed the EPDS screen 7 weeks after the birth of their child.", "The sample size for the current study comprised all the depressed fathers (from the wider prospective cohort study) with diagnosed depression on the SCID (n = 19) who were then individually matched with a non-depressed father.", "First, the sample is described – demographic characteristics of the total sample were examined using means and standard deviations (SD) for continuous data and percentages for categorical data.", "We also examined infant (gender and birth order) and paternal demographic characteristics (age, marital status, educational attainment and ethnicity) in relation to the two study groups.", "Second, descriptive statistics for the interaction dimensions are presented and bivariate associations between these dimensions were tested.", "Third, using the Wilcoxon-signed-rank test for paired data, depressed and non-depressed fathers were compared on the four main interaction dimensions.", "This non-parametric test was used as the sample was small and some data were not normally distributed.", "Finally, where a significant association was found between exposure (paternal depression status) and outcome variables (four interaction domains of paternal playfulness), we additionally took into consideration the infant's affective state, i.e. fretfulness.", "This was based on existing evidence which suggests that parental depression is linked with difficult child temperament (Hanington et al., 2010), and that negative child affect is also linked with less positive parent- child interactions (McBride et al., 2002).", "We therefore wished to try and control for any potential effects of having a more difficult or fretful infant on the interaction, so that we could more confidently ascribe any differences in interaction to the potential effects of depression in the father.", "Our rationale for including infant fretfulness was thus based on the existing literature.", "Fathers (n = 38) had a mean age of 35.89 years (SD = 5.42), 94.7% were white and all were married/cohabiting.", "In terms of highest level of educational attainment, 10.5% had GCSE's, and 21.1% had post-graduate qualifications; 92.1% of the fathers were in full time employment.", "For all but two fathers, English was their first language.", "Of the infants, 18 (47.4%) were first born and 14 (36.8%) second born.", "There were no differences between the two groups on infant (gender and birth order) and paternal demographic characteristics (marital status, ethnicity and employment status) (Table 1).", "Additionally, infant fretfulness did not differ between depressed (M = 3.74, SD = 1.32) and non-depressed fathers (M = 3.94, SD = 1.09); t = −0.50, p = 0.619.", "Descriptive statistics for the interaction dimensions and bivariate associations between them are presented in Table 2.", "Fathers who used more gross motor stimulation in their play tended to touch their infant vigorously (rs = 0.35, p < 0.05).", "Furthermore, fathers who engaged in increased playful excitation touched their infant less vigorously (rs = −0.37, p < 0.05), and their interactions were highly stimulating (rs = 0.53, p < 0.01).", "Depressed and non-depressed fathers did not differ in their use of physicality during play (Table 3).", "Findings on episodes of playful excitation indicate that depressed fathers were less likely to arouse and startle their infants using sudden, emotional, physical or vocal behaviours (M = 0 .71 = SD = 1.16) compared to non-depressed fathers (M = 2.53, SD = 2.03); z = −.280, p = .005, with a medium effect size (r = 0.45).", "There were no differences in the overall duration of touch in the two study groups (Table 4).", "However, depressed fathers were less likely to engage in gentle touch (M = 38.57, SD = 16.11) compared to non-depressed fathers (M = 53.37, SD = 14.97); z = −2.44, p = .015, with a medium effect size (r = 0.40).", "There were no mean differences in the vigorous touch composite between the two study groups (Table 4).", "As shown in Table 3, depressed fathers (M = 2.29, SD = 1.31) compared to non-depressed fathers (M = 3.24, SD = 1.15) were less actively engaged with their infants; z = −2.01, p = .044, with a medium effect size (r = 0.33).", "Next, when adjusting for infant fretfulness, group differences reported on the following dimensions remained largely unchanged: playful excitation (unadjusted: β = 0.48, p = 0.004; adjusted: β = 0.48, p = 0.004); gentle touch (unadjusted: β = 0.46, p = 0.006; adjusted: β = 0.46, p = 0.007); active engagement (unadjusted: β = 0.34, p = 0.044; adjusted: β = 0.36, p = 0.038).", "This study aimed to broaden the focus of measurement of early paternal play behaviours in the context of depression.", "The findings afford new, albeit exploratory, insights into the impact of depression on: (i) physicality in play, (ii) playful excitation, (iii), tactile stimulation, (iv) active engagement in fathers.", "The findings suggest that depressed fathers compared to non-depressed fathers engaged in fewer episodes of playful excitation, less gentle touch and less active engagement.", "There was no evidence of a difference in physical play and in the overall duration of tactile engagement between the two study groups.", "The study does have several limitations, including a small sample size, and limited demographic variability of the participants (predominantly white, well-educated fathers).", "Furthermore, we did not conduct a formal power analysis in advance of the study.", "It was anticipated that the exploratory nature of the study would subsequently allow for reported group differences from the small sample, with clearly defined groups, to be then tested on a larger sample.", "Hence caution should be exercised in interpretation of the findings reported, at least until subsequent research also addresses these important questions.", "In addition there are a number of strengths to the current study.", "First, we developed these specific dimensions of paternal behaviour drawing on the existing literature on child developmental and parenting strategies, to develop relevant constructs.", "Second, the scales were reliably administered and coded by blind coders.", "Third, the study includes two well-matched groups, and participants were diagnosed for depression using a structured interview rather than a questionnaire for symptoms.", "Although, the purpose of matching was to eliminate bias in relation to paternal age and education, future studies, with larger samples should consider additional covariates.", "There are many potential covariates to consider, but some key ones might include infant birth order, birth weight, child gender and maternal depression (Hallers-Haalboom et al., 2017; Vismara et al., 2016).", "The prediction that depressed fathers would engage in less physical play compared to non-depressed fathers was not supported.", "These findings stand in contrast to previous studies of fathers and their infants in play across the first year of life (Clarke-Stewart, 1978; Crawley and Sherrod, 1984; Lamb, 1977; Yogman, 1981).", "We suspect that, at three months, face to face communication in fathers may focus on tactile behaviours and limb movement games rather than active gross-motor stimulation.", "Previous research suggests that only in the sixth month after birth is there a peak in gross-motor stimulation (Pellegrini and Smith, 1998), while other studies seem to suggest low levels of physical play before 1 year (MacDonald and Park, 1986).", "Furthermore, fathers tend to engage in physical play with their infant sons more than their daughters (Bronstein, 1984).", "Hence, the lack of group difference reported on physicality in play, may be due to our sample comprising more female infants overall.", "Moreover, our study may lack the power to further differentiate boys and girls, and hence child gender would be important to consider in future studies with larger samples.", "Although positive arousal in paternal interactions has been consistently reported (Arco, 1983; Clarke-Stewart, 1978; Feldman, 2003; Yogman, 1981), we are not aware of previous evidence on the influence of paternal depression on excitatory features of father-infant interactions.", "The prediction that depressed fathers compared to non-depressed fathers would engage in fewer episodes of playful excitation was supported in the present study.", "It is likely that the display of sudden vocal, motoric and facial expressions in the context of high emotionality may be more susceptible to depressive affect.", "Further, that such behaviours are more likely to be produced as a result of the fathers’ own level of engagement and interest in the interaction, and not in response to a prior behaviour emitted by the infant, suggests they may represent a distinct motivational characteristic of social communication, which is reduced in fathers experiencing depression.", "In contrast to findings from the maternal literature (Herrera et al., 2004; Malphurs et al., 1996a), we did not find a difference in the overall duration of touch between depressed and non-depressed fathers.", "Earlier findings from this study suggest that depressed and non-depressed fathers did not differ in sensitivity in their interactions (Sethna et al., 2015).", "These findings may thus help explain why depressed and non-depressed fathers did not differ on the duration of tactile stimulation.", "Furthermore, in early infancy, touch facilitates the regulation of arousal, attentiveness and interest, and helps soothe the infant (Koester et al., 1989; Tronick and Cohn, 1989).", "Since infants in the two study groups were similar with respect to positive affect and attentiveness, it is likely that fathers did not differ in the overall duration of tactile stimulation necessary to regulate infant behaviour.", "The prediction that depressed fathers compared to non-depressed fathers would engage in more vigorous touch as opposed to gentle touch was partially supported.", "We found that while there were no differences in the use of vigorous touch by depressed and non-depressed fathers, they did differ in their use of gentle touch.", "These results are partially consistent with previous findings from the maternal literature (Cohn et al., 1986; Feldman, 2003; Field et al., 1990; Weinberg and Tronick, 1998), which indicate an association between depression and more negative tactile behaviours as opposed to affectionate ones.", "Finally, depressed fathers compared to non-depressed fathers engaged in fewer episodes of active engagement.", "The few available studies on the impact of depression on paternal behaviour report similar findings with regards to paternal withdrawal and lack of engagement (McElwain and Volling, 1999; Paulson et al., 2006; Zaslow et al., 1985).", "Depression is likely to interfere with the parent's emotional availability to stimulate the infant, evident in laboratory studies that report fewer positive animated faces and voices, and decreased levels of interest (Elgar et al., 2004; Lundy et al., 1996).", "Furthermore, it is also possible that biological symptoms of depression including sleep disturbance, low energy and loss of interest may each contribute to decreased involvement in interactions with the infant.", "This study shows that early playful paternal behaviour with young infants differs between fathers with and without diagnosed depression.", "These behaviours specifically reduced excitatory playfulness, gentle tactile stimulation, and less active engagement could be potential targets for future family interventions, early in life.", "Furthermore, this new evidence of associations between depression and fathers play behaviours with their young infants highlights the need for the continued development and application of father-focussed measures.", "Studying fathers who are experiencing difficulties in their relationship with their infants is an important undertaking." ]	[ "Playful behaviours at 3-months differ between depressed and non-depressed fathers.", "Depressed fathers use less playful excitation, gentle touch and active engagement.", "Coding measures focusing on fathers may accurately detect and classify playfulness.", "Unique features of paternal play could be a target for future family interventions." ]
XRF techniques to quantify heavy metals in vegetables at low detection limits	Sample preparation techniques, measurement routines and custom calibrations were developed and optimized for the quantification of Pb and additional heavy metals in dried and wet consumable vegetable tissues via WD-XRF and/or portable ED-XRF spectroscopy. After mitigating matrix influences fundamental to XRF quantification of heavy metals in a carbon matrix, the custom measurement and calibration routines were capable of detecting and quantifying Pb in dried prepared vegetable samples at concentrations relevant to the World Health Organization. The viability of each calibration was evaluated with measurements of vegetables grown in metals-rich garden soil collected from residential properties. Although robust calibrations for dried vegetables were obtained with WD-XRF, the slight compromise in the precision and accuracy of measurements with portable ED-XRF is offset by the portability of this technology. Heavy metal concentrations in wet coarsely-homogenized raw consumable vegetable tissues were quantified; however, further work is needed to test the viability of ED-XRF calibrations.	[ "AGRI", "CHEM" ]	[ "Arsenic (PubChem CID: 5359596)", "Cadmium (PubChem CID: 23973)", "Chemical compounds studied in this article", "Chromium (PubChem CID: 23976)", "Copper (PubChem CID: 23978)", "Heavy metals", "Lead", "Lead (PubChem CID: 5352425)", "Nickel (PubChem CID: 935)", "Portable energy dispersive X-ray fluorescence", "Urban agriculture", "Vegetable sample preparation", "Wavelength dispersive X-ray fluorescence", "Yttrium (PubChem CID: 23993)", "Zinc (PubChem CID: 23994)" ]	[ "1106", "1602" ]	[ "For lithologic media, the quantification of elements using X-ray Fluorescence (XRF) spectroscopy is well understood and methods of sample preparation, measurement methods, and calibration/quantification are well documented (e.g. Byers, McHenry, & Grundl, 2016).", "In quantifying element concentrations using XRF, photons of energy are generated by an X-ray source (such as a compact Rh X-ray tube) and the photons pass through one or more primary filters to reduce the variability in source X-ray photon energy.", "Source photons then pass into a sample and transfer their energy to an inner-shell electron of an atom within the sample, which slightly displaces the electron from its preferred orbit leaving an unstable atom.", "An electron from an outer orbital then fills the vacancy in the lower orbital and at the same time releases energy in the form of a fluoresced secondary X-ray unique to the element and unique to the energy difference between orbitals.", "Element concentrations are determined by the rate at which fluoresced secondary X-rays are measured by a detector in the spectrometer.", "Due to the non-destructive nature of the analysis, XRF is emerging as a promising method for rapid quantification of heavy metals in vegetables as recent work demonstrates that lead and other heavy metals are taken up and translocated from the soil into consumable vegetable tissues (Clark, Brabander, & Erdil, 2006; Ferri et al., 2015; Finster, Gray, & Binns, 2004; Jolly, Islam, & Akbar, 2013; Lima, Nascimento, Silva, Carvalho, & Filho, 2009; Nabulo, Black, & Young, 2011; Rodriguez-Iruretagoiena, Trebolazabala, Martinez-Arkarazo, Diego, & Madariaga, 2015; Sekara, Poniedzialek, Ciura, & Jedrszczyk, 2005).", "Although human exposure and the resulting health impacts from direct contact with lead-contaminated soil is considered to be a primary pathway for lead exposure in urban agriculture, secondary exposure from chronic consumption of vegetables containing lead could be a significant contributing factor in total lead exposure (Chopra & Pathak, 2015; Ferri et al., 2015; Jolly et al., 2013), especially in high-risk populations, such as low-income, immigrant communities where exposure to lead remains disproportionately high.", "Child exposure to lead leads to a multitude of poor health outcomes (Keller, Faciano, Tsega, & Ehrlich, 2017; Lane et al., 2008; Schnur & John, 2014); therefore, further evaluation of lead (and other heavy metals) exposure is paramount.", "Established methods for measuring elements in vegetable and herb tissues includes a combination of traditional wet chemistry methods such as atomic absorption spectrometry (AAS) (Bozym, Florczak, Zdanowska, Wojdalski, & Klimkiewicz, 2015; Chopra & Pathak, 2015; Lima et al., 2009; Sekara et al., 2005; Song et al., 2012; Yadav et al., 2015), inductively coupled plasma-atomic emission spectrometry (ICP-AES) or inductively coupled plasma-mass spectrometry (ICP-MS) (Bešter, Lobnik, Eržen, Kastelec, & Zupan, 2013; Finster et al., 2004; Murray, Pinchin, & Macfie, 2011; Nabulo et al., 2011; Rodriguez-Iruretagoiena et al., 2015; Wiseman, Zereini, & Püttmann, 2013), wavelength dispersive XRF (WD-XRF) (Andersen et al., 2013; Figueiredo, Fernandes, Costa, Gonçalves, & Brito, 2016), bench-mounted energy-dispersive XRF (ED-XRF) (Anjos, Lopes, Jesus, Simabuco, & Cesareo, 2002; Gallardo et al., 2016; Jolly et al., 2013), and portable ED-XRF (Ferri et al., 2015; Gutiérrez-Ginés, Pastor, & Hernández, 2013; Sacristan, Rossel, & Recatala, 2016; Towett, Shepherd, & Drake, 2016).", "Recent work quantified heavy metals in algae with portable ED-XRF using a fundamental parameter factory calibration for plastics (Bull, Brown, & Turner, 2017; Turner, Poon, Taylor, & Brown, 2017).", "Sample preparation when using AAS or ICP-MS to quantify heavy metals in plants involves ashing plant material in a furnace or with concentrated hydrogen peroxide followed by digestion with acid (HNO3, HCLO4, H2SO4) and/or microwave extraction using one or more concentrated acids.", "These sample preparation techniques are inherently dangerous and generate a significant hazardous waste stream.", "Comparatively, XRF sample preparation techniques preserve the sample matrix and minimize waste generation.", "As noted by many in the literature (e.g. Gallardo et al., 2016), XRF is a promising technique in quantification of lead and other heavy metals in vegetable tissues.", "However, prior reviews of this technology have identified several limiting factors (Marguí, Queralt, & Hidalgo, 2009; Palmer, Jacobs, Baker, Ferguson, & Webber, 2009; Singh et al., 2017).", "The greatest obstacles identified in prior studies using XRF to measure heavy metals are achieving a limit of detection within the range of regulatory thresholds and generating consistent results that can be confirmed with another quantification technology.", "Due to limited commercial availability of reference materials and treatment of XRF spectrometers as “black box”, prior studies involving XRF have largely not controlled for matrix effects by: not matching the reference material matrix to sample matrix; using a multitude of sample preparation techniques; or using XRF standard factory calibrations optimized for non-carbon matrices.", "Further complicating prior XRF work is the quantitation of heavy metals based on the intensities of wavelengths with known peak overlaps.", "By addressing these inconsistencies and mitigating matrix effects, we hypothesize that WD-XRF and portable ED-XRF can be used to accurately and rapidly quantify heavy metals in vegetable samples with limits of detection applicable to health-based regulatory thresholds.", "Although the use of XRF in quantifying heavy metals in plant matrices has been reported previously, this work describes methods to systematically mitigate matrix effects through development of custom reference materials, building matrix-specific measurement and calibration routines, and confirming the efficacy of the XRF methods by comparison to ICP-MS analysis.", "Remarkably, no consumption standards exist in the United States for heavy metals in produce or cereals; therefore, this work relies on World Health Organization (WHO) food standards (WHO, 2018).", "On a broader scale, food security threats are often identified in retrospective studies limited to select food groups, select manufacturers/country of origin, and/or elements; however, with multiple new threats to food security identified each year, use of WD-XRF and portable ED-XRF spectrometry is a promising quality check that could be used at ports of entry by regulators, researchers, and/or growers/manufacturers to identify security risks prior to consumption/exposure.", "The single greatest source of bias in XRF measurements of vegetables is inter-element effects due to secondary absorption/enhancement of target wavelengths.", "Secondary absorption occurs when a fluoresced characteristic X-ray is absorbed by another atom in the matrix rather than returning to the detector, and if the absorbed energy is great enough, the atom will generate additional x-rays characteristic of the atom (direct secondary enhancement).", "Additional characteristic X-rays generated following secondary absorption can either return to the detector or be absorbed by additional atoms in the matrix and further generate characteristic x-rays (tertiary enhancement).", "Therefore, absorption/enhancement of characteristic X-rays can significantly alter the rate at which characteristic x-rays return to the detector such that element concentrations in the sample are not represented by the rate of characteristic x-rays.", "Mitigation of inter-element (matrix) effects therefore is central to all aspects of this experimental design.", "The commutability of reference materials to samples is critical in minimizing measurement uncertainty (Byers et al., 2016) and mitigating matrix effects (absorption/enhancement), but has often been overlooked in prior food studies.", "Commutability is especially critical when using XRF to quantify heavy metal concentrations in homogenized dry and undried (raw) vegetables as the mass attenuation coefficient of the vegetable matrix is very small compared to more common and readily available silicate-based soil reference materials, which were often used in previous food studies using XRF spectroscopy.", "Unfortunately, metals-rich plant-based reference materials are either no longer commercially available (e.g. NIST SRM 1515, NIST SRM 1547, BCR-60, BCR-100, BCR-279) or represent a very limited continuum of metal concentrations (e.g. NIST SRM 1570a, NIST SRM 1575a, BCR-129, BCR-414, BCR-482, BCR-670, ERM-CD281, IRMM-804).", "Because fully developed and verified metals-rich plant-based reference materials are not readily available, custom dried plant-based reference materials were prepared from easily obtainable commercial materials using methods similar to Figueiredo et al. (2016).", "Sample preparation techniques were developed for quantitation via WD-XRF and portable ED-XRF spectroscopy using the custom dried plant-based reference materials.", "Lastly, calibration routines were developed and confirmed with paired ICP-MS measurements of the reference materials.", "Paired XRF and ICP-MS measurements were taken for vegetables grown in garden soil collected from residential properties in the City of Milwaukee, Wisconsin to confirm the viability of the XRF measurement and calibration routines.", "Similar wet plant-based reference materials were prepared as an analogue to raw vegetables in an effort to use portable ED-XRF for quantification of metals in the field.", "Sample preparation techniques for undried (raw) samples and calibration routines were established.", "Paired WD-XRF and portable ED-XRF measurements were obtained from vegetables grown in garden soil collected from residential properties in the City of Milwaukee to evaluate the viability of the wet plant-based portable ED-XRF measurement and calibration routines.", "Twenty-one plastic jars containing 14 g of freeze-dried parsley were purchased from a retail source in Milwaukee, Wisconsin.", "The parsley was mixed in bulk and dried in an oven at 60C for 48 h.", "The powdered parsley was powdered by hand and a 30 g (±1 mg) aliquot of parsley power was added to a rotovap flask containing 200 ml of 18 μmho e-pure water and a pre-determined quantity of liquid ICP metals standard containing 100 mg L−1 of Al, As, Cd, Cr, Co, Cu, Fe, Pb, Mg, Mn, Ni, K, Na, Zn and 600 mg L−1 Y (Instrument Check Standard 7, SpecCerti Prep ®; Metuchen, NJ).", "The flask was swirled gently to hydrate the parsley and attached to a water-cooled rotovap (Heidolph Schwabach, Germany) operated at 80C and 80 RPM under vacuum to hydrate the parsley with the metals-rich solution while removing latent water.", "The rotovap process continued for 2 h or until the mixture was the consistency of a thick paste.", "The parsley was removed from the rotovap, dried in an oven at 60C for 48 h. Dried material was milled in a tungsten carbide shatterbox for 30 s to create a uniform homogeneous dry powder similar to Marguí et al. (2009).", "The process was repeated in a step-wise fashion using varying volumes of metals standard to create a library of plant-based reference materials with nominal heavy metal (Al, As, Cd, Cr, Co, Cu, Fe, Pb, Mg, Mn, Ni, K, Na, and Zn) concentrations ranging from 0.5 μg g−1 to 100 μg g−1 dry weight and nominal Y concentrations ranging from 1 to 600 μg g−1.", "A blank reference was created using the same process, but omitting the addition of the ICP Standard.", "A 1 g aliquot of each powdered reference material was digested by TestAmerica Laboratories, Inc. (Chicago, Illinois) using concentrated HNO3, HCl, and H2O2 per Method SW 846 3050B (USEPA, 1996).", "Heavy metal concentrations in the digestions were measured in triplicate at the UWM School of Freshwater Sciences using a high resolution ICP-MS (Thermo Scientific Element 2) to confirm element concentrations in the reference materials.", "ICP-MS detection limits for heavy metals of concern are less than 0.1 μg g−1.", "Preparation of typical powdered soil and rock samples for XRF analysis involves either fusing samples with a flux (such as lithium tetraborate) at high temperatures or pressing powdered samples with a carbon-based binding agent under high pressure to create uniform pellets (Byers et al., 2016).", "Fusion or ashing is impractical; therefore, uniform pellets were created by pressing dried powdered plant samples at 25 t for 60 s in a 40 mm diameter hydraulic die press.", "The variability in pellet thickness across the urban agriculture literature is large and is likely a source of bias.", "Because matrix attenuation is minimal in vegetable samples, source X-rays entering a vegetable sample can pass entirely through the sample and generate fluoresced X-rays from the entire sample thickness.", "Therefore, the depth of measurement (also referred to as escape depth) for each element wavelength becomes limited by the attenuation of the fluoresced secondary X-rays by the sample matrix, not by the attenuation of the source X-ray.", "For a given element wavelength, if the sample is thicker than the measurement depth, the net intensity of fluoresced X-rays is independent of sample thickness, and the pellet is considered “infinitely thick.”", "If the sample is thinner than the measurement depth for a given element wavelength, the pellet is considered “infinitely thin.”", "If a pellet is infinitely thin with respect to a given element wavelength, the net intensity of fluoresced X-rays is a function of sample thickness (mass) and subject to significant bias between samples unless samples are of uniform thickness.", "Additionally, vegetable samples considered infinitely thin could be subject to significant bias from characteristic fluoresced X-rays generated by the spectrometer shielding/housing passing backwards through the sample matrix and potentially generating secondary or tertiary enhancement of characteristic X-rays from matrix elements or by passing through the matrix and being directly measured by the spectrometer detector.", "The depth of measurement (or escape depth of fluoresced X-rays) is calculated based on the matrix density and mass attenuation coefficient for varying X-ray wavelengths of interest (Towett et al., 2016).", "Unfortunately, the mass attenuation coefficient for plant tissues is unknown and the density of dried vegetables is variable between tissues with reported values ranging from 1.1 g cm−3 to 1.7 g cm−3 (Martynenko, 2014; Rodríguez-Ramírez, Méndez-Lagunas, López-Ortiz, & Torres, 2012).", "We used a nominal density of 1.4 g cm−3 to represent a generic dried vegetable tissue and the mass attenuation coefficient of simple sugar + cellulose (C6H12O6C6H10O5) reported by the National Institute of Standards and Technology (Chantler et al., 2001) for varying wavelengths from 0 to 40 keV (Fig. 1).", "With these conservative model factors, calculations indicate a dried plant-based sample 1.7 cm thick should be infinitely thick with respect to the Pb Lβ1 wavelength (12.614 keV).", "Based on the diameter of the pellet die used in this study, a 1.7 cm thick pellet would require approximately 30 g of dried plant material, which is too much powdered material for the XRF pellet die to process.", "Further, as raw vegetables used in our larger study were 87% water on average, roughly 250 g of raw vegetable would be needed to create a single pellet; which is impractical.", "Knowing Cr is present inside the housing and shielding of the Pioneer S4 WD-XRF used in this study (Bruker AXS, Inc.), pellets had to be infinitely thick with respect to the CrKα1 wavelength (5.415 keV) to prevent bias in the measurements.", "To be conservative, 3.2 g of powdered sample per pellet was used and when pressed at 25 t for 60 s, resulted in a pellet approximately 1.9 mm thick.", "Pellets prepared in this manner are competent, resilient to handling, maintain integrity when stored long-term in a desiccator, and can be analyzed by WD-XRF spectrometry under vacuum without breakage.", "Selecting appropriate wavelengths for quantification of heavy metals with XRF is critical to minimize bias and error in measurement routines.", "Lighter weight elements are commonly quantified based on Kα wavelengths, and due to the keV limitations of the Rh X-ray tube, elements heavier than La are commonly quantified based on Lα wavelengths.", "However, as shown in Fig. 2a, the Pb Lα1 wavelength overlaps the As Kα1 wavelength.", "Therefore, to mitigate the peak overlap with As, we used the Lβ1 wavelength for quantification of Pb.", "The potential for absorption and enhancement in the matrix was evaluated for each element of interest using data provided by Hubbell and Seltzer (2004).", "Enhancement of the Pb Lβ1 wavelength is not expected from elements in the parsley matrix, from elements contained in the ICP Standard, or from elements in the shielding/housing.", "However, as illustrated in Fig. S1 of the Supplemental materials, absorption and secondary or tertiary enhancement of Kα1 wavelengths of Zn, Ni, Fe, Cr, Cu, Co, Mn, and As is possible from elements in the parsley matrix, elements contained in the ICP Standard, and/or elements in the spectrometer shielding/housing.", "Using multiple metal foils, we assessed the measurement depth of each element wavelength of interest and possible matrix effects as described in Section 2.1.4 to confirm we had adequately controlled for possible bias associated with pellet thickness and matrix effects.", "A custom measurement routine was developed in the Bruker AXS, Inc.", "Pioneer S4 WD-XRF Spectra Plus software to measure the intensities under vacuum of lead using the Pb Lβ1 wavelength and Cr, Ni, and Y using Kα1 wavelengths.", "Specific analytical details for each element are provided in Table S1 of the Supplemental materials.", "Each pellet in the reference material library was analyzed with the WD-XRF measurement routine.", "Custom calibration routines for each element were developed by comparing net X-ray intensities to known dry weight element concentrations based on element concentrations in each reference material (including the “blank”) as determined by ICP-MS.", "The routine corrected for rhodium Rayleigh and Compton peaks, matrix effects, and possible contamination of tungsten and cobalt from the shatterbox.", "Reference materials with element concentrations less than the limit of detection determined by the Pioneer S4 software were omitted from the calibration routines.", "The goodness of fit, root mean square error, calibration range, the LOD determined by the Pioneer S4 software, and the LOQ calculated per (Thomsen, Schatzlein, & Mercuro, 2003) are listed in Table 1.", "The coefficient of determination (r2) of each calibration is no less than 0.999 with corresponding single-digit root mean square errors.", "Common garden vegetables were grown in pots of metals-rich soil sourced from residential vegetable gardens in the City of Milwaukee, Wisconsin.", "The vegetables were harvested, scrubbed vigorously under running water, peeled (root vegetables only), chopped, dried in an oven at 60C for 48 h, milled in a tungsten shatterbox for 30 s, and a 3.20 g aliquot of each sample was pressed at 25 t for 60 s in a 40 mm diameter pellet hydraulic die press.", "Each sample was analyzed by WD-XRF using the routine described previously and concentrations determined from calibration curves.", "Infinite thickness calculations were confirmed using the vegetable pellets by comparing the change in net WD-XRF intensities measurements with and without the presence of a metal foil placed behind dried pressed vegetable samples.", "The four separate metal foils used in this evaluation include two 99% pure foils of (1) 0.2 mm Cu and (2) 1 mm Pb and two alloy foils of (3) 0.3 mm “nickel-silver” (consisting of 65% Cu, 18% Ni, and 17% Zn) and (4) 0.4 mm stainless steel foil containing Cr and Mn.", "After measurement with WD-XRF, 23 vegetable pressed pellets representing the range of measured lead concentrations were broken into 1 g aliquots and digested by TestAmerica Laboratories, Inc. using Method SW 846 3050B (USEPA, 1996).", "Heavy metal concentrations in the digestions were measured in triplicate at the UWM School of Freshwater Sciences using a high resolution ICP-MS (Thermo Scientific Element 2) and concentrations converted to dry weight for comparison between the two analytical techniques.", "The paired relationships between ICP-MS and WD-XRF measurements of Pb in vegetables are illustrated on Fig. 3a.", "The WD-XRF used in this study is a bench-mounted spectrometer, which limits the usefulness of this technology in real-time quantification of heavy metals in remote locations, such as agricultural fields, gardens, or ports of entry.", "Portable hand-held ED-XRF is a promising technology for rapid quantification of elements and shows particular promise in the areas of food security/food quality.", "However, older ED-XRF spectrometers often measured intensities of elements from peaks with known overlaps and relied on default factory calibrations.", "Recently developed instrumentation allows the entire X-ray spectrum to be captured, stored, and custom calibrations developed based on distinctive element wavelengths.", "Methods for developing and confirming measurement and calibration routines for quantification of heavy metals in dried pressed pellets with portable ED-XRF are identical to the methods described previously in developing and confirming routines for WD-XRF.", "In addition to confirming the infinite thickness calculations using metals foils, the influence of measurement time on accuracy and the influence of multi-metal primary filters on detection limits were evaluated.", "The dried pressed pellet reference materials used in the WD-XRF measurement routine were placed on the stage of a Bruker Tracer portable ED-XRF spectrometer and the fluoresced XRF spectra captured using the Bruker S1PXF software program.", "Fluoresced X-rays were measured with a silicon drift detector for 120 s in air at 40 keV and 40 μA and used a removable multi-metal primary filter consisting of 25 μm Cu, 25 μm Ti, and 25 μm Al (Cu/Ti/Al filter).", "The average valid photon count rate was 16,815 photons per second with a resolution of 20.04 eV per channel at full height width for the Mn Kα 1 wavelength.", "The X-ray source of the spectrometer is a Rh-tube oriented at a 53-degree angle with respect to the sample.", "As the entire spectrum from 0 to 40 keV was captured by the software, routines were developed to quantify Cr, Mn, Ni, Cu, Zn, Pb, Y, and Cd.", "The captured spectra for the 10 μg g−1 reference material and the net Pb Lβ1 wavelength intensities for six reference materials are illustrated in Fig. 2.", "Calibration routines were developed by comparing net X-ray intensities to known dry weight element concentrations using the Bruker Microsoft Excel plugin (S1CalProcess).", "The peak overlap corrections, goodness of fit, root mean square error, calibration range, the LOD calculated using the background equivalent concentration approach (Thomsen, 2012) and the LOQ calculated (Thomsen et al., 2003) for each element are summarized in Table 2.", "The r2 of each calibration is no less than 0.97 with corresponding single-digit root mean square errors.", "To confirm that the infinite thickness observations made with WD-XRF remained valid for portable ED-XRF, the same reference materials were analyzed for 120 s and net intensities determined.", "A piece of 1 mm 99% pure lead foil was placed behind the pellet and the analysis repeated.", "The Pb foil was removed and a 0.025 mm piece of 99.9% pure Mo foil was placed behind the pellet and the analysis repeated a third time.", "The associated intensities increased with the presence of a metal foil, confirming the pressed pellets are infinitely thin with respect to the Pb Lβ1 and Mo Kα1 wavelengths.", "Prior studies have counted fluoresced X-rays in plant material from 30 to 240 s (Bull et al., 2017; Gutiérrez-Ginés et al., 2013; Sacristan et al., 2016), or did not specify count times.", "To optimize the portable ED-XRF measurement routine and to quantify the improvement in the accuracy with an increase of time, four dried plant-based reference materials were analyzed by ED-XRF as described previously by varying measurement times from 5 s to 300 s.", "The resulting Pb concentrations were compared to the known Pb concentrations and the relative percent differences between each data pair calculated as described in Byers et al. (2016) (Fig. S2 of the Supplemental materials).", "The relative difference neared ± 10% at 120 s and only marginal improvements were noted with longer measurement time, therefore 120 s was selected as the default measurement time for the portable ED-XRF measurement routine.", "Primary multi-metal filters have been designed by ED-XRF manufacturers to reduce variability in the energy of source X-ray photons and reduce background radiation.", "As illustrated in Fig. 2A, the Pb Lβ1 peak is located on the shoulder of the inelastic Compton radiation peak originating from the Rh tube.", "Normally, XRF measurements are normalized to this inelastic scatter (18.5–19.5 keV); however, normalization requires additional steps in the calibration.", "Therefore, the ED-XRF measurement and calibration steps outlined above were repeated using an alternative removable multi-metal primary filter consisting of 25 μm Ti, 50 μm Fe, and 25 μm Mo (Ti/Fe/Mo) designed to reduce the overall Compton radiation.", "The average valid photon count rate was 2931 photons per second with a resolution of 19.98 eV per channel at full height width for the Mn Kα1 wavelength.", "The peak overlap corrections, goodness of fit, root mean square error, calibration range, the LOD calculated using the background equivalent concentration approach (Thomsen, 2012) and the LOQ calculated (Thomsen et al., 2003) for each element summarized in Table S2 of Supplemental materials.", "Although the calibration goodness of fit parameters with the Ti/Fe/Mo multi-metal primary filter are reasonable (r2 values near one with single digits RMSE values), there is a small loss of fit in lower wavelengths.", "The researchers grew common garden vegetables as described in Section 2.1.4 and samples prepared as described in Section 2.1.3.", "Each sample was analyzed by portable ED-XRF using the primary filter as described above.", "After measurement with the ED-XRF, 27 vegetable pressed pellets representing the range of measured lead concentrations were broken into 1 g aliquots and analyzed using ICP-MS as described in Section 2.1.3.", "The paired relationships between ICP-MS and ED-XRF measurements for Pb are illustrated in Fig. 3b.", "To determine if changing primary multi-metal filters improved the applicability of the calibration to vegetable samples with respect to Pb, 35 additional samples of vegetables grown by the researchers were prepared as dried pressed pellets and analyzed using ED-XRF equipped with the Ti/Fe/Mo multi-metal primary filter.", "Concentrations were compared to measurements made using WD-XRF; the paired relationships between WD-XRF and ED-XRF measurements for Pb are illustrated in Fig. 3c. Although the calibrations for Pb using either primary filter are acceptable, the viability of the calibration routine to actual samples is better with the Cu/Ti/Al filter.", "A major limitation to the use of portable ED-XRF in urban agriculture and food security applications is the lack of commercially available metals-rich raw (undried) plant-based reference materials.", "In developing undried reference materials analogous to raw vegetables, additional commercially sourced dried parsley was mixed with the liquid ICP metals standard and processed in a roto-vap as described previously to create a second library of eleven dried/homogenized parsley-based reference materials with Pb concentrations ranging from 0.5 μg g−1 to 100 μg g−1 on a dry-weight basis as confirmed by WD-XRF.", "The mean water content of vegetables grown in metals-rich soil used in this study is 87% with a standard deviation of 4%.", "To prepare a reference library analogous to raw vegetables, a 1.50 g aliquot of dried reference material was added to a glass vial containing 8.50 g of e-Pure water.", "The mixture was gently stirred, the jar lid tightly secured, and the mixture allowed to rest for 12 h.", "The process was repeated in a step-wise fashion to create a library of “wet” plant-based reference materials with Pb concentrations ranging from 0.0 μg g−1 to 15 μg g−1 wet weight.", "A blank reference was created using the same process.", "A second set of reference materials was created with 65% water for comparison purposes.", "The 65% and 85% water content plant-based reference material sets were packed into single open-ended 32 mm diameter (10 ml volume) XRF sample cups (Premier Lab Supply; Port St. Lucie, FL) and secured with 4.0-µm polypropylene film (Premier Lab Supply).", "Each sample cup was analyzed via portable ED-XRF for 120 s using the Bruker S1PXF software program as described previously using the Cu/Ti/Al primary filter.", "The average valid photon count rates for reference materials with 65% and 85% water were 34,684 and 35,324 photons per second, respectively, with resolutions of 20.02 eV per channel at full height width for the Mn Kα wavelength.", "The measurement routine measured fluoresced X-rays for 120 s at 40 keV and 40 μA.", "Calibration routines were developed to quantify Cr, Ni, Pb and Y. Custom calibration routines for each set of reference material were developed in Microsoft Excel using the Bruker plugin (S1CalProcess).", "The goodness of fit, root mean square error, calibration range, the LOD calculated using the background equivalent concentration approach (Thomsen, 2012) and the LOQ calculated (Thomsen et al., 2003) for each element is summarized on Table 3 for both water contents.", "The goodness of fit parameters for each element using the 85% water content reference set are less than those using the 65% water content reference set.", "Although the LOD and LOQ values for each element are similar between the two water contents, when converted from wet weight concentrations to dry weight concentrations, the calibration developed with 85% water content is weaker by comparison.", "Common garden vegetables were grown by the researchers in pots of metals rich soil sourced from residential vegetable gardens in the City of Milwaukee, Wisconsin.", "The vegetables were harvested, scrubbed vigorously under running water, and peeled (if necessary).", "Each vegetable tissue sample was coarsely homogenized in a food processor for 10 s.", "An aliquot of the homogenized slurry was poured into a 32 mm XRF sample cup and the sample cup secured with 4-µm polypropylene film.", "The sample cup was placed on the stage of a Bruker Tracer portable ED-XRF spectrometer and the fluoresced XRF spectra captured and analyzed as described in Section 2.2.2.", "The concentration of heavy metals was calculated based on the 85% water calibration routine.", "After analysis, the wet sample was dried and pressed pellets prepared as described in Section 2.1.2 and analyzed using the dried WD-XRF measurement routine.", "To confirm the measurement depth of raw vegetables with portable ED-XRF, 67 vegetables grown in soil collected from residential vegetable gardens in Milwaukee, WI were coarsely homogenized, packed into sample holders, X-rays counted for 120 s, and net intensities determined.", "A piece of 1 mm 99% pure lead foil was placed behind the sample holder and the analysis repeated.", "The Pb foil was removed and a 0.025 mm piece of 99% pure Mo foil was placed behind the sample holder and the analysis repeated a third time.", "Based on the change in peak intensities, the samples are considered infinitely thick for the Pb Lβ1 wavelength, but not the Mo Kα1 wavelength.", "The r2 statistic is commonly used in bivariate calibration regressions to explain the amount of variability in the dependent variable (concentration) that can be explained by the independent variable (peak intensity).", "In XRF spectroscopy, researchers strive to maximize the r2 value as close to 1 as possible.", "Further, the XRF calibration routines are also evaluated with root mean square error (RMSE) values, which is the standard deviation of the calibration regression residuals.", "Most importantly, the RMSE can be interpreted in terms of measurement units of the dependent variable, which in this study is element concentration expressed in μg g−1.", "Therefore, in refining calibration routines, the r2 values were maximized and RMSE values minimized.", "RMSE values were further used to evaluate the minimum element concentration quantifiable by each calibration.", "If the RMSE is less than the calculated LOQ, then the LOQ represents the smallest concentration that can be quantified by a calibration routine.", "However, if the RMSE is greater than the LOQ, then the RMSE represents the smallest concentration that can be quantified by the calibration routine.", "This approach allows for a greater certainty in the calibration and is more rigorous compared to the more common approach where the calibration range assumed to be represented by the range in concentration of calibration reference materials.", "Evaluation of measurement depth and the infinite thickness assumption.", "Dried pressed pellets of vegetables of consistent mass considered infinitely thick with respect to CrKα1 and considered infinitely thin with respect to Pb Lβ1 were analyzed and the measurement depths confirmed with metal foils as described previously.", "Net intensities of the Pb Lβ1 wavelength increased 4 orders of magnitude when the lead foil was placed behind samples, thus confirming the pressed pellet samples are infinitely thin at 12.614 keV.", "Similarly, the net intensities of the Cu Kα1 fluoresced X-rays increased one order of magnitude confirming samples remained infinitely thin at 8.046 keV.", "When the “nickel-silver” foil was added behind the sample, the mean Ni Kα1 net intensity increased by 11 counts per second, which is a statistically significant increase (t < 0.001; 16 df) and equal to an increase of 77 μg g−1.", "The mean net intensity of the Mn Kα1 wavelength increased by 0.05 counts per second when the stainless steel foil was added, which is a statistically significant increase based on a matched-pair analysis (t < 0.01; 15 df), although the mean increase is equal to an increase of only 1 μg g−1.", "The Cr Kα1 (5.415 keV) net intensities and corresponding concentrations did not increase (t > 0.5; 15 df) when the stainless steel foil was added behind samples.", "This empirical evaluation matches the calculations of infinite thickness illustrated on Fig. 1, and by controlling for matrix effects, confirms that the dried vegetable pressed pellets used in this study are appropriate for use in measuring elements with fluoresced wavelengths less than 5.451 keV.", "Potential enhancement from absorption of characteristic X-rays generated in the housing is controlled.", "Researchers are cautioned to evaluate multiple thicknesses of sample to quantify the impact thickness will have on measurement results for a particular element of interest.", "For example, although the concentration of Mn increased by 1 μg g−1 by adding foil behind the pellet, this increase may be within acceptable bias tolerances in some applications, especially if the target element is not present in the shielding and housing of the spectrometer or not subject to influence from absorption/enhancement by matrix elements.", "Evaluation of the dry plant-based WD-XRF measurement and calibration routine for Pb.", "Measurements indicate Pb is not present in the housing of the WD-XRF, therefore, although the pellets are infinitely thin for the Pb Lβ1 wavelength, the housing of the WD-XRF is not a direct source of bias in these measurements.", "The RMSE value of the Pb WD-XRF calibration routine is greater than the LOD, but less than the LOQ; therefore, the calibration range for Pb varies between 1 and 96 μg g−1 (Table 1).", "Although RMSE and LOQ values from calibrations developed by others are not widely available in the literature, the LOD value for Pb in this study is one or more orders of magnitude less than values reported previously (Andersen et al., 2013; Gallardo et al., 2016; Gutiérrez-Ginés et al., 2013), and more importantly, the LOD (0.3 μg g−1) is equal to the Maximum Level for lead in leafy vegetables (on a dry weight basis) and only 0.1 μg g−1 greater than the Maximum Level for lead in cereal (WHO, 2018).", "The appropriateness of the WD-XRF calibration routine for Pb developed in this study to actual vegetable samples was further confirmed with paired ICP-MS measurements.", "The slope of the bivariate regression between 23 WD-XRF and ICP-MS measurements of vegetables grown in metals-rich soil is 1.08 with an r2 value of 0.96 providing additional support in the accuracy of WD-XRF measurements (Fig. 3a).", "Evaluation of the dry plant-based WD-XRF measurement and calibration routine for Cr, Ni, and Y. ICP-MS measurements confirmed the presence of Cr and Ni in the parsley used in this study.", "However, as RMSE values are less than LOQs and the XRF software calculated LODs are less than the Cr and Ni concentrations in the blank, the Cr and Ni calibrations are considered satisfactory for the element concentrations represented by the reference materials (Table 1).", "Although RMSE and LOQ values for calibrations are largely absent from the literature, our LOD values for these elements are less that previously achieved (Ni; (Gutiérrez-Ginés et al., 2013); Cu; (Gutiérrez-Ginés et al., 2013; Otaka, Hokura, & Nakai, 2014)).", "The concentrations of Y were minor in the vegetable samples; therefore, our evaluation of the WD-XRF routine with ICP-MS was not possible.", "However, measurements indicate Y is not present in the housing of the WD-XRF, therefore, although the pellets are infinitely thin for the Y Kα1 wavelength, the housing of the WD-XRF is not a direct source of bias in these measurements.", "Unfortunately, the concentrations of Cr and Ni are greater than the WD-XRF LOQs for only eight confirmation samples; therefore, our evaluation of the WD-XRF routine with ICP-MS is limited.", "Potential bias from absorption/enhancement due to the matrix and further enhanced by XRF shielding containing mixed metals is critical when evaluating transition metals in a carbon matrix.", "For instance, because the pressed pellets are infinitely thick for Cr Kα1, the presence of Cr in the housing/shielding of the XRF is not considered a direct source of bias in measurements.", "However, fluoresced Fe Kα1 X-rays from the shielding could be absorbed by Cr in the matrix and enhance fluorescence of Cr Kα1 X-rays.", "In addition, measurable quantities of Ni are present in the WD-XRF housing/shielding and the pellets are considered infinitely thin for Ni; therefore, characteristic Ni Kα1 X-rays generated by the XRF housing could return to the detector and serve as a source of direct bias.", "Or, Ni Kα1 characteristic X-rays generated by the shielding could be absorbed by Fe in the sample matrix, which could in turn enhance the generation of Cr Kα1 wavelengths.", "Therefore, researchers cannot ignore increased matrix enhancement due to the influence of shielding.", "To mitigate this potential source of bias, the same shielding/housing must be used for all samples so that critical enhancement influences can be controlled during development of the calibration routine.", "Evaluation of measurement depth and the infinite thickness assumption.", "This study focuses on the most common heavy metals, therefore optimization of ED-XRF calibration focused primarily on the spectrum between Cr Kα1 (5.415 keV) and Pb Lβ1 (12.614 keV) with less interest in the Y Kα1 (14.958 keV) to Cd Kα1 (23.173 keV) range.", "Measurement depths of the dried pellets with portable ED-XRF were evaluated with metal foils as described previously.", "Similar to WD-XRF measurements, the Pb Lβ1 and Mo Kα1 intensities measured by ED-XRF increased significantly with the presence of the associated metal foil.", "Therefore, the pressed pellets are infinitely thin with respect to the Pb Lβ1 and Mo Kα1.", "Evaluation of the dry plant-based ED-XRF measurement and calibration routine for Pb.", "The RMSE, LOD, and LOQ values for the portable ED-XRF calibration for lead with the Cu/Ti/Al multi-metal primary filter (Table 2) are nearly identical to the values for the WD-XRF calibration routine.", "Even more promising is the calibration accuracy that was achieved with the portable ED-XRF even though measurement count times are 5 times less than WD-XRF.", "Further, the confirmation of ED-XRF measurements with 27 paired ICP-MS measurements suggests the ED-XRF calibration routine with the Cu/Ti/Al multi-metal primary filter is accurate (regression r2 = 0.97; slope = 1.02) with no apparent bias (Fig. 3b).", "The variation between the measured concentrations and known concentrations using four reference materials decreases sharply when portable ED-XRF measurement times are increased, with very little difference in RD values in measurements lasting 120 s or longer (Fig. S2 of Supplemental materials).", "Therefore 120 s analysis time is considered sufficient.", "To reduce the Compton peak, the calibration and verification process summarized above was repeated using the a Ti/Fe/Mo multi-metal primary filter.", "Although the valid photon count rates reduced significantly with the second primary filter, the RMSE, LOD, and LOQ values (Table S2 of Supplemental materials) did not increase significantly compared to the first filter.", "However, the confirmation of Pb ED-XRF measurements with 35 paired WD-XRF measurements of vegetable samples indicate the ED-XRF calibration routine with the Ti/Fe/Mo multi-metal primary filter is weak (regression r2 = 0.91; slope = 1.39) with an unexplainable positive bias (Fig. 3c).", "Although the Ti/Fe/Mo primary filter decreased background radiation, use of the Cu/Ti/Al primary filter is preferable for quantification of lead in vegetables.", "Evaluation of the dry plant-based ED-XRF measurement and calibration routine for additional heavy metals.", "Although Pb is the primary metal of interest in this study, the ED-XRF captured a wide spectrum range allowing for quantification of several metals.", "The relatively small RMSE and LOQ values suggest these elements can be accurately measured with ED-XRF down to the single-digit μg g−1 range (Table 2).", "Evaluation of measurement depth and the infinite thickness assumption.", "Based on a matched-pair statistical analysis, adding Pb foil behind the sample cups did not increase the net intensity of the Pb Lβ1 wavelength (p < 0.08, DF = 67) suggesting the coarsely homogenized wet (raw) samples packed into sample cups are infinitely thick for wavelengths less than 12.614 keV.", "However, adding Mo foil behind the sample cups significantly increased the net intensities of the Mo Kα1 wavelength (p < 0.001; DF = 66) confirming that samples are infinitely thin for wavelengths greater than 17.480 keV.", "The RMSE, LOD, and LOQ values on a wet weight basis from the 85% water calibration of Cr, Ni, Pb, and Y (Table 3) are similar to values from the dry pressed pellets indicating X-ray attenuation by the presence of water is minimal.", "However, when converted to a dry weight basis the portable ED-XRF RMSE, LOD, and LOQ values from the wet plant-based calibration routine are comparatively large.", "Although the water in the reference materials does not significantly attenuate fluoresced X-rays, the water present in the matrix dilutes the quantity of heavy metals per unit volume of sample to a point where ED-XRF measurements of wet vegetables are not useful from a regulatory perspective unless the element concentrations are sufficiently large.", "Very few vegetables grown in this study had heavy metal concentrations greater than LOQs, therefore evaluation of the 85% water ED-XRF calibrations with confirmation samples was not possible.", "Increasing the sample analysis time is the most common way to increase the precision and accuracy of XRF measurements and lower XRF detection limits.", "A supplemental ED-XRF calibration was developed using the 85% water content reference set by increasing the count time from 120 s to 300 s; calibration details are summarized in Table S3 of Supplemental material.", "By increasing the measurement time, the relatively small RMSE values from the calibrations converted to dry-weight concentrations based on water content suggests Cr, Ni, and Pb can be accurately measured with ED-XRF down to the single-digit μg g−1 range in raw samples (Table 3S).", "Although we were unable to empirically measure the LOD and LOQ using 300 s measurement time, increasing sample analysis time by a factor of 4 should reduce the detection limit by a factor of 2.", "If this relationship holds true for raw vegetable samples, based on the LOD at 120 s (Table 3), we expect the LOD for Pb with 300 s measurement time to decrease to near 1 μg g−1 (wet weight), which would be equivalent to approximately 7 μg g−1 on a dry weight basis.", "The mean water content of 261 vegetable samples used in this study is 87% with a standard deviation of 4%, therefore, using the 65% water content calibration would be inappropriate to quantify heavy metals in our raw vegetables.", "However, this work suggests that if the water content of food samples is not more than 65% (for example grains or cereals), portable ED-XRF spectrometry could detect Pb in raw samples at concentrations as low as 1 μg g−1 dry weight (Table 3) with 120 s count time.", "Further, this suggests that portable ED-XRF is a viable technology for use in element quantification in prepared foods with low water contents.", "This work has shown that by managing matrix effects, XRF can be a useful tool in quantification of lead and other heavy metals in vegetables.", "In our case, practical limits existed on the preparation of samples of sufficient thickness to retain all source and characteristic X-rays.", "Therefore, in addition to measuring samples and reference materials under the same conditions (e.g. energy, current, filter, count time, atmosphere), the most critical factors we managed in developing measurement routines for quantification of heavy metals in plant tissues with WD-XRF and portable ED-XRF were:", "Developing reference materials with commutability to samples and maintaining consistency with sample preparation/handling (e.g. drying time, milling time, sample mass),", "Selecting proper wavelengths to eliminate peak overlaps and controlling for possible enhancement from within the matrix or from characteristic X-rays generated by the shielding/housing,", "Analyzing samples for long enough to maximize accuracy and precision, and", "Confirming the viability of new routines to actual samples through paired analysis of samples with another quantification technology to provide additional assurance in the measurement and calibration routines.", "As we hypothesized, by addressing these critical factors, this study demonstrates that WD-XRF and portable ED-XRF can be used to accurately and rapidly quantify heavy metals in vegetable samples with limits of detection achieving regulatory thresholds.", "Although the most robust calibration was obtained with WD-XRF, this technology is limited to fixed laboratory-based instruments.", "Slight compromise in the precision and accuracy of measurements with portable ED-XRF is offset by the portability and ease of use of this technology outside of a traditional laboratory setting.", "Quantification of heavy metals in wet coarsely-homogenized raw (undried) vegetable tissues was performed; however, RMSE and LODs on a dry-weight basis are strongly influenced by measurement time and water content which currently limits this technology.", "However, the technology is very promising for analysis of coarsely homogenized wet (raw) foodstuffs with lower water contents, such as grains and legumes and could easily be adopted for prepared foods.", "This work was supported by the Children’s Health Environmental CORE Center at the University of Wisconsin Milwaukee, which is funded through the National Institute of Environmental Health Sciences [Grant No. P30ES004184]." ]	[ "Calibration routines for quantification of Pb in vegetables with XRF are optimized.", "These XRF methods achieved detection limits of 0.3 μg g−1 for Pb in dry vegetables.", "Pb in raw vegetables is quantified at levels as low as 1 μg g−1 with ED-XRF." ]
Complexity, uncertainty and ambiguity: Implications for European Union energy governance	Energy security is an ambiguous concept. Growing academic interest has aimed at defining, conceptualising and measuring energy security, often through indicators. Energy policy in the European Union (EU) is not concerned with energy security's ambiguous conceptualisation, nor does it use energy security indicators, but it refers consistently to security as one of its primary aims. In this paper, by analysing the use of energy security both in scientific publications and in EU policy, we argue that the ambiguity of the concept plays a function in the policy process and is only seen as a problem in the academic literature. Building on the uncertainty literature, we conceptualise ambiguity as the type of uncertainty that emerges from complexity. Complexity leads to the existence of multiple representations of a system, which may serve different purposes in the policy process, generating ambiguity. Uncertainty is mobilised to frame energy policy as a matter of security. This has implications for the science-policy interface: on one hand, the analysis suggests that science's aim of providing holistic assessments and clarifications may not serve its desired instrumental purpose in policymaking; on the other, ambiguity allows for materially ineffective policy measures to persist in the name of energy security.	[ "ENER", "SOCI" ]	[ "Definition", "Energy security", "Indicators", "Policy-making", "Science-policy interface" ]	[ "2102", "2103", "2104", "2105", "3301" ]	[ "Energy security has received growing attention in European Union (EU) and United States (US) public policy since the early 2000s, reviving a concept that had been predominant during the oil crises of the 1970s [1].", "While persisting as a priority in national agendas, its definition has evolved in time.", "Up to World War II, energy security was tied to the supply of fuels for the military [1], linking the concept of security to the military narrative.", "According to Lippman, “a nation is secure to the extent to which it is not in danger of having to sacrifice core values, if it wishes to avoid war, and is able … to maintain them by victory in such a war” [2].", "With the oil embargo of the 1970s, energy security became part of political diplomacy and geopolitical concerns.", "In this context, it referred to the risks of depending on oil imports, and on the vulnerability of oil trade to international diplomacy and political instability in producing countries.", "With the publication of the report “The limits to growth” (Meadows et al. 1972), security was also tied to the possibility of physical scarcity of non-renewable energy sources.", "Starting from the 2000s, the term has been increasingly associated with resilience based on a complex systems perspective [1], with the resource scarcity narrative based on concerns for the depletion of fossil fuel reserves [3], and with renewable energy based on the challenges of a renewable transition [4–6].", "Challenges and solutions offered by specific primary energy sources, such as gas, coal, uranium, shale gas and variable renewable sources have also contributed to the proliferation of definitions of energy security.", "In the EU, security concerns became central to the energy policy agenda following the 2005–09 gas and oil disputes between Russia and Ukraine, and between Russia and Belarus [7].", "As the electrification of energy systems gradually increases to incorporate a higher share of renewable energy, energy security has also evolved to incorporate dimensions that are unique to electricity, such as the security of transnational grids [8].", "The reintroduction of energy security in policy agendas has sparked academic debates that can be grouped under the field of energy security studies.", "The view that energy is a key input to all economic processes is widespread in these studies [9,10], and in disciplines such as ecological economics [11–13].", "Characterisations of energy security range from the availability of primary energy sources [10], to the stability of internal markets [14], to the affordability at consumer level [3].", "While availability and affordability are the two dimensions most commonly associated with energy security [3,10,14–18], it has been linked to a number of other aspects, including for example sustainability [3], sovereignty and resilience [1].", "The ubiquity of energy security, and its context-dependent nature [18,19], have been the object of a branch of security papers exploring the ambiguity of the concept.", "Ciută [9] explains the multiplicity of definitions of energy security as a result of the fact that “energy security is a complexity model with interlocking segments and levels of interaction” [p. 132].", "Energy security studies recognise that the security of modern energy systems has different characteristics than those attributed to the concept in the 1970s, among other factors because of the increased importance of renewable energy, and attempt to handle the multiple dimensions that are associated with this polysemic concept [19], which sometimes mixes technical and colloquial definitions of energy security.", "From a policy perspective, the growing focus given to energy security in EU policy from the early 2000s follows a trend of securitisation of energy issues [20], i.e. “a process where governments frame energy as an existential threat to state interests” [21, p.114].", "This is in contrast with previous framings of the 1990s, when energy had been mostly relegated to the technical domain, as highlighted by Kuzemko [22] for the case of the UK.", "Energy security has long been a recurring theme for many national agendas of countries that are poor in primary energy sources, often used as a justification for disparate policy measures.", "This was highlighted for the US by MIT economist Paul Joskow [23]: “There is one thing that has not changed since the early 1970s.", "If you cannot think of a reasoned rationale for some policy based on standard economic reasoning, then argue that the policy is necessary to promote ‘energy security’” [p. 7].", "From a governance perspective, energy security is used both as a means to mobilise uncertainty and as a material issue.", "The multiple definitions provided by academia and the overflow of information do not lead to paralysis-by-analysis, as proven by the fact that the concept of energy security is recurrently used in policy.", "It is due to the very existence of multiple definitions that one may view energy security as characterised by ambiguity.", "Focusing at the interface between science and policy, there is a clear gap between the conceptualisations and measurements of energy security in academia, and the (non-)issue of ambiguity in policymaking [24].", "Although some authors recognise that ambiguity plays a role in policy-making [25–27], scientific literature largely sees ambiguity as a problem and aims at reducing this type of uncertainty.", "Stirling [27] argues that ambiguity is a type of uncertainty due not to lack of knowledge, but to the existence of “contradictory certainties”.", "He submits that the real challenge at the science-policy interface is the failure to recognise this type of uncertainty and to reduce all scientific advice to risk assessment.", "However, this insight has fallen through and scholars that analyse the science-policy interface still argue for reducing ambiguity.", "We argue that the role of ambiguity in governance is poorly understood and that reducing ambiguity may not always be functional in policymaking.", "This is in contrast with the predominant view that scientists should work towards the clarification of slippery concepts: Wellstead et al. [28], for example, recognise that policy problems and solutions can be framed in different ways, leading to ambiguity, yet argue that scientists should take a normative stand and support a specific framing in order to reduce said ambiguity.", "The aim of this paper is to contribute to a better understanding of ambiguity as distinct from other types of uncertainty, drawing from complexity theory.", "We use energy security as our empirical reference.", "We analyse and compare the multiple representations of energy security in scientific literature and in EU policy documents, and assess the level of uncertainty associated with each representation.", "Even though energy security is recurrently described as an ambiguous concept, to our knowledge no attempts have been made at conceptualising ambiguity in this context, nor to theorise the role of ambiguity in energy governance.", "Because the contribution of this paper is primarily theoretical, we first develop a theoretical background that conceptualises ambiguity as the type of uncertainty created by complexity, and we discuss the implications of ambiguity for the science-policy interface.", "We use text analysis to identify the different types of uncertainty, including ambiguity, mentioned in scientific publications and EU policy documents.", "Section 3 introduces the materials and methods used.", "The results (Section 4) show the multiple scales of analysis used to define energy security and the types of uncertainty associated with it.", "Based on our results, we return to the discussion of how this type of uncertainty does not lead to paralysis in the policy process in the EU context, but rather provides a rationale for materially ineffective policy measures.", "We conclude the paper by arguing that our finding about the (non-)issue of energy security definitions at the science-policy interface has theoretical implications for the role of ambiguity as distinct from other different types of uncertainty in policy processes, which requires a careful consideration of the limits of holistic assessments in guiding policy.", "In general terms, energy security is recurrently linked to risk [26], uncertainty [29] and ambiguity [9,16–19,25,26,29–31].", "Because of the extensive use of these terms, it is important to clarify their meaning.", "In this paper, we draw upon the work of Knight, Stirling and Wynne [32–34] on uncertainty, and of Funtowicz and Ravetz, Zellmer et al., Strand, Kovacic and Giampietro [35–38] on complexity.", "Knight [32] first introduced the distinction between risk and strict uncertainty in economics in his foundational work in the 1920s, defining risk as a situation in which the possible outcomes are known and the probabilities associated with each outcome can be calculated.", "Strict uncertainty is defined as a situation in which the possible outcomes are known but the associated probabilities cannot be calculated.", "We speak of “strict uncertainty” to distinguish the technical definition of uncertainty from the general study of uncertainty, which includes risk, strict uncertainty, ignorance, indeterminacy and ambiguity.", "In the case of energy security, strict uncertainty can be associated with events such as price volatility [26], and disruptive events such as tsunamis [29], which are known to occur, but cannot be precisely predicted.", "The novelty introduced by Knight is that uncertainty is conceptualised as a matter of degrees of (lack of) knowledge1 .", "Building on the idea of degrees of uncertainty, Wynne [34] identifies four types of uncertainty: (i) risk, in which the odds are known, (ii) strict uncertainty, in which “we know what we don’t know”, (iii) ignorance, in which “we don’t know what we don’t know”, and (iv) indeterminacy or systemic uncertainty, in which “causal chains or networks are open”.", "With regard to energy security, ignorance may be associated with geopolitical threats derived from “insecure political and unstable economic environments” [39], as well as with the emergence of new technologies, such as fracking, which introduce unforeseen opportunities and risks.", "Indeterminacy is associated with systemic changes, or what some authors call phase changes.", "“Threats like delivery disruptions or global warming of more than 2 °C can be seen as phase changes, because in addition to having a direct impact on consumers they also change the way in which the system works” [13 p.11].", "In the context of indeterminacy, uncertainty is irreducible, as systemic changes reduce the possibility of knowing.", "Stirling [33] reorganises Wynne’s levels of uncertainty in a 2 × 2 matrix, which ranges from known to unknown outcomes and known and unknown probabilities, and introduces ambiguity as an additional type of uncertainty.", "Once again, risk is defined as known outcomes and known probabilities, strict uncertainty as known outcomes and unknown probabilities, and ignorance as unknown outcomes and unknown probabilities.", "According to Stirling, ambiguity is defined as a situation in which probabilities are known but outcomes are unknown.", "The ambiguity of outcomes is not necessarily due to lack of knowledge, but to the fact that one cannot predict which of the known outcomes will be realised [27], because of divergent and contested perspectives on the justification, severity or wider meanings associated with a perceived threat [40].", "In their historical analysis of the evolution of energy security, Cherp and Jewell [1] discuss how the concept has been used to pursue different outcomes.", "The concept of ambiguity thus suggests that uncertainty may be mobilised to pursue different goals, not because of a lack of information, but because a plurality of representations can be accommodated.", "In the context of uncertainty, the linear model of science speaking truth to power is questioned, not only because science may produce incomplete knowledge, but also because social and political threats indicate that decisions cannot be reduced to rational, utility-maximising, get-the-facts-then-act models [41,42].", "With regard to energy security, distinguishing between different levels of uncertainty is critical for decision-making.", "For example, after the Fukushima accident, experts declared that new designs for nuclear power plants took into account the risk of earthquakes [43].", "Better designs, however, do not solve the uncertainty linked to the unpredictability of earthquakes and tsunamis.", "Safer designs refer to advances in the reduction of operating risk at the level of the power plant.", "Strict uncertainty about earthquake forecasting (higher levels of uncertainty) cannot be factored into new nuclear designs.", "The undistinguished reference to uncertainty with disregard to the type of uncertainty in question may create a false illusion of control, and a diffuse understanding of the role, and limits, of scientific knowledge in decision-making.", "Stirling’s reference to open causality and unpredictability invokes complexity.", "Many authors have studied the implications of complexity for governance and public policy [35,37,44–46].", "In this literature, complexity is often defined in opposition to simplicity [37,45], and as a criticism of the instrumental understanding of the system to be governed through the lenses of reductionism, determinism, predictability and mono-causality.", "Positive definitions of complexity, however, are many and contrasting.", "Strand [37] distinguishes between thin and thick complexity, Geyer [47] speaks of reductionist complexity, soft complexity and complexity thinking as a new epistemology of science, and Salthe [48] provides five definitions of complexity ranging from measurable complexity to ontological complexity.", "Thin and reductionist complexity can be used as a means to recognise “reality” as differentiated and changing.", "A system is defined as complex if the whole presents emerging properties that cannot be inferred by analysing only the components of the system.", "The non-linearity and emerging properties of complex systems challenge predictability.", "Thin complexity departs from the positivist and reductionist use of scientific facts that underpins the linear model of science speaking truth to power.", "In this case, complexity is also used to reject post-modernism [49], and to postulate the existence of a complex “reality” out there, whose complexity is independent of the observer.", "Concepts of thick complexity and complexity thinking, as well as Salthe’s [48] insistence on the role of the observer in all definitions of complexity, create an understanding of complexity as a consequence of analytical choices as much as of what is observed.", "In this view, complexity requires the use of multiple scales of analysis [36,50,51], so that both local rules of interaction between system components and emerging properties of the whole can be observed.", "These multiple observations, however, are not reducible to one another, leading to the existence of non-equivalent descriptive domains [38,52].", "The epistemology of complexity does not refer to knowledge about complex systems, but rather yields a relational understanding of knowledge as the correspondence between what is observed and what is modelled, building on Rosen’s modelling relation [53].", "For this reason, both Salthe [48] and Kovacic and Giampietro [38] refer to Peirce’s semiotics [54], the study of signs as a triadic relation between representation, application and interpretation of experiences.", "That is, knowledge cannot be separated from action.", "As Strand [37] argues “craftsmanship and tacit quality judgments play an integral part in the process of transformation from a scientific finding to a fact.”", "Rather than the study of systems “out there,” complexity is the study of “holons” [55], that is, the relationship between (i) the knowledge generated, (ii) the choice of narratives defining causality between observables [38], and (iii) the choice of a temporal scale of the model, determining how knowledge is updated with experience (as an application of the knowledge itself) [43].", "Similar to uncertainty, complexity poses a challenge to the interface between science and policy.", "Complexity theory is used by many authors to critique the use of reductionist science to guide decision-making [44–46], and is used to encourage more pragmatic, humble, reflexive and adaptive approaches to policy making [37,38,45,56].", "Complexity is also used to describe the policy process as affected by negative and positive feedbacks, attractors and path dependence [44,46].", "In line with our aim of conceptualising ambiguity and its role in governance, we build on the relational understanding of complexity as a means to critically re-think the implications of limited and contradictory knowledge for decision-making.", "Semiotics can be used to argue that science plays a role in the creation of emergent complexity [35].", "For instance, through technology, scientific research is also a form of intervention in the world [37].", "We argue that the contribution of complexity theory consists not so much in the type of science advice that can be derived from the concepts of emergence, non-linearity, open causality, self-organisation, etc.", "Geyer and Cairney [44] correctly remark that these concepts are newish, as the messiness of policy making has been long studied in political science with no need to make reference to complexity.", "Rather, we submit that complexity theory is most usefully deployed in questioning the relationship between science and policy.", "Rather than speaking a more nuanced and complex truth to power, relational complexity makes it possible to analyse the mutual relation between science, scientific truths and power.", "The linear model relies on the rational use of scientific facts produced by supposedly neutral scientists.", "Questioning the linear model requires taking a critical stance also with regard to scientific facts and the role of experts.", "The epistemic pluralism [57] of complexity is an alternative concept to monolithic and unifiable truths of Newtonian science.", "Therefore, the study of ambiguity may lead to insights about the role of experts.", "Building on Stirling’s definition of ambiguity as a type of uncertainty, we conceptualise ambiguity as the uncertainty created by the existence of multiple non-equivalent representations of the same issue.", "Ambiguity is thus not just a matter of different opinions, as may be the case of the rise in the price of oil (perceived as beneficial by oil producers and problematic by oil importers).", "In linking ambiguity to complexity, we argue that this type of uncertainty is caused by the existence of incommensurability in the knowledge base.", "That is, although a lot of information can be produced about energy security, there is no univocal way to combine these representations, as can be seen in the proliferation of energy security indicators [17,25,39] and of academic papers dedicated to the conceptualisation of energy security [26,31].", "The role of ambiguity in policy has been widely analysed in the literature [33,58–61].", "According to Matland [58], ambiguity is necessary to limit conflict (ambiguity of goals), and to define policy when there is uncertainty over the technology needed or over the role that various organisations are to play in the implementation process (ambiguity of means).", "With reference to the EU, Zahariadis [59] explains that ambiguity is an integral part of the policy making process in contexts where there is a plurality of, often contrasting, interests, a multiplicity and high turnover of actors, and highly bureaucratic systems that cause a fragmentation of the policy process.", "Shackley and Wynne [62] suggest that ambiguity may not only be preferred over knowledge in decision-making, but may also provide more robust scientific tools than those generated by a precise knowledge base.", "Understanding the function of ambiguity in the policy process has important consequences for the interface between science and policy.", "Whereas numerous studies argue for more holistic and integrated conceptualisations of energy security [1,25,29], Matland warns against the dysfunctional effects of clarity in policy implementation.", "The literature on energy security is replete with examples of paradoxes and inconsistencies that emerge from the multiple definitions of energy security [1,9,25].", "According to Sovacool and Mukherjee [25], a paradox arises as “energy security is integral to modern society, yet its very ubiquity makes it prone to market failure and under-distribution” [p. 5343].", "That is, complexity is manifested in the multiple, non-equivalent representations, conceptualisations and quantifications of energy security and the resulting heterogeneity of policies.", "Building on our analysis of energy security, we will return to the theoretical discussion of how ambiguity challenges the relationship between science and policy in the discussion section.", "Following our theoretical focus on uncertainty and complexity, we use text analysis to identify how these concepts are articulated with regard to energy security.", "The aim of this analysis is to identify the multiple representations of energy security across scales, and their level of uncertainty.", "Both scientific literature and policy documents are analysed.", "As can be expected, scholarly articles focus predominantly on defining and measuring energy security, and policy documents focus on identifying different policy measures to increase energy security.", "For the literature review, twenty-three papers on energy security were analysed, published between 2002 and 2014.", "The papers are among the most cited on the topic, and provide a comprehensive spectrum of how energy security is assessed in academia.", "They include perspectives from economics, engineering, geopolitics, policy, technology, political science, finance and geology, and were selected using citation indexes from Web of Science and the snowballing technique [63].", "On the policy side, focus is given to the EU, which we chose as case study because (i) energy security has been part of EU policy since the creation of the EU [1,9], (ii) many of the scientific articles analysed aim at informing EU policy [6,26,64–66], and (iii) energy security has been a high priority of the EU policy agenda since the 2008 gas crisis with Russia [1,6,10,14,66].", "Three branches of EU documents are considered: the Energy Security Strategy (2014) [67], the Clean Energy for All Europeans package (2016) [68] and energy directives in force [69–72].", "Additionally, the 2015 State of the Energy Union report [73] is analysed.", "The aim of the policy analysis is to obtain an overview of the role played by energy security in EU policy, of which measures have been proposed as a means to increase security, of which issues are seen as problematic and of how uncertainty is handled.", "Therefore, the policy analysis is not constrained to legal documents, but also includes press releases and fact-sheets [74–77], as they can provide valuable insights into the underlying narratives surrounding energy security in EU policy.", "The texts are coded for definitions of energy security and policy measures, scales and dimensions of analysis, as well as uncertainties, risks and ambiguities.", "The categorisation of energy security into different disciplines, domains, dimensions, pillars and principles is popular in the literature and the terms, often referring to different scales, are sometimes used interchangeably.", "Without delving deep into this categorisation, our interest lies in the different scales and types of uncertainty.", "The term scale is used here to refer to geographical dimensions (e.g. city, province, nation, region, world), temporal dimensions (short-term, long-term), and to hierarchical levels of analysis (energy system, energy sector, renewables, wind power, wind farm).", "There is no 1:1 mapping between geographical dimensions and hierarchical levels of analysis: the energy sector, for example, may refer to a specific country or to the global economy.", "This broad category of scale is used in this paper to capture the multiple levels of granularity used in the literature to describe energy security.", "Having identified scales, definitions or dimensions (for academia) and measures (for policy) linked to energy security, the next section discusses the role that complexity plays in the energy security knowledge base, and the role that the ambiguity arising from complexity plays in policy.", "Results start with the analysis of energy security, as approached in literature.", "Focus is given to the definitions and dimensions used to characterise energy security, as well as the type of uncertainty that is discussed in relation to these dimensions, and to the relevant scales of analysis (Table 1).", "While the first two columns of the table refer to what was found directly in the text of each article, the third column was added in order to discuss the multi-scalar character of the concept of security.", "The distinction between risks, strict uncertainty, indeterminacy and ambiguity follows the levels of uncertainty discussed in the theoretical framework.", "We analyse all types of uncertainty mentioned in the documents in order to show how the general argument for reducing uncertainty is applied indiscriminately to all types of uncertainty, including ambiguity.", "Stirling [27] argues that whereas risk may be usefully described using scientific assessments, strict uncertainty and ambiguity require humility and the recognition of the limits of scientific knowledge.", "Limits are not to be confused with lack of validity, but are a request for more modest claims.", "Responding to our aim of theorising the role of ambiguity in policy processes, we will add to this argument and show that different types of uncertainty have different functions in the policy process.", "Energy security is connected by different authors to a large number of dimensions, from availability and affordability to resilience and technological development.", "These dimensions are linked to different scales, ranging from individual technologies to global economy, climate and reserves.", "The most common type of uncertainty discussed is risk, which is strongly linked to security discourses.", "Ambiguity is also mentioned, mostly as a problem to be solved, and different types of uncertainties are identified by different authors, mostly linked to global resources and supply stability [15,17,64,79] and price volatility [31,79].", "Natural disasters are also mentioned [26,79].", "Following the distinction proposed by Wynne, these events fall under the category of indeterminacy.", "The results of the text analysis of policy documents are shown in Tables 2a and 2b.", "Table 2a refers to direct measures, which are cited in the Energy Security Strategy.", "Table 2b refers to indirect measures present in wider EU energy policy, linked to energy security as one of many aims.", "The uncertainties reported in the tables are not mentioned directly in the policies but were identified based on the type of measure proposed.", "Looking at the characterization of energy security in science and policy, the results of both tables show that: (i) there is interdependence across scales of analysis, (ii) uncertainty is central to energy security and (iii) ambiguity is treated differently in science and policy.", "There is interdependence across scales of analysis in the dimensions of energy security used both in the scientific literature and in policy documents.", "Energy security is framed as the combination of (i) security of supply (availability and reliability), and (ii) security of demand (affordability).", "The need to match supply and demand requires handling the interplay of constraints posed by elements external to socio-economic systems, such as the availability of fossil fuels, solar radiation, water courses, and elements internal to socio-economic systems, such as geopolitical concerns, import dependencies, diversification of suppliers, transportation, infrastructure, technologies, distribution and accessibility issues, and price volatility.", "The external-internal duality permeates energy security definitions.", "Moreover, energy security applies to multiple hierarchical levels of analysis.", "Demand issues refer to the distribution of resources at a global, regional and national scale with regard to energy markets, infrastructure and economic sectors, and at the individual level with regard to consumers.", "As a consequence, the concept of energy security is based on the interdependence between different hierarchical levels of analysis and the external-internal observation duality.", "Uncertainty is central to the concept of energy security.", "Degrees of uncertainty can be seen in the distinction between different threats.", "Threats coming from price volatility and technical problems are defined as risks to the system.", "Reliability of supply and geopolitical concerns may be defined as strict uncertainties (known but not quantifiable).", "Threats such as peak oil, increasing demand and climate change are seen as sources of indeterminacy that may cause changes in the system.", "Uncertainty is seen as a property of the system with regard to the high dependence of non-oil producing countries on foreign oil, the high level of inertia of the energy system, and the ubiquitous use of energy in economic activities.", "Risk, strict uncertainty and indeterminacy have a central role in the definition of energy security.", "Contrary to ambiguity, these types of uncertainty provide a common ground for the definition of energy security for both science and policy.", "In policy documents, the interest lies in the sources of uncertainty.", "Different sources of uncertainty are used as a means to define and classify policy measures.", "Uncertainty of supply is the most recurrent source of uncertainty mentioned.", "It is related to import strategies, to market instability and to technological development.", "Uncertainty of demand is also present and is related to improvements in efficiency, moderating consumption, and reducing carbon emissions.", "Contingency measures reveal an additional source of uncertainty, related to ignorance.", "They include protection and back-up infrastructure, reserves of critical energy sources, and contingency planning.", "Ambiguity plays very different roles in science and policy.", "While ambiguity is mentioned in most of the scientific articles analysed, and is often the central motivation for the work of clarification, conceptualisation and/or classification of energy security, the worry about ambiguity is virtually absent from policy documents.", "Despite the ambiguity of the concept highlighted in the literature, energy security is an important priority in EU policy.", "This finding is consistent with the study of the use of energy security in the UK [87].", "Energy security is mentioned consistently throughout all EU energy policy documents analysed, although less so in the non-legal documents of the Clean Energy for All Europeans package, where, as the name suggests, focus is shifted to consumers.", "Ambiguity is used in EU energy policy in a variety of instances.", "For example, the definition of energy efficiency is linked to a plurality of accounting methods.", "According to the directive, “energy efficiency means the ratio of output of performance, service, goods or energy, to input of energy” [71] [our emphasis].", "The metrics used to measure services (e.g. contribution to GDP) and energy (e.g. megaJoules) are not equivalent to each other and generate ambiguity in energy efficiency indicators.", "Ambiguity is also present in target setting procedures: “Each Member State shall set an indicative national energy efficiency target, based on either primary or final energy consumption, primary or final energy savings, or energy intensity” [71].", "Vagueness can be seen in the renewable energy directive of the EU.", "For instance, the sustainability requirement is defined as: “Biofuel production should be sustainable.", "Biofuels used for compliance with the targets laid down in this Directive, and those that benefit from national support schemes, should therefore be required to fulfil sustainability criteria” [69].", "A distinction should be made between ambiguity and vagueness.", "Ambiguity is the uncertainty that emerges from complexity, while vagueness refers to the lack of clarity or specificity with which a term is used.", "Both may be useful in policy processes, and may help generate consensus, but vagueness is a political decision (e.g. the term energy security is used in relation to geopolitical concerns to avoid explicit mention of specific countries and regions, to which different member states may have different relations), and ambiguity has to do with incommensurability in the knowledge base and the governance of uncertainty.", "Ambiguity makes it possible for a plurality of knowledge claims to be taken into account, extending the political space.", "It should be noted that, although the framing of ambiguity is very different in policy and in science, the uncertainty that arises from the presence of multiple perspectives is seen as problematic in some instances also in the policy realm.", "For example, a high priority for the EU is the coordination of national energy policies, as expressed by the idea of building an ‘Energy Union’ and including energy security in political dialogues (last pillar of Table 2a).", "According to Natorski and Surallés, “attempts to frame energy as a security issue in order to gain support for a Common Energy Policy have been of limited effect, precisely because the security framing contributed to the further legitimisation of EU member states’ reluctance to cede sovereignty in the energy domain” [65].", "This policy measure can be interpreted as the pursuit for the explicit discussion of topics otherwise left vague and ambiguous.", "Following Matland [58], this instance can be interpreted as a case in which ambiguity of goals is accepted (energy security), in order to reduce ambiguity of means (coordination is needed).", "The more one digs into the materiality of energy security, the more trade-offs, bottlenecks and lock-ins emerge.", "With reference to the nuclear power industry in France, Hecht [88] argues that policy effectiveness relies on material effectiveness.", "That is, the ability to deliver on material changes legitimizes policy, and the use of scientific evidence.", "In the case of energy security, this relationship seems to lean not on effectiveness but on uncertainty.", "Material effectiveness is elusive in the context of complexity because of non-linearity and open causality, and policy relies on ambiguity.", "Uncertainty can be explained with reference to the complexity of energy systems.", "Comparing the current state of the EU’s energy system with the measures in EU documents, it becomes clear that (i) some energy security measures play minor roles in the overall energy system, neglecting larger lock-ins, and can be better understood as performing a symbolic role; (ii) measures targeting different components or stages of the energy system may generate important trade-offs and systemic changes.", "For example:", "Technical security: increasing renewables may present challenges for electric grid control due to the higher penetration of variable sources into the electricity system [89];", "Market security: a higher integration of renewables in the electric grid may also lead to increased prices for consumers due to feed-in-tariffs.", "In Germany, for example, an increase in renewable electricity generation led to lower prices for electricity producers and higher prices for consumers [90];", "Nexus security: biofuels may increase energy security but pose threats to food security, as documented extensively in the literature (see, for example, [91]);", "Geopolitical security: decreasing reliance on imports may decrease security threats caused by geopolitical issues, such as the Russian gas halts, but may also reduce diversification of supply routes and sources, making the system more vulnerable in times of unexpected crises.", "This is referred to as import availability [4];", "Environmental security: domestic production of energy carriers may increase local environmental impacts, such as water contamination;", "Technological security: increasing the use of nuclear power may improve the security of electricity supply but pose other concerns, both of plant security and uranium dependence.", "Many of these trade-offs are acknowledged in the policy documents analysed.", "The directives analysed mention grid control problems caused by the increase in renewable energies, the dependence on fossil fuels for transport, and the possible impacts of biofuels on food production [69].", "The recurrent reference to risks, threats and urgency are means through which the lack of effectiveness in energy governance is recast as a security challenge.", "We argue that uncertainty (in the form of risk, strict uncertainty and indeterminacy) is mobilised to frame energy governance in terms of security.", "In this context, the use of a plurality of policy measures, and the inconsistencies or trade-offs that arise from such plurality reinforce the uncertainty element of energy security.", "Ambiguity is thus functional to this mode of governance, and the multiple representations that are produced by the scientific knowledge base reinforce the construction of energy security as a challenge of uncertainty, which requires governing.", "Ambiguity in this context makes it possible to avoid a paralysis in decision-making due to higher level uncertainties, and makes it possible to form coalitions between different actors and discourses.", "By linking all of the above measures to energy security, the complexity of the issue is compressed to allow for decisions to be taken.", "Energy security is almost always bundled up within a mix of justifications which tend to include climate change and economic growth, as can be seen for example in the recast proposal for renewable energy directive of 2016: “Moreover, renewable energy is also emerging as a driver of inclusive economic growth, creating jobs and reinforcing energy security across Europe” [86].", "Here, ambiguity is used both to group measures together (ambiguity/vagueness of means), and to group targets such as security and economic growth (ambiguity/vagueness of goals).", "Recognising the relevance of ambiguity in decision making, the discussion is now directed to how this affects the science-policy interface.", "Clarifying the concept of energy security would mean showing inconsistencies between representations, indicators and associated measures.", "Therefore, reducing ambiguity may make scientific advice less useful to governance.", "This observation runs counter to some of the ethos of science for policy, which is manifested in the goals of increasing clarity [25], providing a holistic view [25,29], putting boundaries on the term [26], distinguishing between different logics of energy security and investigating their political and normative consequence [9].", "A recurrent recommendation is that uncertainty should be analysed and communicated [5,39,78].", "The results presented here, however, suggest that the communication of uncertainty at the science-policy interface needs to take into account what type of uncertainty one is dealing with.", "While risks, indeterminacy and ignorance are flagged as problematic in policy, ambiguity plays a different role.", "Before aiming to reduce ambiguity, it is important to take into account which policy processes may be affected, and even disrupted, by reducing ambiguity.", "“For example, the call for clear, explicit, and consistent goals contradicts much of what is known about how legislation is passed” [52, p.147].", "Matland [58] argues that ambiguity is a means to reduce conflict and hold together coalitions.", "The use of energy security as a recurrent justification for a wide range of measures indicates that the term is used to form coalitions, or mobilise existing ones.", "Meritet [92], for example, highlights how energy security measures and discourses in France are very different to those in other EU countries, given the role played by nuclear power.", "Ambiguity thus helps maintain coherence at the EU level, glossing over national differences.", "The analysis of the role of ambiguity in policy processes shows that ambiguity is not a deficit of knowledge, and that therefore it is not a matter for scientific experts to “solve.”", "These considerations open the debate about the role of science and science advice to policy in the context of complexity and uncertainty.", "Policy recommendations in the energy security literature are varied, ranging from “the institution of a consultative process towards broadly accepted (…) fuel-specific premiums” [31], the provision of a detailed analysis of risk [39], and definitional clarity [19], to the support of “multilateral approaches and concrete cooperation models” [6], “anticipating and assessing the ‘what ifs’” [14], diplomatic and economic dialogues [66].", "While we are sympathetic to these suggestions, we refer to relational complexity to argue that through ambiguity, multiple knowledge claims and multiple sources of expertise are brought to bear on the policy process.", "Policy is informed not only by scientific evidence, but also by political, economic and social considerations.", "Ambiguity makes it possible to maintain a dialogue with a wide range of actors, including but not limited to scientific experts.", "We argue that it is important to take into account who would benefit and who would loose from the clarification of ambiguity, and to assess what is at stake behind different uses of the term.", "In this paper, we refer to energy security as an entry point to theorise the role of ambiguity in governance.", "Energy security is an ambiguous term, with many competing and contradictory definitions.", "We use complexity theory to argue that the multiple definitions refer to non-equivalent representations of energy security, and cannot be reconciled or unified without losing relevant information.", "We conducted a text analysis of academic publications and policy documents to compare which types of uncertainty are mobilised, with a particular focus on ambiguity, defined as the type of uncertainty arising from complexity.", "Many authors have focused on reducing the ambiguity of definitions of energy security and have provided broad definitions that can capture its multiple facets [16,17,26,31].", "On the other hand, the definition of energy security does not necessarily arise as a problem in EU public policy.", "This incongruence between science and policy is not a problem per se.", "Academia has different interests than policy, and what can be interesting from a research perspective (conceptualising an ambiguous term) may not be a priority in policymaking.", "Moreover, the interactions between science and policy are rarely direct (with scientific evidence guiding policymakers), and the effects of conceptualisation literature can be diffuse [9], in helping to advance a field that may eventually have impacts on policies.", "Since we relied only on secondary information through text analysis, the differences in the treatment of ambiguity between science and policy would benefit from further research based on primary data through, for instance, interviews with both scientific experts and policy-makers.", "The results and discussion suggest that it is important to pay attention to what is at stake before clarifying ambiguities at the science-policy interface.", "This critical reflection should by no means be understood as a call for irresponsible politics or post-factual decision-making.", "Rather, it is important to understand the “network of artefacts, knowledges and institutions” [62, p.257] that constitute energy governance before prescribing good practice.", "To this purpose, we rely on a relational understanding of complexity, which relates representation to the institutions and the uses of knowledge.", "Returning to the debate raised by Wellstead et al. [28] about the need to reduce ambiguity, we argue that from a complexity point of view, reducing ambiguity would entail (i) a normative choice of some scientific facts over others, and (ii) a poor understanding of how plural and ambiguous knowledge is used in policy processes, which would widen the gap between science and policy, rather than closing it.", "In contrast, discussing the role of ambiguity in policy can help manage this gap and may foster an environment for better dialogue between science and policy." ]	[ "Energy security is widely used in policy yet it is an ambiguous term.", "The uses of energy security in academia and EU policy are analysed and mapped.", "Ambiguity is conceptualised as the type of uncertainty emerging from complexity.", "The role of ambiguity at the science-policy interface is discussed." ]
Two-dimensional gel electrophoresis data in support of leaf comparative proteomics of two citrus species differing in boron-tolerance	Here, we provide the data from a comparative proteomics approach used to investigate the response of boron (B)-tolerant 'Xuegan' (Citrus sinensis) and B-intolerant 'Sour pummelo' (Citrus grandis) leaves to B-toxicity. Using two-dimensional gel electrophoresis (2-DE) technique, we identified 50 and 45 protein species with a fold change of more than 1.5 and a P-value of less than 0.05 from B-toxic C. sinensis and C. grandis leaves. These B-toxicity-responsive protein species were mainly involved in carbohydrate and energy metabolism, antioxidation and detoxification, stress responses, coenzyme biosynthesis, protein and amino acid metabolism, signal transduction, cell transport, cytoskeleton, nucleotide metabolism, and cell cycle and DNA processing. A detailed analysis of this data may be obtained from Sang et al. (J. Proteomics 114 (2015)). [1].	[ "MULT" ]	[ "Boron-toxicity", "Citrus", "Proteomics", "Two-dimensional gel electrophoresis (2-DE)" ]	[ "1000" ]	[ "Boron (B)-tolerant ‘Xuegan’ (Citrus sinensis) and B-intolerant ‘Sour pummelo’ (Citrus grandis) seedlings were used to investigate the effects of B-toxicity on leaf proteomics.", "Plant culture and B treatments were performed as described previously [1,2].", "Briefly, 13 week-old seedlings of C. sinensis and C. grandis grown in 6 L pots (two seedlings per pot) containing river sand in a greenhouse under natural photoperiod were irrigated every other day until dripping with nutrient solution containing 10 μM (control) or 400 μM (B-toxic) H3BO3 for 15 weeks.", "There were 20 pots per treatment in a completely randomized design.", "At the end of the experiment, fully expanded leaves collected from B-toxic and control plants at noon under full sun were immediately frozen in liquid N2.", "Frozen samples were stored at −80 °C until extraction.", "About 1 g leaves collected equally from five different replicates (one plant per replicate, one leaf per plant) were mixed as a biological replicate.", "There were three biological replicates for each treatment.", "Proteins from control and B-toxic frozen leaves were extracted according to Yang et al. [3] using a phenol extraction procedure.", "2-DE was performed according to You et al. [4].", "Gel image analysis was performed using PDQuest software (version 8.0.1, Bio-Rad, Hercules, CA, USA).", "Spot intensities were subjected to statistical analysis to obtain the differentially abundant protein spots.", "A protein spot was considered differentially abundant between samples when it had both a P-value of less than 0.05 and a fold change of more than 1.5.", "Based on the two criteria, we obtained 32 up-regulated and 28 down-regulated protein spots from B-toxic C. sinensis leaves, and 31 upregulated and 31 down-regulated protein spots from B-toxic C. grandis leaves.", "All these differentially abundant protein spots in B-toxic C. sinensis and C. grandis leaves are presented in Supplementary Tables 1 and 2.", "All theses differentially abundant protein spots were excised from the colloidal Coomassie Brilliant Blue stained gels and subjected to MS analysis, which was performed on an AB SCIEX 5800 TOF/TOF.", "All acquired spectra of samples were processed using TOF/TOF ExplorerTM Software (AB SCIEX) in a default mode.", "The data were searched by GPS Explorer (Version 3.6) with the search engine MASCOT (Version 2.3, Matrix Science Inc, Boston, MA).", "Protein spots with scores greater than 75 were considered statistically significant (P<0.05) and accepted.", "Bioinformatics analysis of proteins was performed according to Yang et al. [3].", "Here, we identified 50 and 45 differentially abundant protein species from B-toxic C. sinensis and C. grandis leaves.", "A detailed data could be obtained from Supplementary Tables 3 and 4.", "To understand the correlation between gene and protein expression levels, we used qRT-PCR to assay the transcript levels of genes encoding 14 differentially expressed protein species in B-toxic C. sinensis and C. grandis leaves (Supplementary Table 5).", "The transcript levels of all these genes were in agreement with our 2-DE data except for genes encoding glutathione S-transferase (GST) GST1 (S17), ribulose bisphosphate carboxylase activase (S39) and ribosomal protein L12 (G50), demonstrating that most of these differentially abundant protein species were regulated in the transcriptional level." ]	[]
Calibration procedure for solar cells exhibiting slow response and application to a dye-sensitized photovoltaic device	Dye-sensitized Solar Cells are generally known to possess a slow response to changes in photocurrent generation by incident light. Electrical power measurements of photovoltaic (PV) devices can only be accurately made if sufficient time is allowed for complete photocurrent generation. The generation time increases from typically a few milliseconds in the case of c-Si devices, over tens to hundreds of milliseconds for technologies like back-contact and hetero-junction solar cells, to even longer times for dye-sensitized and some types of perovskite solar cells. In this work we propose a procedure for calibration of slow responding PV devices based on an accurate evaluation of their response time. Starting from a quantitative analysis of the photocurrent signal versus chopping frequency on the spectral responsivity set-up, the measurement of a dye-sensitized solar cell was performed at 1 Hz chopping frequency. Then current-voltage (I-V) measurements were performed at different sweep-times and directions, in order to determine the correct parameters for I-V characterization. Combining appropriate spectral responsivity (for determination and correction of spectral mismatch) and I-V measurements yielded a reliable calibration of the device including measurement uncertainty estimation. Based on this work criteria for a reliable calibration of slow responding PV devices are formulated, fulfilling all requirements specified in the standards (IEC 60904 series and IEC 60891).	[ "ENER", "MATE" ]	[ "Calibration", "DSSC", "Dye-sensitized solar cells", "I-V measurements", "Photovoltaics", "Spectral responsivity" ]	[ "2105", "2504", "2508" ]	[ "The rapid improvement of dye-sensitized (DSSC) and perovskite (PSC) solar cell performances and the related growing interest for these emerging photovoltaic (PV) technologies [1,2,3] clearly indicate the need for methods and procedures to reliably measure and characterize PV devices known to possess a slow response for photocurrent generation by incident light.", "Electrical parameters measurements of these devices under standard test conditions (STC), as defined in the IEC 60904 series of standards, can be accurately made only if sufficient time is allowed to complete the photocurrent generation.", "This time is usually much longer for DSSC's and PSC's than for typical crystalline silicon devices [4,5].", "Their long response time to the incident light affects both spectral responsivity (SR) and power measurements [6,7,8] resulting in unreliable results, if the measurement time does not take the slow response of the device into account.", "Moreover, different devices can show different response times, i.e. a procedure valid for standard dye-sensitized devices with a liquid electrolyte may be inappropriate for dye-sensitized devices with a solid holes transport material or some perovskite devices.", "IEC 60904-1 [9] contains a generic note warning that I-V measurements may be influenced by the voltage sweep rate and sweep direction and requests an analysis without giving much detail apart from the requirement for optimal overlap of current-voltage sweeps in opposite directions.", "Performance characterization of DSSC's was described previously [10] focusing on the sweep speed and direction and presenting complete SR measurements at different chopping frequencies.", "However, no clear criterion was formulated how to determine the appropriate chopping frequency.", "In a review [6] the shape and distortion of the signal under monochromatic chopped light was presented, but again without a clear quantitative criterion on selection of appropriate chopping frequency apart from simple visual inspection.", "As in [10] full SRs measured at different chopping frequencies were presented as well as IV curves in opposite sweep directions.", "More recently [11] electrical characterization of PSC's was discussed.", "The main feature of these devices was found to be their inherent instability, so that the measurement depended significantly on stabilization and degradation, both depended on the illumination of the devices prior to and during measurement.", "In this work we propose a procedure for precise calibration of these devices: it consists of firstly an evaluation of the response time of the device under test (DUT) and, consequently, a reliable calculation of the parameters to be used when characterizing them.", "The determination of the chopping frequency to be used in SR measurements is the first step of the procedure and is described in the time response analysis (Section 3.3).", "As opposed to previous works [6,10] we analyze the signals of the chopped light quantitatively to give a more objective criterion for the determination of the chopping frequency.", "The quantitative analysis is performed under chopped broadband light, providing a stronger output signal from the device under test.", "This condition allowed to freely and continuously vary the chopping frequency and to determine the device response in short time (5 min).", "Once determined, the SR measurements were performed with the apparatus available at the European Solar Test Installation (ESTI) after necessary modifications and validation of the setup at the low chopping frequency.", "Prior to electrical characterization, I-V measurements were performed at different sweep times and sweep directions to ensure that the device was measured at equilibrium conditions.", "The combination of SR measurements with I-V characterization yielded a reliable calibration and associated uncertainties of the device at STC following IEC 60904 series and IEC 60891.", "The proposed procedure was applied, as a test, to the calibration of a dye-sensitized solar cell with a liquid electrolyte.", "This was one example; however, the procedure presented here and the criteria formulated are generally valid and can be widely applied when the response time of the DUT is unknown.", "Based on this work criteria for a generalized procedure for the calibration of PV devices with slow response are proposed, which may be included in future standards.", "In this work we will focus on two standards: the IEC 60904-1 [9] (measurements of the performance of PV devices), and the IEC 60904-8 [12] (measurements of PV devices spectral responsivity).", "The latter allows for the calculation of the spectral mismatch factor (MMF) as described in IEC 60904-7 [13].", "Once this factor is known, it can be used to correct the electrical performance measured under a specific light source (whose spectrum is measured) to the reference solar spectrum (IEC 60904-3 [14]), taken as universal reference when characterizing terrestrial PV devices.", "The MMF is one of the required corrections to complete the calibration procedure applicable to every PV device.", "For standard c-Si PV devices the procedures to perform reliable measurements are well established among reference laboratories like ESTI [15].", "However, in case of emerging PV technologies like DSSC and PSC, new procedures need to be specifically developed taking into account the device characteristics, e.g. the slow time response.", "In this work the procedure developed at ESTI for the calibration of emerging PV technologies having slow response is presented.", "An example of its application to the particular case of a single DSSC involving a liquid electrolyte is presented in Section 3.", "It consists of an initial quantitative time response analysis needed to determine the chopping frequency suitable for SR measurements and consequently of a set of multiple I-V measurements performed at increasing sweep times and changing of the sweep directions.", "In the time response analysis we propose to illuminate the DUT with broadband light chopped at a specific frequency and measure the generated photocurrent.", "Varying the chopping frequency from low to higher values a threshold point can be found after which the peak-to-peak amplitude variation of the current signal decreases.", "This is the maximum chopping frequency that can be used when measuring the SR to ensure that the DUT is measured under suitable conditions.", "Standard chopping frequency values for DSSC devices are, for instance, around 1 Hz.", "Additionally, SR measurements were performed at three chopping frequencies around the threshold frequency point with different wavelengths of monochromatic lights.", "This was done to check possible differences in DUT response depending on whether the chopped light is broadband or monochromatic.", "If the relative signals are independent of wavelengths, such an effect can be excluded.", "To ensure reliable I-V curves, not influenced by the sweep time, we propose to perform multiple I-V measurements at increasing sweep times and changing the sweep directions.", "Reproducible I-V curves are required in this case and quantitative agreement between measurements performed in opposite sweep directions must be found.", "Three devices were involved in the measurements presented.", "Two reference devices used to measure incident irradiance both in SR and I-V measurements (REF) and one DUT.", "The reference devices consisted of two calibrated c-Si cells with an active area of 400 mm2 and with an internal temperature sensor (PX302C for SR measurements and PX305C for I-V measurements).", "The DUT was a dye-sensitized single cell in a glass-glass construction having an active area of 77.4 mm2 (total device area 550 mm2) (ESTI code PL84).", "The latter was provided by an external laboratory under a sample transfer agreement.", "Electrical wires were glued with silver paste to the electrodes.", "A sample holder was built to attach a PT100 temperature sensor to the rear of the DUT.", "A picture of one REF and DUT taken during SR measurements is shown in Fig. 1.", "The SR of a PV device is one of the key characteristics and represents the device response (in term of generated photocurrent) to quasi-monochromatic light signals at various wavelengths.", "Practically, in the calibration of a PV device the SR is one of the inputs used to calculate the spectral mismatch factor and to correct the measured I-V curves to STC accommodating for any spectral mismatch between the reference spectrum and the spectrum of the light source used for measurement.", "Techniques for SR determination are well known [16,17] and the procedure to perform measurements on a PV device is described in the IEC 60904-8 [12].", "At ESTI the AC method is implemented and a dedicated setup is available for SR measurements of PV devices (cells and mini-modules).", "The schematic of the setup has been described in more details [16,18] and is shown here in Fig. 2.", "The setup consists of a continuous monochromatic illumination generated by a Xenon light source coupled with band-pass filters (mounted on five filter wheels with 16 filters each), covering the wavelength range from 300 to 1750 nm with a band-width from 8 to 20 nm.", "The monochromatic light is chopped and additional continuous broadband bias irradiation is normally used.", "The DUT and REF are placed next to each other (Fig. 1), illuminated with the same light (bias and monochromatic) and measured simultaneously.", "Two lock-in amplifiers are used to distinguish current response due to the chopped monochromatic illumination from the total output current.", "The voltages of DUT and REF, generated by the photocurrent across two calibrated precision shunt resistors (5 Ω for DUT and 1 Ω for REF), are acquired after the stabilization of the signals is reached.", "An in-house LabVIEW software automatically controls the lock-in amplifiers parameters to acquire the signals and to automatically move the filter wheel once the signals are acquired.", "The temperature of the devices is controlled to be 25 °C±2 °C by a water cooled sample stage and is monitored by a PT100 placed behind the DUT and by the internal temperature sensor of the REF.", "The existing SR apparatus runs typically at a chopping frequency around 70 Hz.", "This chopping frequency has been found to be optimal for standard c-Si PV devices, while it is certainly too high for slow responding PV devices.", "In order to investigate and find the optimal chopping frequency for these devices, we propose the time response analysis presented in the next section.", "The time response analysis presented here allowed us to determine the maximum suitable chopping frequency for the DUT.", "The setup implemented at ESTI for these measurements is part of the SR measurements setup described before.", "The filter wheel was removed in this case and REF and DUT were illuminated with broadband chopped light, giving significantly higher signals than with monochromatic light.", "The alternating voltage signals generated by REF and DUT across the shunt resistors were acquired simultaneously with a Yokogawa storage oscilloscope, without the use of lock-in amplifiers.", "No bias light was applied in this case.", "The chopping frequency was varied from 0.5 to 80 Hz in this study (in about 5 min).", "The signals were acquired for at least three periods at each frequency and the peak-to-peak amplitude variation was calculated afterwards over the whole measured frequency range.", "In Fig. 3 the calculated values for REF and DUT normalized to the lowest frequency value for comparison are shown.", "The values of DUT were also divided point-by-point by the REF values (black squares) to compensate for any lamp instability.", "As shown in the figure the c-Si device (REF) reported a constant response in the measured frequency range, independently from the chopping frequency (standard deviation of the values ±1.5% due to lamp instability), while the DSSC device (DUT) showed a strong distortion of the signals above 1 Hz with corresponding reduction of peak-to-peak values due to its slow photogeneration characteristic.", "If we consider acceptable a standard deviation within ±1.5% also for DUT values, we can determine the suitable chopping frequency range for SR measurements below and up to 1 Hz where REF and DUT are both measured at equilibrium (highlighted in the graph with a green pattern).", "To confirm this issue the two signals from REF and DUT were synchronized at low chopping frequency (below 1 Hz) and reported an increasing phase shift at higher chopping frequencies as shown previously [8].", "The time response analysis presented (Fig. 3) suggests that a chopping frequency of 1 Hz is sufficiently low to obtain the full signal amplitude.", "Below this frequency the amplitude reached almost the maximum value and can be considered constant for our purposes.", "Above 1 Hz the DUT is not reacting fast enough to the chopped illumination and will exhibit under-estimated SR values if measured.", "The measurements above were performed with broadband chopped light, due to its higher signal.", "To exclude a possible wavelength dependency of the time response, measurements with chopped monochromatic light were made.", "For this, a series of complete SR measurements were performed at 1.5 Hz, 2 Hz and 10 Hz.", "The ratio of SRs measured at 10 Hz and 1.5 Hz showed a slight wavelength dependency.", "The same ratio of SRs measured at 2 Hz and 1.5 Hz did not show any more this wavelength dependency.", "This confirms that the chopping frequency determined with broadband chopped light by the time response analysis above, is adequate for the measurement of the complete SR.", "Considering the results coming from the time response analysis, the system for SR measurements described in Section 3.2 was modified to work at low chopping frequency (≤2 Hz) by replacing the DC motor, which was limited to frequencies above 2 Hz, with a new stepper motor.", "The parameters of the lock-in amplifiers (time constant and waiting time) controlled by the LabVIEW software were chosen after observing carefully the signals from the lock-in amplifiers during their stabilization.", "The system was validated after modification using a chopping frequency of 1 Hz and comparing two SR measurements of a calibrated c-Si device performed at 78 Hz and 1 Hz [8].", "After validation, the system described here was applied to measure the SR of the DSSC DUT.", "Measured data and interpolated fitting curve are shown in Fig. 4.", "Piecewise Hermite polynomials were used to fit the data and interpolate between the measured points in the wavelength range from 300 to 1200 nm with a step of 1 nm.", "The spectral mismatch factor (MMF) value was calculated following the procedure in IEC 60904-7 [13] as 1.0191 for DUT.", "Taking into account this value, the measured I-V curves of DUT were corrected to STC considering this spectral mismatch between the spectrum of the solar simulator, the reference spectrum, the SR of REF and the SR of DUT [14].", "A WACOM dual lamp steady-state solar simulator WXS 140 Super with an illuminated area of 14 cm by 14 cm and of class AAA (IEC 60904-9 [19]) was used for measuring the I-V characteristics.", "The procedure for measuring current-voltage characteristics of PV devices is described in IEC 60904-1 [9].", "In the standard it is mentioned that depending on the technology, I-V measurements may be influenced by the voltage sweep rate and sweep direction.", "In these cases the effect should be carefully analyzed and problems can be excluded when measurements performed in opposite sweep directions (voltage ramp from short circuit conditions to open circuit and from open circuit to short circuit conditions) overlap optimally.", "Sweep rate dependency happens, typically, with highly capacitive devices like back-contact and hetero-junction c-Si solar cells and with DSSC's [6].", "Following the note included in the standard, the effect of the sweep time on the power measurements of the DUT has been studied here.", "The sweep time was calculated as the total time of the I-V sweep between the crossing-points of the current-axis (short circuit condition: V =0) and the voltage-axis (open circuit condition: I =0).", "The I-V measurements were performed supplying a linear voltage ramp going from voltage values slightly below zero to values slightly above Voc (forward sweep) to ensure the crossing of both axes.", "All the measurements here reported were performed at an irradiance of about 990 W/m2 and corrected to 1000 W/m2.", "In Fig. 5 the electrical parameters extracted from the measured I-V curves of DUT at varying sweep time ranging from 70 ms to 10500 ms are shown.", "At each sweep time multiple measurements were made and their mean (as dots) and their standard deviation (as error bars) are represented in Fig. 5.", "All parameters (Isc, Voc, Pmax and FF) increased up to 1000 ms reaching a plateau for longer sweep times.", "Interestingly the standard deviation of Isc and FF values when measuring I-V curves too fast was significant (about 1% for Isc and 3% for FF below 300 ms).", "At higher sweep time (>300 ms) the repeatability of the measurements increased as expected.", "In some cases at long sweep times the standard deviation bars are not visible in the graph because they are smaller than the dimension of the point.", "The unusual high standard deviation measured on Voc values (about 0.4%) at 10500 ms of sweep time could be linked to temperature effects.", "In such long measurements the device was heated up by the light during the sweep and consequently the Voc varied.", "The set of measurements reported suggested that a sweep time between 1 s and 10 s should be suitable for performing reliable power measurements of the DUT under investigation.", "In order to fulfill the requirements of the IEC 60904-1 [9] a good overlap between measurements performed in opposite sweep directions must be verified.", "I-V measurements performed in opposite sweep directions using three different sweep times (432 ms, 1460 ms, 5760 ms) are presented in Fig. 6.", "Here the forward sweep is defined as the sweep from short circuit Isc to open circuit Voc and the reverse sweep the one scanned in the opposite direction from Voc to Isc.", "The temperature was monitored and controlled to be around 25 °C±0.3 °C during the sweep.", "No correction for temperature, nor for MMF was applied.", "The hysteresis observable at the two lower sweep times disappeared in the third case when the sweep time was above 5 s. Quantitatively the difference in Pmax between measurements performed in opposite sweep directions with the same sweep time decreased from 8.7%, at a sweep time of 432 ms, to 0.5%, at a sweep time of 5760 ms. The electrical parameters extracted from the measured I-V curves shown in Fig. 6 are presented in Table 1.", "The relative difference of the values corresponding to I-V curves measured in opposite sweep directions was calculated for comparison and is reported in Table 1.", "It is noticeable that at short sweep time (432 ms) there is a significant difference between the parameters extracted from I-V curves scanned forward and reverse.", "As the sweep time increases both I-V curves (scanned forward and reverse) converge to an intermediate I-V curve.", "The I-V curves measured in forward direction at any sweep time is closer to the intermediate I-V curve at long sweep times than the respective curve measured in reverse (Fig. 6 and Table 1).", "Nevertheless the difference between forward I-V curve at fast sweep speeds and the one at slow sweep speed suggested that long sweep times were necessary for this type of device.", "A sweep time of about 5 s appeared to be sufficiently long to guarantee reliable I-V measurements.", "Finally three I-V measurements of the DUT performed near STC conditions using a forward sweep time of 5760 ms were selected for the calibration of the device.", "In Fig. 7 the I-V characteristics are plotted (solid lines) together with the power at each point (dashed lines).", "The electrical parameters (Isc, Voc, Imp, Vmp, Pmax and FF) calculated from the I-V curves shown in Fig. 7 are reported in Table 2, as average values and their standard deviations due to repeatability.", "The standard deviations are well below the overall measurement uncertainties of the ESTI apparatus.", "This is expected as the small standard deviations are due to the good repeatability of the measurements as they have small random variations, whereas the overall uncertainty of the measurements is dominated by systematic contributions [20].", "In order to complete the calibration of the DUT and obtain final electrical values corrected to STC, MMF correction was applied.", "The MMF value as calculated in Section 3.3 for the DUT was 1.0191 and the corrected parameters are reported in Table 3 together with their uncertainties.", "The calculated combined expanded uncertainties (k=2) of the ESTI apparatus come from the analysis presented by Müllejans [20].", "The UCs for a c-Si device of the same dimensions would be even smaller.", "But in the case of a DSSC device the following additional UC contributions were considered: difference between actual device junction temperature and temperature sensor (about 1 °C), larger uncertainty of the MMF due to significant difference between the technology and, consequently, the SR's of DUT and REF and the hysteresis effect.", "In this work a general procedure for accurate calibration of slow responding PV devices is described and discussed.", "It allows for a precise evaluation of their response time and, consequently, for a reliable calibration fulfilling all requirements specified in the standards (IEC 60904 series).", "The procedure consists of an initial quantitative time response analysis needed to determine the chopping frequency suitable for SR measurements and then performing this measurement.", "Then a set of multiple I-V measurements performed at increasing sweep times and changing of the sweep direction.", "The procedure has been applied as an example to a single dye-sensitized solar cell incorporating a liquid electrolyte and a demonstration of its practical applicability is given.", "However, the method is not limited to this type of device and can be generally applied, in particular to PV devices which are known to have a slow response or whose response is not known.", "For the time response analysis a set-up needs to be able to operate at sufficiently low frequencies (here below 1 Hz), which in our case required a modification of the hardware of the SR set-up normally operated above 50 Hz.", "The measurement of the response to varying chopping frequencies is rather fast as the signal is high due to the broadband chopped light.", "Therefore the chopping frequency can be continuously varied over a wide range of values and data acquired by a storage oscilloscope.", "The analysis here was done manually, but could easily be performed by a dedicated software so that the result is available immediately.", "Therefore, our analysis is improved from previous work [6,10,11], as it uses quantitative analysis of the signal and is much faster than previous methods which compared essentially complete SR measurements at various chopping frequencies.", "Furthermore, for I-V measurements a light source which provides stable irradiance for sufficiently long times is required.", "For the sweep times required here (5 s) this is only provided by a steady-state solar simulators, as pulsed systems are limited to tens of milliseconds (and in exceptional cases may reach about 1 s).", "For high capacitive c-Si devices attempts have been made to compensate the short pulse duration by acquiring I-V curves in smaller parts by multiple light pulses.", "While this has been shown to work satisfactorily in some cases, it is highly unlikely that such an approach will be successful for DSSC's and some types of perovskite devices, because of two aspects: one is the sufficiently slow sweep time (which can be successfully simulated by multiple flashes), but the second is the DUT stabilization under illumination, which cannot be simulated by multiple flashes.", "Therefore, a steady-state solar simulator is required, which also has an illuminated area larger than the dimensions of the PV device.", "For PV modules there are relatively few of such systems available.", "The use of steady-state simulators poses a challenge in controlling the device temperature during the measurements, as the continuous illumination with the irradiance required by STC inevitably heats up the device.", "While the device can be shielded from the light between measurements, the relatively long measurement times will likely result in device heating.", "This in turn will alter the measurement results, as PV device parameters exhibit temperature coefficients typically of a few per mille per Kelvin.", "Therefore temperature control is mandatory.", "Here this was achieved by an actively cooled sample stage with a thermal mass much larger than that of the DUT, acting as a heat sink.", "For larger DUTs, and in particular modules, this challenge will be larger.", "At the present time it is not clear how to adequately deal with it.", "Based on the work and results presented, we propose the following criteria for determining suitable measurement conditions for slow responding PV devices:", "perform time response analysis (see Section 3.3) using broadband chopped light over a wide frequency range.", "Select a chopping frequency such that the peak-to-peak signal is within 3% of the equilibrium.", "measure SR at least at three representative wavelengths (near the maximum of the SR, at a wavelength around the lower boundary plus 50 nm and at a wavelength around the higher boundary minus 50 nm) selecting the chopping frequency determined in a) and its half.", "Check that the ratio of the SR measured at the two chopping frequencies is constant for the three selected wavelengths; if not choose lower chopping frequency until condition met.", "perform complete SR measurement at the chopping frequency determined in b).", "acquire I-V curves in forward and reverse sweep directions at increasing sweep times.", "For each sweep time analyze the difference of the three main I-V curve parameters (Isc, Voc and Pmax) extracted from I-V curves measured in forward and reverse sweep directions and select the sweep time with the following criteria: ΔPmax <0.5%, ΔIsc and ΔVoc <0.1%", "measure multiple I-V curves at sweep time determined under d) (minimum 3) and check for repeatability with the following criteria: Isc and Voc better than 0.1% and Pmax better than 0.5%.", "Only when all criteria are fulfilled and the respective measurements have been made, a valid calibration of the PV device is achieved.", "In this work appropriate chopping frequency for SR measurement and appropriate sweep-times for I-V curves acquisition were determined and then the respective measurements made.", "Together with the correction for spectral mismatch this constitutes a complete PV device calibration at STC.", "We propose to apply this procedure, specifically developed for devices which have long time response (like back-contact and hetero-junction solar cells, DSSC's and some types of PSC's), also for the calibration of devices with unknown time response.", "If the criteria outlined above are fulfilled, a reliable calibration of the PV device is achieved.", "There will be cases where this approach is not applicable.", "For example in the case of some types of PSC's where the situation might be even more complex [11], requiring additional steps as the devices are inherently unstable and require light soaking which at the same time might already contribute to device degradation.", "Moreover the response in PSC's is highly dependent on the type of perovskite material and also on the device configuration [21]: problems can occur due to the ionic-electronic character of the halide perovskite.", "In these cases different approaches have been tested and proposed in the literature: the simplest is to hold the device at the voltage at Pmax (Vmp) and wait for stabilization [11].", "However, this voltage is not known a priori and therefore the result might deviate significantly from the real maximum power point.", "Maximum power point tracking as proposed by Zimmermann et al. [21] has in our opinion the disadvantage that the algorithm might lead to oscillation, because the device is inherently unstable.", "We suggest to apply in these cases the method proposed by Christians et al. [22], which consists in the measurement of steady-state photocurrent at several voltages around the presumed Vmp and then check the self-consistency with the I-V curves measured at various scan rates in forward and reverse directions and the steady-state Isc and Voc values determined beforehand.", "We propose to do this first starting from voltages below Vmp to above and then to repeat in the opposite direction.", "The two Pmax values obtained in this way should be consistent within 0.5%.", "Following the steps described here will ensure a reliable electrical calibration of slow responding PV devices.", "The criteria proposed should be considered for inclusion of a respective future standard for measurement of such devices." ]	[ "A procedure for accurate calibration of slow responding PV devices is proposed.", "The method relies on an initial quantitative time response analysis.", "A demonstration of its practical applicability is given with calibration of a DSSC.", "Final electrical parameters and corresponding uncertainties are shown." ]
Changes in brain Glx in depressed bipolar patients treated with lamotrigine: A proton MRS study	Background: Lamotrigine is a useful treatment in bipolar depression but requires several weeks of dose titration before its clinical effects can be assessed. Animal experimental studies suggest that lamotrigine lowers glutamate release. The aim of the current study was to assess the effect of lamotrigine on brain glutamate in depressed bipolar patients and to determine whether baseline glutamate could be used to predict clinical response. Methods: We studied 21 bipolar patients who received lamotrigine treatment for a current episode of depression. Before starting lamotrigine and after 10–12 weeks treatment, patients underwent proton magnetic resonance spectroscopy (MRS) scanning at 3 Tesla where levels of glutamate (measured as Glx) were determined in anterior cingulate cortex (ACC). Results: Overall, lamotrigine treatment had no significant effect on Glx levels in ACC. However, in patients who responded clinically to lamotrigine treatment Glx concentrations were significantly increased. Baseline levels of Glx did not predict response to lamotrigine. Limitations: The main limitation of the study was the modest sample size. Most patients were medicated which may have modified the effect of lamotrigine on glutamate activity. MRS at 3T cannot give a reliable estimate of glutamate separate from its main metabolite, glutamine, and thus changes in Glx may not give a precise estimate of effects of lamotrigine on glutamate itself. Conclusion: Lamotrigine does not appear to have a direct effect on glutamate levels in ACC in bipolar patients. However, therapeutic improvement during lamotrigine was associated with increased Glx, suggesting that alterations in glutamatergic activity might be related to recovery from bipolar depression.	[ "MEDI", "PSYC" ]	[ "Bipolar depression", "Bipolar disorder", "Glutamate", "Glx", "Lamotrigine", "MRS" ]	[ "2738", "3203" ]	[ "Recurrent depression is the one of the main burdens experienced by patients with bipolar disorder.", "The treatment of bipolar depression is often difficult, with conventional antidepressants generally not recommended, being relatively ineffective and also carrying the risk of mood destabilisation (Bowden and Singh, 2016).", "The anticonvulsant drug, lamotrigine, has evidence of efficacy in both acute bipolar depression and in the longer-term prevention of depression in bipolar disorder (Solmi et al., 2016).", "However, as with other pharmacological treatments in bipolar disorder, lamotrigine is effective in only a proportion of patients.", "Moreover, the risk of Stevens-Johnson syndrome associated with lamotrigine treatment mandates a particularly slow initiation of dosing (Wang et al., 2015).", "Hence assessment of clinical response may take many weeks.", "There would therefore be significant utility in the identification of a biomarker that could predict therapeutic response to lamotrigine treatment in bipolar patients.", "Development of such a biomarker could be based on knowledge of the mode of action of lamotrigine to alleviate depression in bipolar illness.", "Lamotrigine's pharmacological actions are complex and effects on ion channels and various neurotransmitters have been reported.", "However, an action of potential relevance in bipolar depression is reduction in glutamate release probably secondary to inhibition of voltage-sensitive sodium channels leading to stabilization of presynaptic neuronal membranes (Ketter et al., 2003).", "Glutamate is the main excitatory neurotransmitter in the brain, whose removal from the synapse depends on the integrity of glial cells.", "A number of lines of evidence have implicated glutamate in the pathophysiology of mood disorders.", "For example, in addition to lamotrigine, a number of drugs acting on glutamate mechanisms have antidepressant effects, most notably the NMDA receptor antagonist, ketamine.", "Currently employed mood stabilising agents such as lithium and valproate modify glutamate release and glutamate receptor function.", "Further, post-mortem studies of mood disorders patients have reported loss of glial cells together with changes in expression of glutamate receptors (see Sanacora et al., 2008; Yuksel and Ongur, 2010).", "Glutamate levels in the human brain can be measured non-invasively by proton magnetic resonance spectroscopy (MRS) (Yuksel and Ongur, 2010).", "While the findings are somewhat inconsistent a meta-analysis suggested that glutamate concentrations are increased in frontal brain regions, in bipolar illness (Gigante et al., 2012).", "In this study we therefore predicted that lamotrigine treatment would lower MRS glutamate levels in bipolar patients.", "We also predicted that higher baseline levels of glutamate would be associated with a better clinical outcome of lamotrigine treatment because high baseline glutamate might identify a specific subgroup of patients who would be helped by the glutamate-lowering effect of lamotrigine.", "Participants with bipolar disorder were patients of the Oxford Health NHS Foundation Trust, identified by their treating clinicians as being depressed and for whom treatment with lamotrigine was clinically indicated.", "Patients about to start lamotrigine were approached by a member of the research team and asked if they would give consent to an MRS examination before and during the course of lamotrigine treatment.", "All participants gave full, written informed consent to the study which was approved by the Hampshire A Research Ethics Committee.", "Twenty-nine patients with bipolar disorder according to DSM-IV were recruited into the study, 16 females (mean age 33 years, range 19–56) and 13 males (mean age 36 years, range 22–58).", "Fifteen patients had the diagnosis of bipolar type 1, and fourteen patients bipolar type 2.", "All patients also met criteria for Major Depressive Episode.", "Patients were started on lamotrigine as a part of their clinical regime.", "The design involved two visits: before lamotrigine treatment started and after 10–12 weeks, when clinical response was assessed.", "An MRS scan was performed at each visit.", "The lamotrigine was prescribed by a clinician responsible for management of the patient according to clinical guidelines and the patient's response to treatment.", "Of the 24 patients who were scanned at baseline, in nine lamotrigine was their only drug therapy; in the other fifteen patients lamotrigine was added to other drug treatments, including citalopram, sertraline, fluoxetine, duloxetine, venlafaxine, mirtazapine, aripiprazole, quetiapine, lithium, and sodium valproate.", "This drug treatment continued unchanged for the duration of the study.", "Clinical response to lamotrigine treatment was assessed by the Hamilton Rating Scale for Depression (HAM-D).", "HAM-D ratings were taken at baseline and at the second scan.", "Response to treatment was defined as a 50% decrease in HAMD score from baseline.", "Baseline MRS data were available for twenty-four patients with response status known for twenty-two patients and MRS datasets for both the baseline and follow-up scan were available for twenty-one patients.", "Five patients were unable to complete the baseline MRS protocol due to high levels of anxiety in the scanner (four patients) and restless legs syndrome (one patient).", "Three patients did not return for the follow-up scan: one patient because of eye surgery, one where lamotrigine was stopped by their treating clinician, and one due to practical issues.", "Spectra were acquired using the Siemens Trio 3-Tesla whole-body MRI scanner and a 32-channel coil at the OCMR (University of Oxford).", "A high-resolution T1-weighted MP RAGE image was acquired for accurate MRS voxel placement and subsequent structural analyses (TR = 2040 ms, TE = 4.7 ms, flip angle = 8°, 192 transverse slices, 1-mm isotropic voxels).", "B0 shimming was achieved using a GRESHIM, available as a work-in-progress package on the Siemens system.", "Spectra were measured from the pregenual ACC voxel (2 × 2 × 2 cm3) (Fig. 1) with a semiadiabatic localization by adiabatic selective refocusing (SEMI-LASER) sequence (TE = 28 ms; TR = 3 s; 64 averages) with variable power radiofrequency pulses with optimized relaxation delays (VAPOR) water suppression and outer volume saturation (Tkac and Gruetter, 2005).", "Unsuppressed water spectra acquired from the same voxel were used to remove residual eddy current effects and to reconstruct the phased array spectra (Natt et al., 2005).", "All MRS spectra were visually inspected for quality and between scan differences before inclusion.", "Mean full width at half maximum (FWHM) at the baseline scan was 0.080 (SD = 0.009), and at the follow-up scan 0.077 (SD = 0.016).", "Signal to noise ratio (SNR) at the baseline scan was 30 (SD = 0.6.4), and at the follow-up scan 31 (SD = 7.4).", "Cramér Rao Lower Bounds (CRLB) was 4.6 for both scans (SD, respectively, 1.7 and 1.1).", "Metabolites were quantified with LCModel (Provencher, 1993) using the unsuppressed water signal as reference.", "Model spectra of metabolites were generated based on previously reported chemical shifts and coupling constants by the GAMMA/PyGAMMA simulation library of VeSPA (Versatile Simulation, Pulses and Analysis) (Tkac, 2008; Soher et al., 2011).", "Metabolites quantified with Cramér-Rao lower bounds (CRLB, estimated error of the metabolite quantification) >20% were classified as not detected.", "T1-weighted structural images were brain extracted and tissue-type segmented using the Brain Extraction Tool of FMRIB Software Library (FSL) and Statistical Parametric Mapping (SPM) (Smith, 2002).", "The percentage of cerebrospinal fluid (CSF) within the MRS voxel was calculated from the resulting images, and used to correct metabolite concentrations for CFS fraction.", "Statistical analysis was performed using SPSS version 23.", "The effect of lamotrigine treatment on glutamate (Glx) levels in responders and non-responders was assessed with a two way repeated measures analysis of variance (ANOVA).", "Other comparisons were carried out with paired and unpaired t-tests and the Chi square test.", "Correlations were performed using Pearson's test.", "After 10–12 weeks’ treatment with lamotrigine, fifteen patients (68%) were classified as responders and seven (32%) as non-responders on the HAM-D.", "At this point, three patients were taking 100 mg, 150 mg, and 300 mg lamotrigine daily, as prescribed by their clinicians; all other patients were taking 200 mg lamotrigine daily.", "There were no significant differences between responders and non-responders with respect to age, gender, age at bipolar disorder onset and length of illness (Table 1).", "The mean ± SD HAM-D score at baseline was 20.5 ± 6.6 and did not differ between responders (20.8 ± 8.3) and non-responders (19.3 ± 4.6) to lamotrigine.", "At the time of the second scan the mean HAM-D score had fallen significantly (8.3 ± 5.9, t = 7.22, p = 0.001) (Table 1).", "Sixteen patients had valid MRS scans on both scanning occasions.", "The ANOVA showed no main effect of lamotrigine treatment on Glx levels (F = 0.49, df = 1,14, p = 0.49); however, there was a significant interaction between response status and lamotrigine treatment (F = 7.51, df = 1,14, p = 0.016).", "Patients who responded to lamotrigine (n = 10) showed a significant increase in mean ± SD Glx levels over treatment (14.0 ± 0.90 vs 14.9 ± 0.99 µmol/g, t = 3.12, p = 0.012) while non-responders (n = 6) showed no significant change (14.8 ± 1.3 vs 14.3 ± 0.98 µmol/g, t = 1.11, p = 0.31).", "Overall there was no significant change in Glx level in ACC in the whole patient group (14.3 ± 1.1 vs 14.7 ± 0.99 µmol/g, t = 1.20, p = 0.25).", "There were no differences in baseline Glx level between patients who went on to respond to treatment and those who did not, though responders showed a trend to have lower values (14.8 ± 1.2 vs 14.1 ± 0.93 µmol/g, t = 1.38, p = 0.19).", "There was no significant correlation between baseline Glx and change in HAM-D score over treatment (r = −0.31, p = 0.21).", "The main finding of this study is that lamotrigine, given over a period of about 12 weeks, significantly increased Glx in the ACC of depressed bipolar patients who responded to treatment, as defined by a 50% decrease in HAM-D core.", "However, there was no significant change in Glx levels in patients who did not respond.", "Also, lamotrigine did not change Glx levels in the patient group overall when baseline and follow-up scans were compared.", "These findings are clearly at variance with our hypotheses as stated in the Introduction.", "Studies in animals suggest that lamotrigine decreases glutamate release, probably as a consequence of sodium channel blockade (Ketter et al., 2003).", "However, two previous MRS studies of lamotrigine in bipolar patients have not confirmed these animal experimental data.", "For example, Croarkin et al. (2015) observed a significant increase in Glx in the ACC of 15 bipolar patients after 12 weeks of lamotrigine treatment, though change in Glx did not correlate with therapeutic response.", "In contrast, Frye et al. (2007) found no change in ACC Glx levels in 23 depressed bipolar patients following 12 weeks lamotrigine.", "Again, there was no correlation between change in Glx levels and clinical response.", "Both the latter investigations were carried out at relatively low MR field strength (1.5 Tesla) which makes it difficult to interpret individual components of the Glx signal.", "The notion that lamotrigine might lower ACC Glx in bipolar depressed patients is consistent with the view that bipolar depressed patients have raised Glx levels prior to treatment.", "While findings from different studies are not entirely consistent, meta-analyses have suggested that bipolar disorder is associated with increased brain Glx levels and specifically that bipolar depression might be distinguishable from unipolar depression on this basis (Gigante et al., 2012; Taylor, 2014).", "However, the latter possibility was not confirmed in a recent direct comparison between unipolar and bipolar depressed patients where both patient groups had lower glutamate levels than healthy controls (Wise et al., 2018).", "Because our study lacked a healthy comparison group we cannot comment on whether Glx levels in our patients prior to lamotrigine treatment differed from healthy controls.", "However, the fact that clinical improvement correlated with increased Glx might suggest that in these patients increased Glx was a consequence of the amelioration of depression rather than a direct result of lamotrigine treatment.", "In this study we focused on the ACC, a brain region frequently used as a voxel in MRS studies of patients with mood disorders (Yuksel and Ongur, 2010) and in other MRS studies of lamotrigine treatment (see above).", "While the ACC has been strongly implicated in the pathogenesis of mood disorder (Cerullo et al., 2009) other relevant regions, such as thalamus and striatum, would also be worth investigating, as glutamatergic changes may be limited to individual structures.", "Hence it is possible that lamotrigine could alter Glx levels in brain regions other than ACC.", "The strength of the study is its application of MRS to measure the effect of lamotrigine on Glx in real world patients in a within-subject design.", "The limitations are the modest sample size, the fact that patients were also maintained on other medications, the lack of placebo control and that the MRS procedure was unable to separate glutamate and glutamine.", "One methodological issue for our study was that – as according to clinical practice - in most cases lamotrigine was added to pre-existing medication.", "Thus effects of these latter medicines on glutamatergic mechanisms might have modified or suppressed effects of lamotrigine.", "In particular other mood stabilisers such as lithium have been reported to influence glutamate neurotransmission (Sanacora et al., 2008; Yuksel and Ongur, 2010) perhaps making it difficult for a specific effect of lamotrigine to be detected.", "Future studies would gain from the use of ultrahigh field MR (for example 7 Tesla) which yields clear delineation of glutamate and glutamine and thereby allows a better assessment of glutamatergic activity than is provided by the composite measure of Glx (Godlewska et al., 2017, 2018).", "Such studies should also include a larger initial number of patients, to increase the reliability of findings and to allow better for treatment drop-outs and technical failures with MRS imaging.", "Also a placebo control group would enable effects of lamotrigine to be more clearly separated from those relating to clinical change.", "Our study shows no major effect of several weeks of lamotrigine treatment on Glx levels in the ACC in bipolar depressed patients.", "However, therapeutic improvement during lamotrigine was associated with increased Glx, suggesting that alteration in glutamatergic activity might be related to recovery from bipolar depression.", "Beata R. Godlewska substantially contributed to conception and design of the study, acquisition of the data, data analysis and interpretation; she has drafted the manuscript.", "Uzay Emir substantially contributed in the area of methodological aspects regarding MRS data acquisition and analysis; he participated in drafting the manuscript.", "Charles Masaki contributed to data acquisition and analysis.", "Theodoras Bargiotas had substantial contribution to data acquisition.", "Philip J Cowen substantially contributed to conception and design of the study, data analysis and interpretation, and writing of the manuscript.", "All authors gave their final approval of the version to be published.", "This work was supported by Oxfordshire Health Services Research Committee (grant number 1104) and Medical Research Council (grant number MR/K022202).", "CM was a Rhodes Scholar.", "The authors also acknowledge support from the NIHR Oxford Cognitive Health Clinical Research Facility.", "The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health." ]	[ "MRS was used to measure determine brain glutamate (measured as Glx) in depressed bipolar patients before and after 12 weeks of lamotrigine.", "Overall, lamotrigine treatment had no significant effect on Glx levels in anterior cingulate cortex (ACC).", "However, clinical improvement with lamotrigine was associated with an increase in Glx.", "Baseline levels of Glx did not predict response to lamotrigine." ]
Characterizing the vertical distribution of chlorophyll a in the German Bight	Coastal and shelf seas display strong variability in the horizontal and vertical distributions of chlorophyll a (CHL). Detailed data are required to identify the processes that drive the observed spatio-temporal dynamics. A high-resolution, vertically resolved transect data set for biogeochemical and physical properties was collected in the inner German Bight (GB) from 2009 to 2011 on a seasonal basis. We used fluorescence as an indicator for phytoplankton biomass via the CHL concentrations. We classified profiles into different types by evaluating the heterogeneity of CHL vertical distribution and identifying vertical location (upper mixed layer, subsurface layer, bottom mixed layer of water column) of high CHL concentration in each profile. We analyzed the spatio-temporal occurrences of the different CHL vertical distribution types in the context of the hydrodynamic environment. More than half (68.7%) of all profiles showed vertically homogeneous CHL distributions. A smaller subset (3.2%) of all profiles showed subsurface CHL maximum layers (SCMLs) in the vicinity of the pycnocline, co-varying with strongly stratified conditions in deeper water. Profiles with highest concentration of CHL in the upper part of the water column (HCU) were observed in 11.5% of all profiles. Profiles with highest concentrations of CHL in the lower part of the water column (HCL) comprised 16.6% of all profiles. HCL profiles were extensively observed during the decay phase of the spring bloom and were associated with resuspension and erosion from pre-existing SCMLs, which could be driven by tide; photosynthetic activity below the pycnocline could also contribute. Under moderate weather conditions, tidal currents were the main driver of resuspension. This study highlighted the occurrence of SCMLs and HCL patterns in vertical CHL profiles in shallow shelf seas, such as the GB.	[ "AGRI", "EART" ]	[ "German Bight", "Resuspension", "SCANFISH", "Stratification", "Subsurface chlorophyll maximum layers", "Turbulent mixing", "Vertical chlorophyll-a profiles" ]	[ "1104", "1907", "1910" ]	[ "Temporal and spatial variability in vertical chlorophyll a (CHL) distribution has long been recognized in temperate ocean waters (Boss and Behrenfeld, 2010; Herdman, 1923; Russell, 1927).", "The vertical distribution of CHL is shaped by a combination of biological processes, including the vertical patterns of growth and grazing influenced by the availability of light, nutrients and predators, and physical processes, such as mixing, resuspension and advection of plankton biomass (Riley, 1942).", "Surface sampling, e.g., via satellite images, limits the accurate description of the whole system (Charnock et al., 1993) and hampers the mechanistic understanding of the relation between environmental conditions and CHL distributions.", "Since the method of continuous vertical sampling of fluorescence as a proxy for CHL was introduced (Lorenzen, 1966), previously unappreciated features of the vertical distribution of CHL have been revealed.", "More than 20 years ago, strong heterogeneity in vertical CHL profiles, such as the occurrence of subsurface CHL maximum layers (SCMLs), has been found in the central and northern North Sea (Nielsen et al., 1993; Richardson and Pedersen, 1998).", "In the northern Dogger Bank area, recurrent and persistent SCMLs were suggested to significantly contribute to primary production (Fernand et al., 2013).", "The occurrence of SCMLs was attributed to bottom fronts, where injection of nutrients by tidal pumping supports primary production (Richardson et al., 2000; Weston et al., 2005).", "In stratified areas without frontal systems, hotspots of SCMLs were found to be related to local tidal mixing resulting from specific bottom bathymetry (Scott et al., 2010).", "We would expect considerable production in shelf seas contributed by SCML.", "However, limited estimations are able to confirm this based on available data.", "First, CHL:C (Chlorophyll a to carbon) ratio varies significantly in different time and spatial scales (Jakobsen and Markager, 2016; Wang et al., 2009).", "Second, phytoplankton tends to fluoresce more in autumn, when productivity is less due to lower light levels.", "and increasing light limitation (Kalaji et al., 2017).", "Third, SCML production also dependents on injection of nutrients, which would be driven by physical process such as tidal stirring (Zhao et al., 2018) and baroclinic circulation (Pedersen, 1994).", "In the southeastern North Sea, especially in the German Bight (GB), the vertical distribution of CHL has seldom been addressed since, as water depths are less than 40 m, tidal and wind-driven mixing were thought to homogenize the vertical CHL distribution at most times of the year.", "However, observations and simulations have shown stratification in the water column of the German Bight, which displayed an evident variability in extent, duration and intensity (Haren and Howarth, 2004; Pohlmann, 1996a, 1996b; Schrum, 1997).", "Apart from stratification, which potentially favors the development of SCMLs, other characteristic processes of the inner GB, such as the accumulation of settled detritus (Westernhagen et al., 1986) and low turn-over rates of organic matter (Beusekom and Brockmann, 1999), should further modify the vertical distribution of CHL and make it different from that in deeper areas.", "We herein investigate to which degree vertical heterogeneous CHL profiles develop and attribute their characteristics to coastal hydrodynamics and biogeochemical cycles.", "Here we will employ high resolution transect data from the Coastal Observing System for Northern and Arctic Seas (COSYNA) (Baschek et al., 2017).", "Fluorescence profiles sampled in the COSYNA project throughout spring to autumn were used as proxy for CHL concentrations and we focused on vertical distribution pattern of CHL which are quantified within each profile.", "We are aware of the uncertainties resulting from varied fluorescence: CHL ratio; however, in general, the quantification of relative distribution of CHL within each profile, would not be influenced by those uncertainties.", "Afterwards, we characterize the seasonally variable vertical CHL patterns in the GB and examine potential mechanisms responsible for the observed vertical patterns.", "Our study thus provides a reference for the vertical CHL distribution in the GB.", "It will support the interpretation of observations and challenge coupled hydrodynamical-biogeochemical models for the GB in the future.", "In this study, we attempt to provide answers to the following questions: (1) To what extent does the vertical CHL distribution show heterogeneity in the inner GB?", "(2) Are there any temporal and spatial patterns in the observed vertical CHL distribution that are related to the hydrodynamic environment or phytoplankton growth dynamics?", "Therefore, we first classified the vertical CHL profiles depending on whether relatively high concentrations of CHL were present in the upper or lower part of the water column and on the existence of a SCML.", "Next, we analyzed the spatial-temporal appearances of different vertical CHL profile types in the context of the seasonal dynamics of phytoplankton and stratification.", "Additionally, we investigated the potential biological and physical mechanisms responsible for the maintenance of observed SCMLs mainly via an empirical analysis.", "The potential factors responsible for the wide-spreading of higher CHL in the lower mixed layer, such as resuspension and erosion from a pre-existing SCML, were also explored.", "In this section, we first review the general hydrodynamic and biogeochemical conditions in our study area.", "Secondly, the data set and sampling methods and the pre-processing of the observational data are described.", "Thirdly, the detection of SCMLs and the classification of different types of vertical CHL distributions are laid out.", "In the fourth part, the characteristics of the CHL profiles, such as resuspension signals and CHL gradients in the deeper parts of profiles, are specified.", "To further analyze the occurrence of types of vertical CHL distributions with respect to the environmental setting, the irradiance, wind speed, and simulated hydrodynamic setting are introduced in the fifth part.", "To assess the tidal currents, which were not measured simultaneously, simulation results from a 3D hydrodynamic model were incorporated into this study after validation.", "The hydrodynamic processes potentially influencing the vertical CHL distribution pattern, such as tidal phases and stratification intensity were quantified.", "The GB is a shallow area located in the southeastern part of the North Sea, with average water depth of approximately 25 m.", "The hydrodynamics are dominated by tides and wind forcing.", "The tidal amplitudes in the GB range from 1.8 to 3.4 m, with tidal currents of up to 0.6–1.0 m s-1 (Dietrich, 1950).", "In the near coastal areas of the GB, the water column is continuously mixed by tidal and wave forcing, which act against the seasonal thermal and haline stratification which is supported by the surface buoyancy induced by surface heat fluxes, riverine fresh water fluxes and tidal straining (Simpson et al., 1991).", "Further offshore, the surface heat flux competes with tidal stirring and generates seasonal thermal stratification (Czitrom et al., 1988).", "However, seasonal stratification can be interrupted by wind-driven mixing, even in summer.", "The interplay between the surface heat flux, wind/tidal mixing and river run-off regulates the mixing/stratification status in the GB (Dippner, 1993).", "The initiation and duration of continuous mixing and stratified conditions show high inter-annual variability (Schrum et al., 2003a, 2003b).", "The inner GB exhibits high primary production (Joint and Pomroy, 1993).", "Driven by tidal and wave forces, resuspended materials are more frequently observed and remain in suspension longer in the southern part than in the northern part of the North Sea (Eisma and Kalf, 1987).", "The interactions between produced particulate organic matter and suspended sediments enhance aggregation and sinking (Maerz et al., 2016).", "The GB was suggested to be a temporal deposition area for organic matters, due to its high local productivity (Lohse et al., 1995) and its location in the northeastward transport routs of organic matter in the North Sea (Gehlen et al., 1995).", "Physical factors, such as frontal systems and estuarine circulation (Krause et al., 1986), also support primary production and confine the high turbidity, high nutrient concentrations and high primary production to the coastal area.", "The resuspension of settled organic matter containing CHL (Duineveld and Boon, 2002) due to shifts in the stratification/mixing status in the frontal zone (thereby modifying the intensity of vertical mixing) would further complicate the distribution pattern of CHL.", "High-resolution vertical transect data on biogeochemical and physical properties were provided by the observation project ‘Coastal Observing System for Northern and Arctic Seas’ (COSYNA) (Baschek et al., 2017).", "This observation project aimed at mapping interactions between physical and biological processes in the GB, where natural variability is influenced by anthropogenic and environmental changes.", "The vertical transect campaigns were repeated from 2009 to 2011 and covered most of the German Exclusive Economic Zone in the inner GB beyond 10 m water depth throughout spring to autumn.", "The observational data were sampled on ten cruises between 2009 and 2011 using the undulating towed system ScanFish Mark III™ (SCANFISH) connected to the RV Heincke (https://www.awi.de/en/expedition/ships/heincke.html) as part of the project “Coastal Observing System for Northern and Arctic Seas” (COSYNA: http://www.cosyna.de, Baschek et al., 2017).", "The research vessel sailed along a designated grid of east-west and north-south transects in the area of 3.5–8.3°E and 53.5–55.8°N that covered the German Exclusive Economic Zone in the GB (Fig. 1) and captured the across-shore gradients both off the East Frisian (north-south direction) and North Frisian (east-west direction) coast.", "The operation of the ship and the SCANFISH instrument required water depths deeper than 10 m and wind speeds less than 14 ms-1.", "Sometimes, weather conditions prevented the completion of observations; the coverage of each cruise is listed below (Table 1).", "Further analysis was confined to profiles with valid profile depth no less than 10 m.", "The ten cruises covered the growing seasons (spring, summer and autumn) in three consecutive years (Table 1), with similar spatial coverages in the majority of the cruises.", "Percentage of profiles measured in each campaign among all available profiles were laid out in the brackets behind the profile numbers.", "Thus, these data are suitable for investigating the seasonality using measured CHL profiles.", "Tidal phase was not considered in the planning of the cruises.", "The largely moderate wind conditions during the SCANFISH operation may have created a bias in stratification/mixing status, which is considered in the interpretation of the results.", "The SCANFISH undulated 200–250 m behind the vessel and recorded vertical profiles along continuous V-curve paths, while maintaining safety margins of 3–5 m from the surface and the bottom.", "The vessel sailed at a speed of 6–8 knots.", "With a vertical speed of the SCANFISH of 0.4 m s-1 and a sampling frequency of 11 s-1, the vertical spacing between measurements was approximately 0.04 m.", "The horizontal span of each V curve ranged from 250 to 400 m, varying with the sailing speed of the vessel and the water depth.", "This sampling strategy provided simultaneous horizontal and vertical observations at a spatial resolution sufficiently high for the purposes of this study.", "Among the multiple outputs provided by the sensors mounted on the SCANFISH, our analysis considered water pressure (PA-7, Keller AG, Switzerland), water temperature (PT100, ADM Elektronik, Germany - cruises 303–319; NTC sensor, ISW Wassermesstechnik, Germany - cruises 325–365), conductivity (Conductivity Sensor, ADM Elektronik, Germany), optical turbidity (Seapoint Turbidity Meter 880 nm, Seapoint Sensors Inc., USA), oxygen saturation level (Shallow Water Dissolved Oxygen Micro-Sensor, AMT Analysenmesstechnik GmbH, Germany), chlorophyll a fluorescence (TriOS MicroFlu-chl, TRIOS Inc., Germany).", "For the chlorophyll a fluorescence instrument, light source ultra-bright blue LED has peak wavelength at 470 nm; detection of peak wavelength is at 685 nm, with 20 nm of full width at half maximum.", "Optical attenuation coefficient was computed from transmission measurement at 660 nm (C-Star Transmissometer, WETLabs Inc., USA).", "Salinities and potential water densities (σt) were calculated using the international thermodynamic equation of seawater (McDougall et al., 2010; Millero et al., 2008).", "The sensitivity of the TRIOS fluorescence signal to turbidity was corrected as described in Maerz et al. (2016).", "TRIOS Inc. delivers the output of the TriOS MicroFlu-chl from raw fluorescence readings that were transferred to physical units calibrated a lab chlorophyll-a standard by a first order polynomial equation.", "The ratio between fluorescence and chlorophyll pigments is known to vary (Kiefer, 1973) by up to a factor of two or even more (Petersen et al., 2011).", "In the subsequent data analysis we used fluorescence as a proxy for phytoplankton biomass via the CHL concentrations.", "For the conversion we assumed a constant ratio between fluorescence and CHL (we call it F: CHL hereafter and use arbitrary unit of CHL as the unit of fluorescence signal).", "In general, we focus on the relative distribution of CHL in the water column; in another words, we quantified the vertical shape of each CHL-profile in terms of CHL concentration in one segment of water column being “higher than”, “lower than” or “comparable to” to other parts (upper, middle (pycnocline) and lower parts) of the same profile.", "No comparison was made among different profiles using absolute value.", "We are aware of the uncertainties resulting from varied fluorescence: CHL ratio and CHL: C ratio (Babin et al., 2008; Claustre et al., 1999).", "Relevant calibration and estimation of these uncertainties has been conducted to prove that the results would not be influenced by those uncertainties.", "183 HPLC measured CHL data were sampled simultaneously during these campaigns.", "We made use of the available HPLC measured CHL data to estimate the varying range of F: CHL, in each season, each year and between different vertical segments (Appendix A).", "Influence of quenching on fluorescence profile in the surface water column has also been adjusted (Appendix A).", "Generally, F: CHL mainly lies in the range between 1 and 2; the seasonal and inter-annual variability also vary no more than a factor of 2.", "In addition, around 60% of the F: CHL’s variabilities between surface and subsurface segments of water column, are no more than factor of 2.", "In the following analysis, we took a variation of factor 2 into account.", "For the profile classification we also choose this criteria, which exceeds the range of the uncertainty in the F:CHL ratio, to ensure that the difference in fluorescence reflects the difference of CHL, rather than resulting from the varied F: CHL ratio.", "The data sets were binned along the observation curve into regular vertical blocks of 10 cm to avoid unevenly sampling.", "The consecutive data sets were split into up and down profiles.", "For all parameters except those relevant to oxygen, we used only downward profiles to keep the lag effects of the sensors systematically coherent.", "A comparison of subsequent downward and upward profiles of oxygen saturation exhibited a substantial lag in the oxygen signal, suggesting a time constant of several seconds in campaigns taken after 2009.", "This temporal lag corresponds to approximately 5 m in the vertical signals.", "Rather than detecting the accurate vertical location of the oxygen maximum, we focused on the relative positions of the oxygen maximum and the SCML, under the assumption that the lag effects are stable.", "Downward profiles covered a horizontal span ranging from 125 to 200 m.", "We assumed that the lateral change within this horizontal span was negligible and treated the V curves from downward profiles as being perpendicular to the seafloor.", "As the upper mixed layer generally extended 10 m or more downwards, any disturbances attributable to the ship’s hull or quenching effects of CHL in the most uppermost meters should have a negligible impact on the profile classification.", "All the binned data sets underwent low-pass Butterworth filtering (Butterworth, 1930) to remove short-term variations.", "Half of the resolution of the binned vertical step was set to be the higher constraint of the filter to avoid aliasing (Grenander, 1959).", "This process also damped out most of the artefacts in the computed salinities and water densities at the pycnocline caused by the thermal lag of the conductivity cells.", "To evaluate and compare the heterogeneity of vertical CHL profiles in a systematic way, we classified the measured CHL profiles into 6 characteristic types depending on whether a SCML was detectable and whether the CHL concentrations differed between the upper part and the lower part of the water column (Fig. 2).", "Following Dekshenieks et al. (2001), a SCML is defined as a subsurface CHL layer whose amplitude is distinctly higher than adjacent water columns and the thickness is relatively thin compared to whole water column; the characteristics in amplitude and thickness display persistence in reasonable temporal and spatial scales.", "Considering the transient and turbulent environmental conditions, as well as the notable variability in vertical CHL concentrations in our system, we modified these criteria to capture the layer features and to reflect the general character of SCMLs in our system.", "To select potential candidates for SCML in the profiles, we applied three steps: First, we identified points where the first-order derivative of the CHL profiles switched from positive to negative (Fig. A.3.", "b).", "Second, to identify the edges of a potential SCML, within 5 m of the potential layer peak, the local maxima in the second-order derivative of a CHL profile above and below the peak were assigned as the upper and lower edges of the SCML, respectively (Benoit-Bird et al., 2009) (Fig. A.3.", "c).", "Third, to evaluate the significance of the potential SCML, the amplitude of the peak was compared to the background value (Fig. A.3.", "a).", "We estimated the local background value by linearly interpolating the upper and lower edges’ values to the depth of the peak.", "A SCML was only recognized if the candidate satisfied the 3 following threshold criteria.", "Criterion 1: To discard peaks resulting from local fluctuations, the peak amplitude of a potential SCML was required to be 2.5 times larger than the standard deviation of the CHL profiles within a 5 m extent upward and downward from the peak.", "Criterion 2: The peak value was required to be 2 times higher than the background value (Fig. A.3.", "a).", "Criterion 3: To exclude CHL layers with weak gradients, the relative CHL gradient above and below the peak was required to exceed 0.125 m-1.", "Here, we used the relative gradient instead of the absolute gradient to exclude the effects of spatial variability in CHL amplitudes and to make it possible to process all profiles with the same threshold criteria.", "The relative gradient was calculated by dividing the CHL gradients between the peak and edges by the peak value.", "Under the condition that a SCML was present, we divided the whole water column into 3 sublayers: an upper layer, a middle layer corresponding to the SCML and a lower layer.", "Otherwise, we divided the water column into sublayers depending on the difference in CHL concentrations between the upper and lower parts of the water column (Fig. A.4).", "We selected the depth corresponding to the maximum absolute difference between averaged CHL concentration above and below to split the whole water column into upper and lower parts (Fig. A.4.", "b).", "The difference in the CHL concentrations among the sublayers was quantified by the ratio between the sublayers (Fig. A.4.", "a).", "By applying this method, the spatial variability in CHL was also excluded and we could process all profiles using the same analysis criteria.", "After we divided the water column into 3 (SCML present) or 2 (no SCML present) sublayers, we classified the CHL profiles into 6 groups depending on the CHL ratios among the sublayers.", "A complete flow chart of the profile classification method is presented in Fig. A.5.", "If the averaged CHL ratio between two sublayers was larger than 2 (Appendix A), the difference between the two sublayers was regarded as significant.", "The 6 types of vertical CHL profiles (Fig. 2) were then defined as follows.", "Type 1, SCM: A SCML was present, and the mean CHL concentration of the SCML was notably higher than that of the upper and lower parts of the water column.", "No significant difference in the mean CHL value between the upper and lower parts of the water column was present (Fig. 2a).", "Type 2, higher CHL concentrations in the lower layer (HCL): Relatively high CHL concentration was present in the lower part of the water column without the appearance of a SCML (Fig. 2b).", "Type 3, higher CHL concentrations in the upper layer (HCU): Relatively high CHL concentration was present in the upper part of the water column without the appearance of a SCML (Fig. 3c).", "Type 4, well-mixed (WM): Neither a SCML nor a significant difference in CHL concentrations between the upper and lower parts of the water column was found.", "In many profiles with a SCML, the upper or lower parts were not symmetric with respect to the subsurface layer; in other words, the mean CHL value in the SCML was significantly higher than that in one part but not the other.", "These profiles were classified as two transient types as follows (Fig. 2d).", "Type 5: The mean CHL value in the SCML was higher than that in the upper part of water column but not that in the lower part (SCM-HCL) (Fig. 2e).", "Type 6: The mean CHL value in the SCML was higher than that in the lower part of water column but not that in the higher part (SCM-HCU) (Fig. 2f).", "To investigate the seasonality of vertical CHL distribution patterns, we binned profiles into 4 segments depending on water depth: 10–15 m, 15–25 m, 25–35 m, 35–45 m.", "We grouped the 10 campaigns into 4 growing seasons: early spring (319: March 2010; 353: April 2011), late spring (303: May 2009; 325: May 2010), summer (308: July-August 2009; 331: July 2010; 359: June 2011), and autumn (312: September 2009; 336: September 2010; 365: September 2010).", "The spring season was divided into early and late spring regarding to whether the spring bloom decayed, with reference to the surface CHL concentration estimates from remote sensing data (ESA Ocean Colour Climate Change Initiative (OC-CCI; http://www.esa-oceancolour-cci.org/) (Müller et al., 2015).", "We performed several sensitivity analyses on our selection criteria.", "For the detection of SCMLs, we assessed the sensitivity of the second and third criteria introduced above.", "Among all profiles with SCML candidates (the first-order derivative of the CHL profile crosses 0 in water columns deeper than 5 m), the proportion of confirmed SCMLs stabilized at approximately 20% when the threshold of peak-background ratio was set between 1.125 and 1.7 and the relative gradient ranged from 0.05 to 0.2 m-1.", "Fewer SCMLs are recognized with stronger criteria (with higher value), and vice versa.", "Based on the uncertainty analysis regarding the varied F: CHL, we have to choose the peak-background ratio as 2 to exclude the condition that the higher CHL indicated by fluorescence signal is due to the increase F: CHL in subsurface layer.", "When applied this criterion of peak-background ratio, among all potential SCMLs, 10% satisfied the threshold criterion.", "It indicates that the chosen criteria guaranteed the separation of SCMLs from background noise and, at the same time, ensured that the analysis remained selective.", "To select the optimal critical ratio between sublayers, we varied the ratio of averaged CHL concentrations among sublayers from 1.5 to 2.5, with a step of 0.1.", "More profiles were classified as WM when higher critical ratios were used.", "Even if SCMLs were present, the profiles were more frequently identified as transient types, i.e., SCM-HCL and SCM-HCU, when higher critical ratios were applied.", "When we increased the critical ratio values to 1.8, the number of profiles classified into the 4 major types (SCM, HCL, HCU, and WM) was insensitive as it was for smaller critical values. ,", "with the number of profiles changing by no more than 9%.", "Notably, the appearance of a SCML would not necessarily classify the profile as SCM if the mean CHL value in the SCML was not distinguishable from that of the other sublayers.", "To investigate the source of high CHL concentrations in the lower mixed layer, we detected CHL peaks near the bottom (CPB) and CHL gradients in the deepest 5 m (CG5) in the CHL profiles to identify resuspension.", "There were two criteria for the detection of CPB: 1) The peak value in the bottommost 5 m should be at least 1.5 times higher than the average value in the lower layer.", "The mean value in the lower layer was used as a substitute for defined background values, which we have used for the detection of SCMLs since the layer bound detection possessed high uncertainty near the bottom.", "2) The relative gradient had to be larger than 0.0625 m-1.", "The calculation of the relative gradient was done in a similar way to that of SCMLs.", "CHL gradients in the deepest 5 m in CHL profiles (CG5) were calculated as follows: the averaged CHL value in the deepest 1.25 m in the profile was subtracted from the averaged CHL value in the deepest 5–7.5 m in the profile and then divided by the depth interval, which is 5 m here.", "Here, we adopted the average value of depth intervals rather than single points to minimize the bias caused by local spikes and discontinuities in profiles.", "Since many profiles presented an increasing trend downward instead of a sharp increase at the bottom, we also calculated the mean gradient of CHL in the lower part of the water column (in the SCM-HCL, HCL and SCM profiles), referred to as CGL for short, to represent the general CHL variation trend with depth.", "If the CHL concentration increased with depth, the gradients (CG5, CGL) mentioned above possess positive values, and if the CHL concentration decreased with depth, the opposite is true.", "To further investigate the mechanism driving resuspension, we examined the occurrence of resuspension with respect to tidal phases.", "We counted the number of profiles belonging to tidal phase segments and the occurrence of resuspension signals (e.g., CPB) in the profiles.", "By dividing the number of profiles possessing resuspension signals by the number of profiles measured in each tidal-phase segment, we obtained the relative occurrence of resuspension for each tidal phase.", "Euphotic layer depth: Euphotic layer depth is an indicator to quantify how deep the irradiance can penetrate and sustains photosynthesis.", "Here, we calculated the euphotic layer depth to examine whether irradiance is able to influence the SCML depth.", "We used short-wave radiation records and profiles of attenuation coefficients to calculate the irradiance profiles in the water column according to the Lambert-Beer law (Swinehart, 1962).", "Daily mean values of two surface short-wave radiation data time series were used to represent the average irradiance conditions for the GB.", "These two measuring stations for surface short wave radiation are located on Norderney and List on the island of Sylt (Fig. 1) at the southern and eastern border of the GB, respectively (DWD, 2015).", "The value of 7 W/m2 was chosen (Brand and Guillard, 1981) as the lowest amount of irradiance necessary for the growth of phytoplankton, and we defined the euphotic layer depth as the depth where absolute irradiance values hit this threshold (Banse, 2004).", "To better interpret the vertical distribution of CHL with respect to the hydrodynamic setting, we calculated some proxies to quantify the counteracting processes between mixing and stratification that may help initiate, maintain or undermine the vertically heterogeneous CHL structures.", "Here, we briefly outline the definitions and calculations of these proxies for the turbulent status, as used in previous analytical work (Steinbuck et al., 2010).", "The critical gradient Richardsen number is Ri = 0.25.", "For Ri < 0.25 the flow is likely to remain turbulent (Kundu et al., 2016).", "Reynolds number: When the Reb is smaller than 15, stratification dominates.", "In contrast, turbulence is unaffected by stratification when the Reb is greater than 100.", "When the Reb ranges from 15 to 100, stratification is affected by turbulence (Monismith et al., 2018).", "The Simpson-Hunter parameter indicates the tendency for a formerly stratified water column to become mixed.", "The larger the value is, the stronger the rates of tidal energy dissipation per unit mass are, and the easier the stratification can be disrupted.", "In accordance with our criteria, among all analyzed downward profiles 54% were stratified.", "Stratification mainly emerged in campaigns 303 (May 2009), 331 (July 2010), and 359 (June 2011) when stratification was present in at least 80% of the profiles.", "The seasonality of stratification has been revealed by the proportions of stratified profiles (Fig. 3).", "As noted by previous studies, the onset of thermal stratification occurs in late spring/early summer (Pohlmann, 1996a, 1996b).", "In the study area, haline stratification could superimpose mixing in regions with freshwater inflows (Simpson et al., 1991).", "In early spring, the proportion of stratified profiles was not more than 25%.", "The proportion of stratification peaked in June and began to drop in August.", "In autumn (September), proportion of stratification decreased down to approximately 50% of the profiles in deeper areas and only approximately 20% of the profiles in shallow areas.", "Due to the rather moderate weather conditions during the sampling periods of the cruises, which is not necessarily representative (Geyer et al., 2015) in terms of the climatological conditions in longer time scales, we expected that the stratified proportions should be higher than the mean climatological conditions.", "Approximately 68.7% of all profiles showed a vertically homogeneous CHL distribution.", "The HCU type were observed in 11.5% of all profiles and the HCL type accounted for approximately 16.6% (Fig. 4).", "Only a small subset (1.9%) of all profiles were identified as the SCM type.", "The transient types SCM-HCL and SCM-HCU only represented a tiny proportion in the typology analysis, together accounting for 1.3%.", "We therefore merged these two transient types with the SCM type (in total accounting for 3.2%) in the following analysis of the seasonality of the vertical CHL profiles.", "We only separate these two transient types when transformation among different distribution types is discussed (cf. Section 3.4).", "The proportions of the WM type corresponded to the seasonal stratification/mixing cycle (Fig. 3).", "They decreased starting in late spring, reached the lowest level in summer and increased again in autumn.", "More profiles displaying defined vertically heterogeneous CHL distributions were found in deeper areas.", "The SCM type only emerged under strong stratification, in summer, its proportion increased with water depth, from 2.0% in the 15–25 m region to 20.0% in regions deeper than 35 m. No SCM type appeared in areas shallower than 15 m.", "In other seasons, SCM profiles were rarely detected, with proportions no larger than 1.0%.", "In late spring, the HCL type dominated except for WM type, particularly in areas shallower than 15 m.", "In autumn, in addition to the increased occurrence of WM, the HCU type dominated in all depth segments except WM type, accounting for 26.7% in areas deeper than 35 m and approximately 2.2–15.2% in shallower areas.", "The occurrences of the SCM and HCL profiles decreased in autumn.", "The occurrences of the HCU and HCL types also displayed substantial variability, especially in spring.", "The upper layer of the water column normally favors of phytoplankton growth in spring, due to higher availability of irradiance compared to that of the lower layer.", "However, higher CHL concentrations in the upper layer were not necessarily detectable in our observations because of the dilution resulting from vertical mixing in the water column.", "In addition, the proportions of HCU and HCL profiles in the late spring also depended on the variability in the decay phases of spring blooms in specific areas and onset time of stratification, even if we have grouped campaigns into seasonal groups based on the general flourishing patterns from satellite images.", "The CHL that sank down can remain suspended in the lower part of the water column and barried below the pycnocline.", "In a comparison of the stratification patterns among different water depth segments and seasons, the occurrence of SCMLs was basically in line with stratification, and the SCM type was detectable in summer and in deeper water, where stratification was observed in at least 66.8% of the profiles.", "The most extensive and notable SCMLs were observed in summer during campaigns 331 (July 2010) and 359 (June 2011).", "In these campaigns, SCMLs were present in 16.0–20.9% of the stratified water columns, with horizontal extents ranging from 50 km to 250 km.", "More than 90% of the SCMLs exhibited a thickness in the range of 4–6 m.", "In the SCMLs, the averaged CHL concentrations were primarily 3–9 a.u. and approximately 10% reached 10 a.u.", "The CHL concentration in the upper mixed layers was lower than that in the SCMLs, and more than 90% of surface layer concentrations were less than 2 a.u.", "In the calibration process, we found that the variation range of F: CHL is no more than factor 2.", "Considering the F: CHL’s variation in the same profile, 66.7% of F: CHL(subsurface/surface) lies in between 0.5 and 2 (Table A1).", "If considering this variation, the peak value of fluorescence in Fig. 5a lies no more than 2 (a.u.), the indicated peak value of CHL should be no more than 4 (a.u.).", "The disparity is not as large as the raw fluorescence data has indicated but we can still expect that the CHL value in the SCML is higher than that in the upper layer.", "The averaged CHL concentration in the lower mixed layer was distinctly higher than that in the upper mixed layer, which we will address further in Section 3.4.", "In addition to the physical conditions, we compared the vertical locations of SCMLs with the pycnocline center and other biochemical parameters to explore the dependence of SCMLs on environmental factors.", "The CHL peaks in SCMLs were closely co-located with the center of the pycnocline, which we defined as the location of the highest squared buoyancy frequency (Fig. 6a).", "The locations of CHL peaks in SCMLs were mostly shallower than the euphotic layer depth and were only weakly correlated with the euphotic layer depth (Fig. 6b).", "Within the euphotic zone, the potential for active photosynthesis is expected, as confirmed by the high correlation between the vertical locations of SCML peaks and highest oxygen saturation values (Fig. 6c).", "This correlation proved that the SCMLs identified here were photosynthetically active rather than being merely sinking remains.", "Photosynthesis in SCMLs has also been observed in the Baltic Sea (Bjornsen et al., 1993; Kononen et al., 1998), which were suggested by the consumption of nutrients instead of oxygen, in such an anoxic environment.", "In areas with seasonal stable stratification, the CHL concentration has been previously related to physical factors.", "For example, the CHL concentration decreases with depth (Brown et al., 2015) or increases after nutrients are entrained by wind-driven mixing events (Carranza and Gille, 2014).", "Based on our data, there were no obvious instantaneous correlations between the vertical CHL distribution and other physical or biological factors, except that the spatial pattern of integrated CHL concentrations in the euphotic zone, i.e. their local maxima, were associated with the occurrence of fronts (not shown here).", "Time lag correlations could not be studied based on only the snapshots from the SCANFISH data.", "Our data are not suitable to resolve the revolution process of stratification and the development of SCML, especially in such a shallow and energetic system as the GB, where stratification can be interrupted by strong wind induced mixing, even in summer.", "In contrast to the southern North Sea, the deeper northern North Sea possesses more stable stratification in summer.", "The SCMLs in the northern Dogger Bank area have been confirmed to have long durations and to be highly consistent based on observations and simulations (Fernand et al., 2013).", "The representativeness of our SCANFISH sampling data for the physical and biological characteristics in the GB merits attention.", "Considering that the weather condition is representative in the specific years and locations but not necessarily represent the climatological conditions in longer time scales (Geyer et al., 2015), we expected that, the GB should be more turbulent and less stratified than the conditions revealed by our data sets.", "If continuous data sets covering longer time scales are available, profiles with SCMLs are likely to account for lower proportions.", "Complementary to the deduction from the statistical analysis, the vertical CHL distribution pattern associated with the hydrodynamic environment has been discussed in a case study.", "Two transects were sampled twice in July 2010 during campaign 331, i.e., on July 14th and 16th, before and after a significant wind event.", "The western branch transect extended from 54.2ºN to 55.0ºN in the longitudinal direction, and the northern branch transect extended from 6.3ºE to 7.5ºE in the latitudinal direction (Fig. 1).", "Since wind mixing events triggered by wind, as pointed out by former studies (Rumyantseva et al., 2015; Williams et al., 2013), are able to re-erode SCM in pycnocline and supply nitrate into pycnocline or even reach the surface water column.", "We used these sections to explore the conditions under which SCMLs persist or decay during a wind mixing event and how the vertical CHL distribution evolves with changes in the hydrodynamic setting.", "According to the re-analysis product from the NCEP Climate Forecast System (Saha et al., 2010), the weather conditions were mild before and during the first sampling event.", "The wind speed did not exceed 5.38 m s-1 during the 24 h before the sampling began (July 13th, 4:00) or during the sampling.", "In the early morning of July 15th, wind speeds increased and reached 13.77 m s-1 at 13:00 on July 15th and dropped to 7.7 m s-1 at 16:00 on July 16th.", "From the early morning of the 15th until noon on the 16th, more than half of the hourly averaged wind speed values were greater than 12 m s-1.", "The second sampling of the western branch was performed during the diminishing phases of the wind event.", "For the northern branch, the second sampling was performed after the wind event, when the wind speed was comparable to the earlier calm conditions.", "On July 14th before the storm, stable stratification was observed in both transects.", "A double-pycnocline structure (Fig. 7) was present and contained an intermediary zone with CHL concentrations that were higher than those in the upper mixed layer.", "A well-structured SCML was present at the base of the lower pycnocline, with average values of 8.1 a.u. in the western branch (Fig. 7a) and 10.0 a.u. in the northern branch (Fig. 7e).", "In the western branch, below the second pycnocline, the lower mixed layer showed an average CHL concentration of 4.8 a.u., which was around 5 times higher than that in the surface mixed layer (Fig. 7a).", "Compared to the western branch, there was no evidence for resuspension in the northern branch, and the CHL concentration in the lower mixed layer was comparable to that in the upper mixed layer (Fig. 7e).", "At the eastern end of the northern transect, the water depth is less than 20 m. Here, CHL concentrations are enhanced in the surface mixed layer, even if a high subsurface CHL concentrations are detectable (Fig. 7e).", "Based on a comparison of the observations before and after the storm event, the upper pycnocline vanished as a result of wind-driven mixing, but the lower pycnocline persisted in most areas.", "In the western branch, the SCML at the base of the lower pycnocline in the first sampling event disappeared (Fig. 7b), except in areas between 54.4°N and 54.5°N where the subsurface CHL layer was more visible than before.", "Resuspension was prominent in the middle part of the western branch.", "At the southern end (54.2–54.4°N) of the western branch, the pycnocline broke down, and the CHL subsurface layer decayed (Fig. 7b).", "In the northern branch, most of the subsurface CHL layer persisted except at the eastern end, where both the pycnocline and the subsurface CHL layer were absent in the second sampling (Fig. 7f).", "The persistence or decay of the pycnocline and the corresponding spatial variability was in line with the stability of stratification as quantified by the Simpson-Hunter number (Fig. 7d, h).", "A higher Simpson-Hunter number indicates stronger tidal mixing, making the stratification more susceptible to mixing.", "The decay of the pycnocline demonstrated the sensitivity of the stratification to wind events in the shallow southern North Sea, which has been illustrated in previous studies involving simulations (Schrum et al., 2003a, 2003b; Schrum, 1997) and observations (Carpenter et al., 2016; Schultze et al., 2017).", "The spatial persistence and decay of the stratification was basically in line with hydrodynamic characteristics, whereas the variation of SCMLs was more complex.", "Most SCMLs decayed during this wind event.", "Because of the weakening of the double pycnocline structure after the wind event, the upper boundary of the whole stratified interior (including the whole double pycnocline structure in the first sampling) deepened by approximately 10 m. Furthermore, considering that the SCML was located at the base of the lower pycnocline, we compared the upper and lower boundaries of the lower pycnocline in the first sampling to those in the second sampling.", "The upper boundary of the lower pycnocline deepened after the wind event in most parts of the two branches (Fig. 7c, g).", "The variation in the lower boundary of the stratified layer fluctuated around 0, but remained mainly positive, indicating that most of the lower boundary deepened.", "It is likely that the accumulated CHL was mixed up during the deepening of the pycnocline.", "Although, it cannot entirely be excluded that biological processes which could contribute to the changes, it is rather unlikely because these are usually not effective enough on such short time scales.", "The variation in CHL concentration throughout the whole water column and in each sublayer divided by the pycnocline was in the range of the natural growth of phytoplankton, but again, natural growth in low light conditions would be an unlikely candidate for the changes.", "The increase in CHL concentration in the lower mixed layer in the western branch could also come from resuspension (Fig. 7b).", "The increase in CHL concentration in the upper mixed layer could be either mixed up from a pre-existing SCML or produced following the entrainment of nutrients from lower layers due to the deepening of the pycnocline.", "The decrease in CHL concentration in the stratified interior could also be induced by enhanced grazing due to suspended zooplankton during wind events, but again this can be considered as rather unlikely due to short timescale and substantial turbulence during the wind event, which would limit capturing success of zooplankton.", "The HCL-type profiles occur frequently, especially in campaigns performed in late spring (Fig. 4).", "Thus, an exploration of the mechanisms leading to high CHL in the bottom mixed layer is necessary.", "It is possible that this phenomenon results from different directions of residual currents below and above the pycnocline, which bring phytoplankton from different source regions to the same area (McCandliss et al., 2002).", "This might be the case especially for floating and non-sinking species, such as phaeosystis which also dominates in the German Bight in spring (Iriarte et al., 1991) (together with diatoms).", "If the local vertical mixing plays a role, there are two related hypotheses: 1) the HCL type profiles involves CHL from layers above either because of sinking or erosion from pre-existing SCMLs or 2) the HCL type involves CHL from below because of re-suspension effects.", "In this section, we first test these two hypotheses by investigating CHL gradients near the bottom and in the lower layers of water columns.", "Afterwards, potential hydrodynamic drivers and responsible biochemical factors will be discussed.", "Resuspension has been quantified by the presence of a CHL peak in the bottom layer (CPB) and a corresponding CHL gradient in the upper boundary of the CPB.", "Among the HCL profiles, 30% had detectable CPBs, whereas only 5.7% of SCM profiles and 15% of SCM-HCL profiles had CPBs.", "The relative upward gradients in the CPBs in HCL profiles were generally larger than those of SCM and SCM-HCL profiles.", "The higher occurrence of resuspension signals and higher gradients in the upper branch of the CPBs indicate the importance of resuspension in the formation of HCL-type profiles.", "In addition to the CPB, the CHL gradients in the lower layer (CGL) can be used to approximate the CHL variation with depth across a relatively long vertical segment.", "In all profiles classified as SCM, CHL continually decreased from the base of the SCML to the bottom.", "Furthermore, 76% of SCM-HCL profiles also showed higher CHL concentrations at the base of the SCML, indicating that CHL was mixed down or sank from the subsurface layers.", "However, in the remaining 24% of SCM-HCL profiles, the CHL concentration showed that more CHL accumulated towards the bottom.", "The CHL concentration increased with depth in the lower layers in almost all HCL profiles.", "By examining the CHL gradients in the bottom mixed layer and the existence of CPBs, we infer that a considerable proportion of the high CHL concentrations come from the bottom in HCL profiles and some SCM-HCL profiles, probably caused by resuspension.", "To address the relevance of tides, the dominant hydrodynamic feature in this study area (Otto et al., 1990), to resuspension in the observed profiles, herein we explore the occurrence of resuspension with respect to the tidal cycle, which was retrieved from model simulations.", "To improve the statistical significance, only campaigns with more than 400 CPBs in total and more than 10 CPBs in each tidal phase segment were included (Fig. 8).", "We also identified resuspension signals in terms of turbidity as a supplemental forcing indicator, assuming that the resuspension of other materials would not be resource limited, unlike that of CHL.", "The seasonal difference in the significance of resuspension signals in CHL profiles could be attributed to seasonal changes of availability of CHL at the bottom.", "Instead of only using the occurrence of CPBs, which occur in only a small subset of the profiles, we calculated the CHL gradient in the deepest 5 m (CG5) in all the profiles and summarized the increases or decreases in CHL with depth (Fig. 9).", "The occurrence of positive CG5s, which indicated more CHL available near the bottom than in the adjacent layer above, showed a distinct seasonal pattern.", "The CG5% increased from approximately 50% in early spring to more than 75% in late spring during the decay of the spring bloom.", "Thereafter, the percentage decreased throughout summer (30%) to autumn (approximately 20%).", "The CG5 seasonality indicated that the resuspension processes of CHL were resource limited in summer and autumn.", "This pattern was also revealed in the seasonality of vertical CHL distribution patterns (Fig. 4): the proportion of HCL type decreased in summer and especially in autumn, when near-bed CHL had been depleted.", "This seasonal pattern in the availability of resuspension resources at the bottom revealed in our study is similar to previous results for benthic fluff layers.", "Formed by the sinking of phytoplankton cells, fluff can easily be resuspended and transported (Jago et al., 2002).", "The amount of fluff resources is greatest after the spring bloom and decreases in other seasons due to resuspension and predation, leading to resource limitation with respect to further resuspension ( Jago and Jones, 1998).", "We have to stay cautious when we derive CHL from fluorescence signal which showed high values near bottom, since fluorescence signals could be influenced by the presence of other organic particles near the bottom, especially in areas with high turbidity, such as the GB.", "However, the high CHL signals observed near the bottom confirm former measurements of high CHL near bottom at the German Bight and in nearby areas close to the European continent.", "In these studies, CHL data were derived from water sample directly (Jago and Jones, 1998), from calibrated fluorometer (Millward et al., 1998) or Reineck’s box-corer (Dauwe et al., 1998; Jenness and Duineveld, 1985) taken from sea bed.", "They all confirmed that high CHL concentration can be detected below the pycnocline after the onset of stratification due to phytoplankton sinking or trapped by the pycnoline (Jones et al., 1998).", "The high concentrations are mostly detectable in late spring because produced organic matters in spring bloom settled down (Boon and Duineveld, 1996; Jago et al., 1993).", "Compared to other stations (Fourteens, Frisian Front and Skagerrak), the GB showed the strongest predominance of surface deposition and strongest surface accumulation of organic matter at the sea floor, mainly due to longer residence time of water in the GB and the intense local production (Dauwe et al., 1998).", "Given sufficient settled materials, resuspension driven by tide and waves is detectable (Jago et al., 1993; McCandliss et al., 2002).", "Due to sinking and aggregation with suspended particulate matter (SPM), the CHL-rich materials in the bottom mixed layer form fluff layers which are also sensitive to resuspension given strong bottom shear stress and sink down in low energy conditions (Jenness and Duineveld, 1985; McCandliss et al., 2002).", "It is likely that production is able to persist given enough irradiance penetration below the pycnoline (Peeters et al., 1995).", "However, light limitation plays an important role in controlling primary production in the shallow North Sea, typically in spring and in windy conditions in summer and autumn (Su et al., 2015), since light penetration is very sensitive to resuspension events (Jones et al., 1998).", "Separating the resuspension process from sinking based on our data is challenging.", "It would be problematic to categorize all positive CHL gradients in the bottom mixed layer (increasing CHL concentrations with water depth) as a resuspension signal since many phytoplankton species have a higher densities than water and tend to sink (Smayda, 1971).", "Especially in the decaying process of the spring bloom, after the nutrients in the surface layer are exhausted, massive amounts of phytoplankton cells sink down.", "However, the sinking speed of some cells slows once the nutrient status improves and the buoyancy of the cells increases (Steele and Yentsch, 1960).", "Additionally, some cells may also become neutrally buoyant because of the increasing density with depth.", "In addition to CHL accumulating near the bottom, high CHL concentrations in the lower layers may also come from overlying SCMLs, as 76% of SCM-HCLs displayed higher CHL concentrations at the base of the pycnocline than that of segments further below.", "Considering physical processes, removal of CHL from a pre-existing SCML can result in high CHL concentration at the base of the pycnocline (Sharples et al., 2001).", "If the elevated CHL levels in the pycnocline are eroded by tidal currents, periodic downward pulses of biomass should be resolved in the time series.", "This hypothesis has already been corroborated in simulations of frontal systems in which periodic mixing/stratifying processes and the consequent biomass distribution were shown (Schrum et al., 2006; Sharples, 2008).", "When the turbulent mixing energy in the lower layer reaches the pycnocline, the high gradient of CHL at the base of a SCML can be smoothed or the whole SCM structure can be eroded.", "A SCM profile can be transformed into an HCL-SCM profile or even an HCL profile when the subsurface layer structure is eroded and disappeared.", "This assumption was further supported by comparable vertically integrated CHL concentration in the water segment including pycnocline and lower mixed layer in different CHL vertical distribution types (SCM-HCL, HCL, SCM), under conditions that these different CHL vertical distribution type profiles were detected in adjacent areas (Table B1).", "Notably, tidal mixing processes from the bottom are not always detrimental to the persistence of SCMLs.", "In addition to mixing organic matter from an SCML down to the bottom of the water column, tidal mixing can also inject nutrients into the pycnocline, triggering the production and maintenance of photosynthetic activity there (Sharples et al., 2007; Zhao et al., 2018).", "The vertical mixing rates can also influence the persistence of sinking species in the bottom mixed layer by returning phytoplankton cells to depths where irradiance is high enough to sustain photosynthetic activity (Huisman et al., 2002).", "The light availability is also sensitive to the existence of SCMLs but also to increasing turbidity by mixing.", "In the GB system, most SCMLs are shallower than the euphotic layer depth (Fig. 6b), which also indicates that high CHL below the pycnocline has the chance to stay photosynthetic active.", "This mechanism can further support the formation of HCL and SCM-HCL.", "This is an interpretation besides the vertical mixing mechanism since phytoplankton biomass or CHL are not conservative.", "Light penetration below the pycnoline has higher occurrence in summer when the self-shading is not as strong as in spring (Table 2)", "Limited by the data availability (no nutrient data and no particulate organic carbon data), we do not have the chance to further investigate biological factors in the vertical CHL distribution.", "Moreover, the measured CHL concentrations can not be converted directly to biomass and quantitative estimates are hardly possible, since the CHL:C ratio is variable among species and sensitive to the environment and season, particularly in coastal areas (Alvarez-Fernandez and Riegman, 2014).", "Given adequate nutrients and low irradiance levels, which meet the lower threshold for sustaining photosynthetic activity, phytoplankton cells tend to use nutrients to generate more pigments to harvest more light energy instead of building biomass (Geider et al., 1997; Macintyre et al., 2000).", "In reality, the measured CHL-rich patches may consist of multiple species with different light and nutrient requirements.", "More nutrient-efficient competitors should dominate the upper part of the SCML, whereas superior light competitors should mainly inhabit the lower part of the SCML (Huisman et al., 2004).", "Oscillations in physical mixing, which modify the dispersion rates of phytoplankton and the upward flux of nutrients, could further promote the phytoplankton diversity (Huisman et al., 2006).", "Implementing a variable allocation of internal resources, simulations suggest that the vertical CHL distribution in the GB critically depends on the cellular storage of nutrients and pigmentary materials (Kerimoglu et al., 2017).", "By performing an in-depth analysis of vertical CHL profiles in the inner German Bight (GB), we studied the heterogeneity in vertical CHL distribution using measured high-resolution vertical transects.", "Our analysis provided the first quantitative description on heterogeneity & homogeneity of vertical CHL distribution in the GB and explores potential mechanisms.", "More than half of profiles show vertical homogeneous distribution of CHL; extrapolating this to the full range of weather conditions, this proportion would further increase considering that the averaged hydrodynamic background is more turbulent than in our observations which mainly encountered calm whether conditions.", "However, heterogeneous vertical distribution of CHL, especially in stratified season, merits more attention.", "After the onset of stratification in late spring, higher CHL in the bottom mixed layer than that in the surface layer has been observed.", "Several mechanisms may lead to this phenomenon, such as diatoms’ sinking, water mass advection and phyto-acclimation.", "Due to the shallowness of the GB, in more than half of the profiles with high CHL concentration below the pycnocline, the euphotic layer depth is deeper than the lower limit of the pycnocline, which indicates that phytoplankton cells in the lower layer have the chance to access enough irradiance to keep photosynthesis active.", "However, the frequently observed high bottom concentrations can hardly be explained by local growth; resuspension was identified as an important candidate to explain this phenomen.", "Our study revealed that the vertical distribution of CHL in a shallow and tidally energetic system can be attribute to both physical processes (i.e. resuspension and turbulent mixing) and biological processes (i.e. growth and sinking of phytoplankton cells).", "The large fraction of near-bottom high CHL regimes in the stratified season indicates the importance of the benthic-pelagic coupling in tidally energetic shallow areas, such as the German Bight and points to the importance to consider this adequately in theory and modelling studies in tidally influenced shallow seas, which has implications for estimates of carbon flux (Bauer et al., 2013) and nutrients cycling (Beckmann and Hense, 2017).", "In such a shallow area where small proportion of organic matters will be mineralized in the water column (Lutz et al., 2002; Suess, 1980), the interface of water column and sediments acts as buffer to release nutrients slowly long after the most productive season (Provoost et al., 2013).", "Particularly in the southern North Sea, the resuspension process (Thompson et al., 2011), the activity of macrobenthos and bacteria (Franco et al., 2010) further complicate the benthic-pelagic coupling (Zhang and Wirtz, 2017).", "Our study and high resolution observations like those presented here, can help to assess model tools and identify structural limitations.", "For example, a validation of some new ecosystem models using vertical transects of CHL shows that even ecosystem models specifically developed for shallow seas, which reproduce well SCM type profiles might have difficulties to do so for the HCL type (Baschek et al., 2017; Kerimoglu et al., 2017).", "This points to the need to improve process descriptions and parametrizations in modelling tools for coastal areas.", "Moreover, since seasonality modulates the vertical heterogeneity of CHL distribution, our study suggests refined in-situ monitoring, with more vertical sampling required in deeper areas during the stratified season (Fernand et al., 2013).", "Besides, our study points also to the need to develop new approaches to conclude on productivity from remote sensing for stratified shallow shelf sea, since subsurface CHL maxima in those regions may provide a reasonable source of error for remote sensing based estimates (Stramska and Stramski, 2005; Yacobi, 2006).", "We drew conclusions in this study mainly based on statistical analysis.", "To analyze the vertical distribution of CHL in a simultaneous bio-physical way, coinciding measurements of vertical CHL profile, organic matter and physical settings are necessary (Ediger et al., 2005; Liu et al., 2017; Sharples et al., 2007).", "For example, adding observations of particulate organic carbon and CHL data sampled by HPLC method in future studies will provide reliable data sets to investigate physiological contributions to the CHL profiles and evaluate the exchange of organic matter between the upper layer and the bottom layer (McTigue et al., 2015; Smith et al., 2012)." ]	[ "The vertical distribution of chlorophyll (CHL) is mainly influenced by stratification, additionally by biogeochemical cycle.", "In more than half of all measured transects, CHL is vertically well mixed.", "Subsurface chlorophyll maxima layers are detectable in 3.2% of all measured profiles.", "They are photosynthetically active.", "Profiles with high CHL in the bottom mixed layer highlight the benthic-pelagic coupling in the energetic shallow area." ]
Mitotic stopwatch for the blast fungus Magnaporthe oryzae during invasion of rice cells	To study nuclear dynamics of Magnaporthe oryzae, we developed a novel mitotic reporter strain with GFP-NLS (localized in nuclei during interphase but in the cytoplasm during mitosis) and H1-tdTomato (localized in nuclei throughout the cell cycle). Time-lapse confocal microscopy of the reporter strain during host cell invasion provided several new insights into nuclear division and migration in M. oryzae: (i) mitosis lasts about 5 min; (ii) mitosis is semi-closed; (iii) septal pores are closed during mitosis; and (iv) a nucleus exhibits extreme constriction (approximately from 2 μm to 0.5 μm), elongation (over 5 μm), and long migration (over 16 μm). Our observations raise new questions about mechanisms controlling the mitotic dynamics, and the answers to these questions may result in new means to prevent fungal proliferation without negatively affecting the host cell cycle.	[ "BIOC", "IMMU" ]	[ "Cell cycle", "Open mitosis", "Time-lapse confocal microscopy" ]	[ "1311", "2404" ]	[ "The filamentous ascomycete Magnaporthe oryzae causes an economically important blast disease on rice (Oryza sativa).", "On the rice leaf surface, a three-celled conidium (one nucleus per cell) produces a germ tube that differentiates into an appressorium, which mechanically breaches an epidermal rice cell and subsequently produces invasive hyphae (IH) that secrete effectors and proliferate inside host cells (Khang et al., 2010; Khang and Valent, 2010).", "Cell cycle regulation has been shown to be important for the appressorium development and IH proliferation.", "The conidial nucleus, which gives rise to a nucleus that migrates into the appressorium, must enter S-phase for the initial differentiation of the appressorium and subsequently G2/mitosis for further appressorium maturation (Saunders et al., 2010a, 2010b; Veneault-Fourrey et al., 2006).", "Once inside the rice cell, IH cell cycle is under control of a transketolase-mediated metabolic checkpoint (Fernandez et al., 2014).", "IH colonize host tissues by filling the first-invaded cell and then moving into neighbor cells via IH pegs that likely pass through plasmodesmata (Kankanala et al., 2007).", "To facilitate the study of the cell cycle, we developed a fluorescence-based mitotic reporter strain of M. oryzae that expresses GFP-NLS together with H1-tdTomato.", "Using four dimensional (4D) confocal microscopy of the reporter strain, we provide evidence that M. oryzae IH undergo semi-closed mitosis accompanied by septal pore closure and also that the mitotic nucleus exhibits extreme constriction and elongation during migration through the narrow IH peg.", "During live cell imaging of IH expressing mCherry fused to a nuclear localization signal (mCherry-NLS), we serendipitously observed that the nuclear-localized fusion protein dispersed into the cytoplasm and subsequently re-localized into two newly divided nuclei (Fig. 1A).", "A similar pattern has been observed in Aspergillus nidulans, in which fluorescent protein (FP)-NLS localizes in nuclei during the interphase but disperses into the cytoplasm at mitotic entry and remains there until mitotic exit (De Souza et al., 2004; Shen et al., 2014).", "This led us to reason that the dynamic localizations of FP-NLS can be used as a stopwatch to study mitosis in M. oryzae.", "We thus generated an M. oryzae mitotic reporter strain, named CKF1962, expressing GFP-NLS together with histone H1 fused to tdTomato (H1-tdTomato) that remains associated with DNA throughout the cell cycle.", "Using 4D confocal microscopy (images taken every ∼13 s over 17 min; 1.5 μm z-sections over 30 μm) of CKF1962 growing inside a rice cell, we observed three nuclear divisions, two of which were complete rounds as one had already begun to divide at the start of imaging (Video 1 and Fig. 1B).", "Based on the fluorescence patterns, we defined the following sequential stages (Fig. 1C): (1) co-localization of H1-tdTomato and GFP-NLS in pre-mitotic nuclei, (2) initial dispersion of GFP-NLS into the cytoplasm, (3) separation of the tdTomato-tagged chromosomes, (4) initial re-localization of GFP-NLS into mitotic nuclei, and (5) complete co-localization of H1-tdTomato and GFP-NLS in the two divided nuclei.", "Given that beginning of dispersion or re-localization of FP-NLS marks mitotic entry or exit, respectively, the first and last stages (1 and 5) represent late G2 and early G1, respectively, and the three middle stages (2–4) represent mitosis (Fig. 1C and D).", "Based on the three nuclear divisions (Video 1; Fig. 1B and C), we estimated the duration of mitosis to be approximately 5 min, which was consistent with another observation of three nuclear divisions at a different infection site (Fig. 1D) and is comparable to that in A. nidulans (Bergen and Morris, 1983).", "Time-lapse movie of CKF1962 IH growing within the first invaded rice cell at 31 hpi.", "CKF1962 expresses GFP-NLS (green) and H1-tdTomato fusion proteins (red).", "Movie consists of 79 images taken every ∼13 s over a duration of 17 min 38 s. Original images contained 20 z-sections (covering 30 μm).", "Increasing evidence suggests that various forms of open mitosis occur in many fungi that were previously considered to undergo completely closed mitosis (De Souza and Osmani, 2007).", "For example, in the basidiomycete Ustilago maydis, as the mitotic spindle forms in the daughter bud, the nuclear envelope (NE) breaks down locally with most of the NE remaining in the mother cell (Straube et al., 2005).", "During mitosis, A. nidulans undergoes partial disassembly of nuclear pore complexes (NPCs) (De Souza et al., 2004).", "Nuclear-localized FP-NLS disperses into the cytoplasm during mitosis when NE permeability increases due to partial NPC disassembly (De Souza et al., 2004).", "Similarly to A. nidulans, our FP-NLS data (Fig. 1A–C) suggests that M. oryzae maintains a partially disassembled NPC during mitosis, resulting in the dispersal of fluorescence into the cytoplasm.", "Consistent with this, cytoplasmically-expressed FP was shown to equilibrate between the cytoplasm and nucleoplasm during mitosis (Bourett et al., 2002).", "Furthermore, DiOC6 staining revealed that the mitotic NE remains intact (Saunders et al., 2010b).", "Taken together, we hypothesize that M. oryzae undergoes a form of semi-closed mitosis where the NE remains intact, but NPCs partially dissociate during mitosis.", "Interestingly, we noticed that when FP-NLS dispersed throughout apical cells during mitosis, it did not diffuse into adjacent cells (Fig. 1A–C), suggesting the mitotic closure of septal pores.", "We also noticed non-synchronized mitosis between the apical cell and its adjacent cell (the first two nuclear divisions in Fig. 1B and Video 1).", "Shen et al. (2014) observed a similar phenomenon in A. nidulans and suggested that opening and closing of septal and nuclear pores are under cell cycle regulation to be out of phase with each other during cell cycle progression.", "This provides an explanation of how the mitotic state of apical cells does not affect the cell cycle in adjacent cells in A. nidulans (Shen et al., 2014).", "Our results suggest that M. oryzae (one nucleus per cell) shares similar mitotic dynamics, namely, semi-closed mitosis and mitotic closure of septal pores, with A. nidulans (multiple nuclei per cell).", "It will be interesting to continue to investigate how such dynamics are regulated in these and other fungi.", "M. oryzae IH undergo an extreme constriction when spreading into adjacent host cells (Kankanala et al., 2007).", "This raises an intriguing question of how the nucleus (∼2 μm diameter in our measurement) migrates through the constricted IH peg (∼0.5 μm diameter reported in Kankanala et al., 2007) (Fig. 1E).", "To answer this question, we documented the nuclear dynamics during IH cell-to-cell movement by utilizing our mitotic stopwatch strain CKF1962.", "This strain allows us to predict the timing of the nuclear division and subsequent migration.", "That is, the separation of the tdTomato-tagged chromosomes occurs within approximately 3 min from the initial dispersal of nuclear-localized GFP-NLS into the cytoplasm (Fig. 1B–D).", "Thus, we first identified an IH cell that had grown into the adjacent host cell and exhibited dispersed GFP-NLS without a tdTomato-tagged nucleus, and then we initiated high temporal and spatial resolution 4D confocal imaging (images taken every ∼20 s; 1.5 μm z-sections over 13.5 μm; Fig. 1F and Video 2).", "Consistent with the prediction, within 3 min we observed the separation of the tdTomato-tagged chromosomes in the IH cell residing in the initially invaded cell.", "One nucleus then squeezed through the constricted region of the IH peg and stretched to over 5 μm in length.", "Subsequently, the elongated nucleus began to round up at the leading edge, becoming spherical as it moved to its final location 16.9 μm from the site of chromosome separation.", "To our knowledge, this is the first report of extreme stretch and long migration of a fungal nucleus during mitosis.", "It is an exciting possibility that nuclear dynamics studies with additional FP reporters, such as for cytoskeleton/motor proteins and NE/NPCs, will provide further insight into mechanisms of nuclear migration and plasticity as well as infection-related development in M. oryzae.", "Time-lapse movie of CKF1962 IH growing in a neighboring rice cell at 39 hpi and undergoing mitosis.", "Movie consists of 17 images taken every ∼20 s over a duration of 5 min 32 s. Original images contained 9 z-sections (covering 13.5 μm).", "In summary, we provide the first detailed analysis of nuclear division and migration in M. oryzae during host invasion and also describe a FP-based reporter system that can be used to study mitosis and nuclear dynamics in other fungi.", "Our studies raise new mechanistic questions concerning the mitotic dynamics, and the answers to these questions may provide new targets to prevent fungal proliferation without negatively affecting the host cell cycle.", "M. oryzae wild-type strain O-137 was transformed with binary vector pBV1078 to generate transformant CKF1468 or sequentially with two binary vectors pCK1287 and pCK1288 to generate transformant CKF1962 using Agrobacterium-mediated transformation (Khang et al., 2005).", "pBV1078 was produced by cloning of NLS (three tandem repeats of the nuclear localization signal from simian virus large T-antigen) at the C terminus of mCherry under the BAS4 promoter in binary vector pBHt2 (hygromycin selection marker; Mullins et al., 2001).", "pCK1287 was produced by cloning of hH1 (histone H1 from Neurospora crassa) at the N terminus of tdTomato under control of the constitutive promoter from the M. oryzae ribosomal protein 27 gene (RP27) in binary vector pBHt2.", "pCK1288 was produced by cloning of NLS at the C terminus of 3XEGFP under the RP27 promoter in binary vector pBGt (G418 selection marker; Kim et al., 2011).", "Rice strain YT16 was grown and inoculated as previously described (Jones et al., 2016).", "Confocal microscopy was performed on a Zeiss Axio Imager M1 microscope equipped with a Zeiss LSM 510 META system using Plan-Apochromat 20×/0.8 NA and Plan-Neofluor 40×/1.3 NA (oil) objectives.", "Excitation/emission wavelengths were 488 nm/505–530 nm (GFP), and 543 nm/560–615 nm (tdTomato).", "Images were acquired using LSM 510 software (Version 3.2).", "Images were analyzed and processed using a combination of the Zen software, Adobe Photoshop, and ImageJ (http://imagej.nih.gov/ij/)." ]	[ "Development of a fluorescence-based mitotic reporter strain of M. oryzae that expresses GFP-NLS together with H1-tdTomato.", "M. oryzae is hypothesized to undergo semi-closed mitosis accompanied by closure of septal pores.", "A nucleus undergoes extreme constriction and elongation during migration through the narrow invasive hyphal peg." ]
Wallerian Degeneration Is Executed by an NMN-SARM1-Dependent Late Ca2+ Influx but Only Modestly Influenced by Mitochondria	Axon injury leads to rapid depletion of NAD-biosynthetic enzyme NMNAT2 and high levels of its substrate, NMN. We proposed a key role for NMN in Wallerian degeneration but downstream events and their relationship to other mediators remain unclear. Here, we show, in vitro and in vivo, that axotomy leads to a late increase in intra-axonal Ca2+, abolished by pharmacological or genetic reduction of NMN levels. NMN requires the pro-degenerative protein SARM1 to stimulate Ca2+ influx and axon degeneration. While inhibition of NMN synthesis and SARM1 deletion block Ca2+ rise and preserve axonal integrity, they fail to prevent early mitochondrial dynamic changes. Furthermore, depolarizing mitochondria does not alter the rate of Wallerian degeneration. These data reveal that NMN and SARM1 act in a common pathway culminating in intra-axonal Ca2+ increase and fragmentation and dissociate mitochondrial dysfunctions from this pathway, elucidating which steps may be most effective as targets for therapy.	[ "BIOC" ]	[]	[ "1300" ]	[ "Axon degeneration is an early event contributing to symptoms in a range of age-related neurodegenerative disorders, including Parkinson's disease, Huntington's disease, Alzheimer's disease, motor neuron disease, and multiple sclerosis.", "Axonal damage also underpins peripheral neuropathies and is a limiting side effect of cancer chemotherapeutic agents (Conforti et al., 2014).", "Maintaining axon structure and function is therefore a promising therapeutic goal for these disorders.", "Two genetic modifications can delay by 10-fold axon degeneration after an acute injury, a process known as Wallerian degeneration (Waller, 1850), highlighting the existence of a regulated and active axon death pathway.", "First, expression of the neuroprotective factor Wallerian Degeneration Slow (WLDs), a chimeric protein which fuses the nuclear isoform of the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) with an N-terminal sequence that relocates this enzyme from the nucleus to the cytoplasm and axons (Avery et al., 2009; Conforti et al., 2000, 2009; Mack et al., 2001).", "WLDs is effective in mice, rats, Drosophila, zebrafish and in primary human neuronal culture (Adalbert et al., 2005; Kitay et al., 2013; Lunn et al., 1989; MacDonald et al., 2006; Martin et al., 2010), indicating strong evolutionary conservation of the axon degeneration pathway with which it interferes.", "WLDs also ameliorates axon pathology and symptoms in some disease models (Conforti et al., 2014), highlighting shared mechanisms and translational potential.", "Remarkably, loss of the Toll-like receptor adaptor protein Sterile alpha and TIR motif containing 1 (SARM1) has the same robust protective efficacy of WLDs.", "Similar to WLDs, the protective effect of SARM1 deletion is evolutionarily conserved in mice and Drosophila (Osterloh et al., 2012).", "It has been proposed that WLDs delays axon degeneration by substituting for the endogenous but short-lived axonal NMNAT isoform NMNAT2, whose enzyme activity is required for axon growth and maintenance.", "NMNAT2 is synthesized in the cell bodies, actively transported down the axons, and rapidly degraded after injury or block of axonal transport.", "WLDs, which retains the same enzymatic activity than NMNAT2 but is much more stable, prolongs axon integrity (Gilley et al., 2013; Gilley and Coleman, 2010; Milde et al., 2013).", "Our previous data support a model in which WLDs/NMNAT activity maintains axon integrity by preventing the accumulation of its substrate, the pro-degenerative NAD precursor nicotinamide mononucleotide (NMN).", "NMN increases after injury likely as a consequence of NMNAT2 depletion and its pharmacological or genetic inhibition strongly delays Wallerian degeneration (Di Stefano et al., 2014).", "The robustness of axon protection phenotype conferred by SARM1 deletion resembles that obtained by scavenging of NMN.", "Crucially, SARM1 depletion blocks NMNAT2 loss-induced axon degeneration but does not reduce NMN, indicating that SARM1 influences the same pathway of axon death downstream of NMN, or a convergent pathway, while other weaker modulators may join onto this pathway (Conforti et al., 2014; Gilley et al., 2015; Yang et al., 2015).", "NMN increases early after injury (Di Stefano et al., 2014), leaving ample scope for downstream events, which could include an effect on intra-axonal Ca2+.", "A key role for Ca2+ in the late steps of degeneration, likely acting by activating Ca2+-dependent proteases, has long been recognized (George et al., 1995; Wang et al., 2012).", "Ca2+ could reach the axons from the extracellular environment or originate from intra-axonal organelles (Barrientos et al., 2011; Villegas et al., 2014).", "Recent studies suggest instead that Ca2+ rise may occur early after injury (Avery et al., 2012), possibly acting upstream of SARM1 (Summers et al., 2014).", "Mitochondria have been reported as central executors in Wallerian degeneration through mitochondrial permeability transition pore (mPTP) opening, which could result from a crosstalk with the endoplasmic reticulum (ER) (Barrientos et al., 2011; Villegas et al., 2014).", "Augmented mitochondrial motility in WLDs could increase Ca2+ buffering capability and contribute to protection (Avery et al., 2012).", "However, Wallerian degeneration is only modestly delayed in axons devoid of mitochondria in some models and mitochondria are dispensable for WLDs protection (Kitay et al., 2013); therefore, the role of mitochondria in Wallerian degeneration and the WLDs mechanism as well as the relationships among NMN increase, SARM1, Ca2+, and mitochondrial dynamics remain unresolved.", "Here we report that intra-axonal Ca2+ elevation after axotomy lies downstream of NMN and immediately precedes axon fragmentation.", "We found that SARM1 is required for NMN to initiate axon degeneration and that Ca2+ rise is a common downstream step.", "Both NMN rise inhibition and SARM1 deletion fail to prevent changes in mitochondrial dynamics occurring after axotomy, despite delaying degeneration.", "We also found that the rate of Wallerian degeneration is not affected by mitochondrial depolarization.", "By clarifying the temporal relationship between NMN rise, SARM1, Ca2+, and mitochondrial dynamics, our results identify critical steps of Wallerian degeneration where pharmacological intervention could be most successful.", "To address events downstream of NAD-precursor NMN (Figure S1A), which we proposed to induce axon degeneration after injury (Di Stefano et al., 2014), we first tested whether NMN accumulation evokes changes in intra-axonal Ca2+ concentration.", "This is important since WLDs, which blocks NMN accumulation, has been suggested to protect degenerating axons by inhibiting Ca2+ increase or enhancing its buffering (Adalbert et al., 2012; Avery et al., 2012).", "To analyze the temporal correlation between Ca2+ rise and axonal fragmentation, we microinjected superior cervical ganglia (SCG) neurons with plasmids encoding the Ca2+ indicator GCaMP5 and the fluorescent marker DsRed2.", "One day after microinjection, the axons were separated from the cell bodies and changes in GCaMP5 fluorescence signal in distal stumps were measured and normalized to their corresponding uncut controls (Figure 1; Figures S1B and S1C).", "Consistent with previous studies (Adalbert et al., 2012), we found a marked increase in Ca2+ levels in distal injured axons starting 2–3 hr after axotomy (Figures 1A and 1B; Movie S1).", "This Ca2+ rise just preceded the appearance of morphological damage and axon integrity was lost 10–25 min after Ca2+ started to increase (Figures 1A and 1C).", "To test whether this Ca2+ rise was induced by NMN accumulation, we treated axons with FK866, a potent and specific inhibitor of NMN-synthesizing enzyme NAMPT (Figure S1A).", "FK866 completely abolished the Ca2+ rise and, consistent with our previous report (Di Stefano et al., 2014), preserved axonal integrity for the entire duration of the experiment (Figures 1A–1C; Movie S1).", "Co-administration of NMN fully reverted the effects of FK866, restoring Ca2+ rise and axonal fragmentation (Figures 1A–1C; Movie S1).", "We previously showed that scavenging NMN by expressing the bacterial enzyme NMN deamidase (Figure S1A; Galeazzi et al., 2011) remarkably delays axon degeneration after injury (Di Stefano et al., 2014).", "We then asked whether this genetic intervention to reduce NMN could also block intra-axonal Ca2+ rise and found that expressing NMN deamidase completely abolished this increase and preserved axonal integrity for the entire duration of the experiment (Figures 1D–1F; Movie S1).", "Thus, NMN increase after injury evokes a late intra-axonal Ca2+ rise, which can be blocked by NAMPT inhibitor FK866 or by genetically scavenging NMN.", "Because injured Sarm1−/− nerves have high NMN levels but do not degenerate (Gilley et al., 2015), it is likely that SARM1 is required for NMN to induce axon death.", "We tested this possibility by adding NMN to transected Sarm1−/− axons and found that they remained intact for at least 4 days, even in the presence of high concentrations of exogenous NMN (Figures 2A and 2B), which we even tested up to 5 mM (data not shown).", "In contrast, FK866-protected wild-type neurites degenerate within 3 hr when exposed to concentrations of NMN 200-fold lower (Di Stefano et al., 2014).", "Thus, NMN requires SARM1 to induce axon degeneration.", "We then asked whether SARM1 is also required for the increase in intra-axonal Ca2+ after cut.", "This is important in view of a recent report that SARM1 acts downstream of Ca2+ (Summers et al., 2014).", "However, in our experimental conditions we did not find any increase after axotomy in Ca2+ signals in the distal stumps of Sarm1−/− axons (Figures 2C and 2D; Movie S2), which remained similar to the uninjured neurites (Figures S1D and S1E).", "In parallel, axonal integrity was preserved for the entire duration of the experiment (Figures 2C and 2E).", "NMN administration failed to induce Ca2+ rise in Sarm1−/− injured axons (Figures 2C–2E; Movie S2).", "Thus, SARM1 is required for NMN to induce Wallerian degeneration and Ca2+ increase represents a common downstream step.", "An increase in intra-axonal Ca2+ in proximity to the cut rapidly follows the transection injury, and it is most likely due to an influx from the cut site (Adalbert et al., 2012).", "This immediate and localized Ca2+ rise leads to rapid degeneration of a short axonal segment adjacent to the site of cut, an event known as acute axon degeneration (Kerschensteiner et al., 2005).", "We recorded an early rise in Ca2+ levels in Sarm1−/− axons near the cut site, followed by degeneration of short axonal segments.", "This was also observed in wild-type controls untreated or treated with FK866 or with FK866+NMN and in NMN deamidase-expressing axons (Figure S2) and it is consistent with direct influx of Ca2+ from the injury site.", "This suggests that Sarm1−/− and NMN deamidase axons, as well as axons treated with FK866, retain the capability to degenerate in response to Ca2+, but that this early, proximal Ca2+ rise is distinct from the delayed increase in the distal stump that occurs just before frank fragmentation (Figure 1).", "To assess the relationship between NMN and intra-axonal Ca2+ in vivo, we used zebrafish larvae 48–54 hours post-fertilization (hpf), transiently expressing DsRed along with the calcium sensor GCaMP-HS in trigeminal and Rohon–Beard somatosensory neurons.", "These were treated with FK866 for 6 hours prior to two-photon laser axotomy (Figures 3A and 3B).", "As we previously reported, FK866 strongly delays axon fragmentation in this model organism (Figures 3Bm–3Bo and 3C; (Di Stefano et al., 2014)).", "Consistent with our in vitro observations, we detected a marked Ca2+ increase within axons distal to the injury site (Figures 3Be–3Bh and 3D).", "The first signs of axon damage appeared as soon as 4 min after Ca2+ rise (Figures 3Ba–3Bd and 3E; Movie S3).", "FK866 remarkably delayed this increase in intra-axonal Ca2+ (Figures 3Bp, 3Br, 3D, and 3E; Movie S4) and the subsequent axon fragmentation (Figures 3Bm–3Bo, 3C, and 3E).", "Thus, NMN synthesis inhibitor FK866 blocks the increase in Ca2+ that immediately precedes axonal fragmentation in vivo in a vertebrate model organism.", "Because both extracellular influx of Ca2+ and its release from intracellular stores have been implicated in Wallerian degeneration (Wang et al., 2012), we asked what is the primary source of the Ca2+ rise induced by NMN.", "SCG explant axons were cut and immediately treated with FK866.", "In order to exclude intracellular conversion of NMN to NAD mediated by any residual axonal NMNAT2, we added NMN to FK866-protected neurites 12 hr after cutting (Figure 4A), a time when little or no NMNAT2 remains (Gilley and Coleman, 2010).", "In these conditions, NMN induces complete degeneration of FK866-protected axons within 3 hr from its addition (Figures 4Be–4Bl and 4C; (Di Stefano et al., 2014)).", "We used EGTA to chelate extracellular Ca2+ and saw a remarkable delay of axon degeneration after NMN addition (Figures 4Bm–4Bp and 4C).", "Similar results were obtained using L-type Ca2+ channels blockers verapamil (Figures 4Bq–4Bt and 4C) and nifedipine (Figures S3A (i–l) and S3B).", "In addition, also KB-R7943, an inhibitor of the reverse Na+/Ca2+ exchanger, significantly delayed NMN-induced degeneration, although its effect was weaker (Figures 4Bu–4Bx and 4C).", "EGTA was protective also in dorsal root ganglion (DRG) cultures (Figure S4).", "A release of Ca2+ from axonal ER, leading to mPTP opening and release of intra-mitochondrial Ca2+ has been observed in the execution phase of Wallerian degeneration (Villegas et al., 2014).", "Therefore, we tested whether this sequence of events is induced by NMN.", "We observed that ryanodine receptor antagonist ryanodine significantly delayed NMN-induced degeneration, although its effect was weaker than that obtained with EGTA and the other Ca2+-channel blockers used (Figures S3A (m–p) and S3B; Figures 4B and 4C).", "We also found that cyclosporine A and ruthenium red, which block mPTP opening (Barrientos et al., 2011), more modestly influence the rate of NMN-induced axon degeneration in our model (Figures S3C (i–p) and S3D).", "These results indicate that the main source of intra-axonal Ca2+ stimulated by NMN is the extracellular environment, with a weaker contribution from intracellular organelles.", "To investigate the temporal relationship between Ca2+ influx and its internal release and to detect any Ca2+ increase in areas surrounding mitochondria, Ca2+ buffering organelles which have been proposed to release Ca2+ during Wallerian degeneration (Avery et al., 2012; Barrientos et al., 2011; Villegas et al., 2014), we microinjected SCG neurons with GCaMP5 and the mitochondrial marker TagRFP-mito.", "We then cut the axons and immediately added FK866 and NMN.", "As expected, NMN administration induced a diffuse intra-axonal Ca2+ increase in FK866-treated axons, rapidly followed by the formation of Ca2+-rich axonal swellings which started to appear as early as 10 min after Ca2+ rise (Figures 4Da, 4Db, 4Dd, and 4E; Figure S5A; see also Figure 1).", "Eighty percent of these swellings contained mitochondria (Figures 4Db and 4Dd)).", "The relative Ca2+ fluorescence intensity inside axonal swellings was significantly higher compared to the signals measured inside adjacent axonal segments (Figures S5A and S5B), suggesting an accumulation of Ca2+ at the sites of morphological damage.", "EGTA addition drastically reduced NMN-induced Ca2+ rise (Figures 4De and 4E) and blocked axon degeneration (Figures 4Bm–4Bp and 4C).", "However, we still observed a weak increase in Ca2+ levels confined in some axonal areas where mitochondria were present (Figures 4De and 4E).", "This localized Ca2+ rise was considerably weaker than that observed following extracellular Ca2+ influx (Figure 4E); small swellings also appeared within the same confined axonal regions; nevertheless, frank degeneration of the distal axon stump was delayed for many hours (Figures 4Dg and 4Dh).", "Together, these results indicate that NMN stimulates a late influx of Ca2+ from the extracellular environment.", "A more modest Ca2+ release from intracellular stores also occurs, but this is not sufficient to trigger fragmentation.", "Changes in mitochondrial function and structure have been previously shown to occur after injury (Sievers et al., 2003; Villegas et al., 2014); however, it is still unclear whether mitochondria are major players, modest regulators, or simply markers of degeneration.", "As we previously reported early changes in mitochondrial membrane potential, an index of functionally active mitochondria, in injured neurites of young DRG cultures (Sievers et al., 2003), we asked whether these changes also occur in our SCG model of NMN-induced degeneration.", "Surprisingly, using the mitochondrial membrane potential indicator tetramethylrhodamine methyl ester perchlorate (TMRM), we found that mitochondrial membrane potential remained stable for at least 4 hr after axotomy, a time point where axons still appear intact in SCG explant cultures.", "Similar results were observed after FK866 treatment and when NMN was re-added to FK866-treated axons (Figures 5A and 5B).", "A visible decrease in TMRM signal only appeared 6 hr after injury, when clear signs of degeneration were already present.", "FK866 abolished the drop in TMRM signal visible at this time point, which was then re-established by NMN re-addition (Figures S5C and S5D).", "We also found that SARM1 deletion delayed the drop in TMRM signal after injury, which remained stable up to 32 hr after axotomy (Figures S5E and S5F).", "Mitochondrial morphological changes have been reported to be among the earliest detectable alterations during Wallerian degeneration (Vial, 1958; Webster, 1962).", "We thus tested whether NMN accumulation after injury could influence mitochondrial morphology.", "No significant change was observed among the different treatment groups in the size distribution of mitochondria immediately after cut (Figures 5C and 5D).", "We found a small but significant increase in the number of shorter mitochondria 4 hr after injury only in the sample treated both with FK866 and NMN.", "However, no change was detected in the untreated sample, or the sample treated with FK866 alone (Figures 5C and 5D).", "Conversely, we observed a dramatic reduction in mitochondrial length in Sarm1−/− axons 48 hr after injury (Figures 5E and 5F), long before frank fragmentation occurs (Figures 2A and 2B).", "Interestingly, we also found a significant difference (K-S p = 0.0005) in the size distribution of mitochondria between Sarm1−/− and wild-type axons immediately after cut (compare Figures 5D and 5F), perhaps reflecting a direct influence of SARM1 on the maintenance of normal mitochondrial shape.", "Thus, in this experimental system, a decrease in mitochondrial membrane potential marks the late stages of degeneration.", "Furthermore, while mitochondrial size is subject to modifications following an injury, these changes do not appear to correlate with the extent of degeneration.", "WLDs was suggested to confer axon protection after injury by maintaining mitochondrial axonal transport, although this is a modest and short-lived effect in some studies (Avery et al., 2012; O’Donnell et al., 2013).", "Therefore, we next investigated whether a reduction in mitochondrial motility could underline NMN-induced axon degeneration.", "We tested whether blocking NMN synthesis with FK866 had an impact on mitochondrial dynamics after injury.", "To this purpose, we labeled mitochondria with the mitochondrial marker mitotracker red (Figure 6A).", "Consistent with previous reports (Avery et al., 2012; O’Donnell et al., 2013), we found that axotomy caused a marked reduction in the percentage of motile mitochondria 4 hr after cut in SCG explant cultures (Figure 6B; Movie S5).", "We also observed a decrease in the number of motile mitochondria in axons treated with FK866, similar to that in untreated cut axons (Figure 6B; Movie S5), despite FK866-treated axons maintaining their integrity during this time (Figure 6C), and for at least another 30 hr (Di Stefano et al., 2014).", "A similar reduction in mitochondrial motility was also observed in axons treated with both FK866 and NMN (Figure 6B; Movie S5).", "Interestingly, FK866-treated axons showed an almost complete arrest of mitochondrial transport 24 hr after axotomy, a time point when they still have overtly normal morphology (Movie S6).", "We also analyzed mitochondrial transport in Sarm1−/− SCG neurites, where protection against Wallerian degeneration is even stronger than that achieved with FK866.", "Remarkably, a substantial reduction in mitochondrial motility was also detected as early as 4 hr after injury, when Sarm1−/− axons are intact (Figures 6D and 6E; Movie S7); mitochondrial transport completely stopped 48 hr after injury (Movie S8), long before axons fragment (Figures 2A and 2B).", "Thus, axon protection by FK866 and SARM1 deletion is not mediated by an effect on mitochondrial dynamics.", "Next, we asked whether direct damage to mitochondria could affect the rate of Wallerian degeneration.", "This is possible because damaged mitochondria could release mediators of cell and axon death that could cause the axon demise after injury (Court and Coleman, 2012).", "Therefore, we treated axons with the protonophore Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which causes an uncoupling of the proton gradient and disruption of mitochondrial membrane potential (Ly et al., 2003).", "When exposed to 50 μM CCCP, mitochondria in axons immediately lost membrane potential, as shown by a remarkable, instantaneous decrease in TMRM fluorescent signal (Figure 7A).", "Despite this, frank axonal fragmentation only occurred 32–48 hr after CCCP addition (Figures 7A and 7B).", "This is a much slower time course than that of Wallerian degeneration, which in our experimental system is completed within 8 hr.", "This remarkable time difference enabled us to test whether mitochondrial depolarization affects the rate of Wallerian degeneration.", "We added CCCP to SCG explants immediately prior to axotomy (Figure 7C) and then followed the time course of axon degeneration.", "Despite the much earlier, almost instantaneous loss of TMRM signal, cut axons treated with CCCP underwent Wallerian degeneration at the same rate as cut, vehicle-treated axons (Figures 7Da and 7E).", "Furthermore, CCCP treatment did not affect axon protection conferred by FK866 or reversion by NMN administration after axotomy (Figures 7Db, 7Dc, 7F, and 7G).", "Similarly, the rate of axon degeneration in transected Sarm1−/− axons treated with CCCP did not differ from that of the untreated axons, despite the rapid depolarization of Sarm1−/− mitochondria upon CCCP addiction, as expected (Figures S6A and S6B).", "Together, these results dissociate the loss of mitochondrial function from the onset of Wallerian degeneration.", "Our proposed model of Wallerian degeneration predicts that axonal integrity is lost as a consequence of the accumulation of the pro-degenerative molecule NMN (Di Stefano et al., 2014), whose levels in axons are normally limited by the NAD-biosynthetic enzyme NMNAT2 (Gilley and Coleman, 2010).", "Here, using pharmacological and genetic experimental approaches in in vitro mammalian primary neurons and an in vivo vertebrate model organism, we show that NMN initiates a Ca2+-mediated execution program of axon degeneration after injury, which requires the presence of the recently identified pro-axon death protein SARM1.", "Furthermore, inhibition of NMN synthesis and SARM1 deletion both fail to prevent early alterations in mitochondrial motility after cut and disrupting mitochondrial membrane potential does not affect the rate of injury-induced axon degeneration; thus, a change in mitochondrial homeostasis appears to be a marker of Wallerian degeneration rather than a causative event.", "Although our NMN-induced axon degeneration model is supported by pharmacological and genetic studies in vitro and in vivo (see Figures 1 and 3 and Di Stefano et al., 2014), some recent studies find instead a beneficial effect of NMN, explained by its intra-axonal conversion to NAD (Wang et al., 2015).", "The difference in our experimental settings (mouse intact explants instead of rat dissociated neurons) precludes a direct comparison.", "However, in our hands, NMN shows a rapid pro-degenerative effect when added up to 12 hr (SCG explants) or 24 hr (DRG explants) post-axotomy (see Figure 4, Figure S4, and Di Stefano et al., 2014); at these time points, NMN conversion to NAD is prevented by the complete degradation of NMNAT2, whose activity could remain in the experiments performed by (Wang et al., 2015).", "Careful comparison studies will need to address these discrepancies to reconcile these apparent contradictions.", "Intra-axonal Ca2+ increase after axotomy follows a biphasic pattern, showing a first rise soon after and near the site of injury followed by second, delayed one along the whole axon length; however its temporal relationship with axonal fragmentation is still debated (Adalbert et al., 2012; Avery et al., 2012; Gerdts et al., 2011; Villegas et al., 2014; Yang et al., 2013).", "Our finding that NMN induces a late intra-axonal Ca2+ increase after injury, which is rapidly followed by axonal degeneration, are consistent with a model in which the late Ca2+ rise is the central and causative event marking the execution phase of degeneration on which several other mediators may converge.", "Consistent with a recent report that SARM1 is required for degeneration downstream of NMNAT2 loss (Gilley et al., 2015), our data suggest that Ca2+ lies downstream of both NMN and SARM1 in a main, unique axon death pathway (Figure S6C; Conforti et al., 2014).", "A recent study proposed that SARM1 acts downstream of intra-axonal Ca2+ increase (Summers et al., 2014).", "The early Ca2+ rise has been suggested as a causative step in Wallerian degeneration (Avery et al., 2012).", "Our data, however, indicate that the early Ca2+ increase in the proximity of the cut site and its associated acute degeneration occur indistinguishably in wild-type rapidly degenerating axons as well in Sarm1−/−, NMN deamidase and FK866-treated axons, all conditions where Wallerian degeneration is dramatically delayed.", "Similarly, WLDs does not prevent the early rise of Ca2+ near the cut site (Adalbert et al., 2012).", "Furthermore, Ca2+ blockers delay degeneration induced by NMN in axons injured as long as 12 hr earlier but maintained intact by FK866.", "Crucially, WLDs-sensitive Wallerian degeneration can be induced without injury in vitro or in vivo by NMNAT2 selective depletion (Gilley and Coleman, 2010; Hicks et al., 2012) and does not require an influx of Ca2+ from the cut site to initiate it.", "All these observations dissociate initial Ca2+ influx from degeneration.", "Two ways of preventing Ca2+ influx from the extracellular environment confer strong protection from NMN-induced axon degeneration: (1) sequestering extracellular Ca2+ with EGTA, and (2) blocking Ca2+ influx via Ca2+ channels.", "While strongly implicating extracellular Ca2+ in the late execution stages of Wallerian degeneration, these observations suggest that the Ca2+ sensitive step could be at a point where morphologically protecting axons may not restore their function.", "Therefore, upstream mediators of the axon death pathway may prove therapeutically more useful targets.", "Extracellular Ca2+ influx may be due to a dysregulation of Ca2+ channels following a depolarization of the axonal membrane, which itself could be a consequence of energy depletion (Mishra et al., 2013; Wang et al., 2005).", "This could be triggered by activation of a MAPK signaling pathway by SARM1 (Yang et al., 2015) or even by a downstream effect on NAD levels (Gerdts et al., 2015).", "Consistent with previous reports (Barrientos et al., 2011; Villegas et al., 2014), our data also indicate a small contribution of intracellular organelles to the total intra-axonal Ca2+ rise after injury, noticeable following EGTA treatment (Figures 4D and 4E).", "Despite being insufficient to trigger fragmentation on its own in the rapid timescale of Wallerian degeneration, this could nevertheless contribute to the degeneration process.", "Our observation that mitochondria localize at the sites of Ca2+ accumulation suggests they could be one of the sources of this weaker Ca2+ increase.", "Consistently, we found a protective effect when interfering with mPTP opening (Figures S3C and S3D), even if more modest to what previously reported ex vivo (Barrientos et al., 2011), where Schwann cells and, more specifically, an effect of the pharmacological treatments on Schwann cell mitochondria, could have a stronger influence on axonal survival.", "Whether mitochondria play a central role in Wallerian degeneration, and whether reported changes in mitochondrial shape, dynamics, and membrane potential (Avery et al., 2012; Sievers et al., 2003; Villegas et al., 2014) are a cause or consequence of degeneration or are general markers of compromised axons remain unclear.", "In our model of NMN-induced axon degeneration, we find that mitochondrial membrane potential drops after cut, but only when morphological damages to axons are already present, and may even be a consequence of Ca2+ influx.", "Crucially, disrupting mitochondrial membrane potential before injury does not alter the rate of degeneration or the effect of FK866 and SARM1 deletion.", "Consistent with our data, Wallerian degeneration occurs also in axons devoid of mitochondria and WLDs protection is independent of axonal mitochondria (Kitay et al., 2013; Rawson et al., 2014).", "The depletion of other axonal proteins after injury may explain the effects on mitochondrial homeostasis.", "For instance, SCG10, which is involved in the regulation of mitochondrial dynamics, is also rapidly lost after injury and its overexpression confers a modest protective phenotype (Shin et al., 2012), raising the possibility that mitochondrial changes may follow the depletion of such a regulator.", "In addition, mitochondrial arrest could also be a response to local Ca2+ changes (Wang and Schwarz, 2009).", "Mitochondria depend on the integrity of a nuclear-mitochondrial communication pathway regulated by NAD metabolism (Fang et al., 2014), and could be affected when this control is lost as consequence of the injury.", "Intriguingly, axonal mitochondria appear shorter and more static in the absence of SARM1.", "While supporting that Wallerian degeneration is independent of mitochondrial shape and motility, these observations raise a series of questions on the role of SARM1 in mitochondria.", "Indeed, SARM1 contains an N-terminal mitochondrial localization signal and is, at least in part, associated with mitochondria (Osterloh et al., 2012).", "SARM1 mitochondrial localization may be dispensable for the execution of Wallerian degeneration; however, it is required for viral-induced cell death (Mukherjee et al., 2013).", "The relevance of mitochondrial homeostasis in SARM1 biological function therefore needs to be further explored.", "In our hands treatment of axons with CCCP to induce mitochondrial depolarization does not affect the rate of Wallerian degeneration, but it does initiate a slower axonal degeneration process in intact axons.", "A previous study showed that CCCP toxicity in uninjured axons was insensitive to WLDs (Ikegami and Koike, 2003), which may appear in contrast to our result.", "However, we used CCCP at a lower concentration and, most importantly, in the context of axotomy where CCCP action could differ.", "Intriguingly, other reports show that mitochondrial dysfunctions activate a pathway of degeneration sensitive to regulators of Wallerian degeneration (Press and Milbrandt, 2008; Summers et al., 2014).", "This mechanism could be different from the axon degeneration induced by depletion of axonal mitochondria (Fang et al., 2012; Rawson et al., 2014), which in contrast is insensitive to NMNAT/WLDs pathway (Fang et al., 2012).", "Furthermore, in C. elegans mitochondrial and transport defects leading to axon degeneration are observed as a consequence of α-tubulin acetyltransferase gene mec-17 loss (Neumann and Hilliard, 2014).", "Explaining the mechanistic links between mitochondrial dysfunction-induced axon degeneration and Wallerian degeneration will be important in view of the causative role of mitochondrial dysfunction in a variety of neurodegenerative disorders characterized by axonal degeneration (Conforti et al., 2014).", "In summary, our data identify crucial steps of an axon death pathway initiated by NMN accumulation.", "We show that NMN requires SARM1 to activate a destruction process where extracellular Ca2+ influx is the main executor (Figure S6C).", "We reveal an additional, minor contribution of mitochondrial Ca2+ release but our data do not support a central role for mitochondria in NMN-induced axon degeneration.", "Because Ca2+ appears to be temporally distant from the point of NMN accumulation, leaving time for signaling events, it is now important to clarify what are these signals and how NMN interacts with SARM1.", "These studies should help identifying effective targets to delay axon degeneration in injury and disease.", "All studies conformed to the institution’s ethical requirements in accordance with the 1986 Animals (Scientific Procedures) Act.", "C57BL/6 or CD1 (referred to as wild-type, Charles River, UK) and Sarm1−/− (kindly provided by Dr. Michael Coleman, the Babraham Institute) mouse SCG explants, were dissected from P0-2 mouse pups and DRG explants were dissected from E16 mouse embryos.", "Explants were cultured for 6–7 days and then axotomized as previously described (Di Stefano et al., 2014) and as detailed in the Supplemental Experimental Procedures.", "Wild-type and Sarm1−/− SCG dissociated neurons were obtained as described (Di Stefano et al., 2014) and as detailed in Supplemental Experimental Procedures.", "Neurites were allowed to extend for 3 days in culture before microinjection and were axotomised the following day using a scalpel.", "We refer to time 0 as the first time point acquired immediately after cut.", "In our experimental system (Di Stefano et al., 2014), neurites originating from wild-type SCG explants degenerate within 8 hr after cut.", "For the purpose of comparison, no morphological changes occur in wild-types within the first 4 hr after cut; clear morphological damages and frank fragmentation occur at 6–8 hr after cut.", "Neurites originating from dissociated cells degenerate within ∼3 hr after cut; this difference could be due to the different time in culture (Buckmaster et al., 1995).", "Dissociated cells were microinjected as described in (Di Stefano et al., 2014) and in the Supplemental Experimental Procedures.", "Wild-type and Sarm1−/− dissociated SCG neurons were cut and then imaged in a controlled environment (37°C and 5% CO2).", "Treatments were applied 30 min before cut or immediately after cut and time-lapse fluorescent images and bright-field images were acquired analyzed and quantified as detailed in the Supplemental Experimental Procedures.", "Zebrafish larvae were grown, transfected, axotomised, and imaged as described (Di Stefano et al., 2014) and as detailed in the Supplemental Experimental Procedures.", "Wild-type SCG neurites were cut and drugs were administered as described in (Di Stefano et al., 2014) and in Supplementary Methods.", "Bright field images were acquired and analyzed as detailed in the Supplemental Experimental Procedures.", "Wild-type and Sarm1−/− SCG explants were pre-incubated with 100nM Mitotracker red CMXRos (Invitrogen) for 15 min at 37°C before axotomy.", "Drug treatment and image acquisition is detailed in the Supplemental Experimental Procedures.", "Quantification of mitochondrial motility was performed using the ImageJ plugin Difference tracker (Andrews et al., 2010).", "Differences in contrast between each movie were minimized by applying automated contrast normalization to all images using ImageJ.", "For mitochondrial membrane potential, wild-type and Sarm1−/− SCG neurons were incubated with the cell-permeant dye TMRM (VWR International) for 30 min at 37°C before axotomy and imaging.", "When used, 100 nM FK866 and 1mM NMN were administered immediately after axotomy.", "For mitochondrial length, wild-type and Sarm1−/− SCG explants were incubated with 100 nM Mitotracker red CMXRos (Invitrogen) for 15 min at 37°C before cutting.", "Drug treatment, image acquisition and image analysis are detailed in the Supplemental Experimental Procedures.", "Uncut and cut wild-type or cut Sarm1−/− SCG neurons were treated with 50 μM CCCP or vehicle (DMSO) just prior to imaging.", "To check that CCCP treatment was triggering full depolarization of axonal mitochondria neurons were incubated for 30 min at 37°C with TMRM.", "Data are expressed as mean ± SEM or SD.", "Statistical analysis was performed using ANOVA, Kolmogorov-Smirnov test, or Student’s t test with p values less than 0.05 being considered significant for any set of data.", "L.C. and A.L. conceived and designed the experiments.", "A.L. conducted most experiments, collected data, and conducted the analyses with the help of L.C. M.D.S. performed some of the in vitro experiments on Sarm1−/− neurons.", "M.G. assisted with the zebrafish model.", "L.C. supervised and coordinated the research.", "A.L. and L.C. co-wrote the manuscript." ]	[ "NMN stimulates an intra-axonal Ca2+ rise shortly preceding axonal fragmentation", "NMN requires SARM1 to induce Ca2+ rise and axon degeneration", "The extracellular environment is the main source of NMN-induced axonal Ca2+ rise", "Mitochondrial dynamic changes are not causative in NMN-induced degeneration" ]
RNA-based adjuvant CV8102 enhances the immunogenicity of a licensed rabies vaccine in a first-in-human trial	Background: We report the first-in-concept human trial of the safety, tolerability and immunogenicity when a novel TLR 7/8/RIG I agonist RNA-based adjuvant, CV8102, was administered alone or mixed with fractional doses of a licensed rabies vaccine (Rabipur®) as model antigen. Methods: The primary objective was to assess the safety and reactogenicity of various dose levels of CV8102 alone or mixed with Rabipur® in healthy 18–40 year-old male volunteers. A secondary objective was to assess the immune-enhancing potential of bedside-mixes of CV8102 with fractional doses of Rabipur® by measuring induction of rabies virus neutralising titres (VNTs). Results: Fifty-six volunteers received 50–100 μg CV8102 alone (n = 11), bedside-mixed CV8102 and Rabipur® (n = 20), or Rabipur® alone (n = 25; control). When given alone or mixed with Rabipur® CV8102 caused mostly Grade 1 or 2 local or systemic reactogenicity, but no related SAEs. As 100 µg CV8102 was associated with marked CRP increases further dose escalation was stopped. Combining 25–50 µg of CV8102 with fractional doses of Rabipur® significantly improved the kinetics of VNT responses; 50 µg CV8102 also improved the magnitude of VNT responses to 1/10 Rabipur® but caused severe but self-limiting influenza-like symptoms in 2 of 14 subjects. Conclusions: Doses of 25 and 50 µg CV8102 appeared safe and with an acceptable reactogenicity profile while significantly enhancing the immunogenicity of fractional doses of rabies vaccine. EudraCT No. 2013-004514-18.	[ "BIOC", "IMMU", "MEDI", "VETE" ]	[ "Adjuvant", "Immunostimulatory", "Prophylactic vaccines", "RNA", "Rabies", "Safety" ]	[ "1313", "2400", "2725", "2739", "3400" ]	[ "Novel highly purified or subunit vaccine candidates have improved safety and tolerability but are often poorly immunogenic [1].", "Adjuvanted formulations increase the immune response to these vaccines, but a number of adjuvants are currently included in licensed vaccines, most of which are vehicle adjuvants including aluminium salts (alum) or oil-in-water emulsions such as MF59 [2].", "Improved understanding of how innate immunity shapes adaptive immune responses has led to the development of novel adjuvants [3].", "In this context, toll-like receptor (TLR) ligands have received extensive evaluation and appear to be important for the induction of optimal Th1 and Th2 immune responses [4,5].", "The TLR4 agonist monophosphoryl lipid A (MPL) is a component of the AS01 adjuvant in RTS.S malaria [6] and recently approved herpes zoster (Shingrix) vaccines, and also in the AS04 adjuvant in the HPV vaccine, Cervarix.", "Another TLR9 agonist adjuvant, CpG 1018, is used in the recently approved hepatitis B vaccine (Heplisav-B™) [7].", "Single-stranded RNA is a ligand for endosomal TLR7/8, induces innate immune responses via TLR7/8 [8,9] and can activate the RLH (RIG like helicase pathway) if it contains 5-triphosphate [10].", "We developed the RNA-based adjuvant CV8102 (RNAdjuvant®, CureVac AG), a TLR 7/8 agonist and RIG I pathway activator [10], to enhance immunogenicity of poorly immunogenic antigens.", "It consists of non-coding, uncapped, polyU repeats-containing single-stranded RNA with a 5-triphosphate modification, complexed with a polymeric carrier, a small arginine-rich disulfide-crosslinked cationic peptide (CR12C) [10,11].", "In preclinical studies CV8102 increased humoral immunity to licensed influenza vaccine in mice and domestic pigs, and to rabies vaccine in mice (Curevac data on file).", "Combining CV8102 with recombinant protein and antigen-derived peptides (T-cell epitopes) induced strong humoral and cellular immune responses [11].", "These preclinical data support CV8102 as an adjuvant candidate for prophylactic and therapeutic vaccines, particularly for conditions where both Th1 and Th2 immune responses are required.", "We report the first-in-human evaluation of the safety and reactogenicity of increasing doses of CV8102 administered alone and mixed with licensed rabies vaccine as model antigen, and enhancement of humoral responses to this viral antigen using the serologic correlate of protection in use at the time of the study [12].", "This phase I, proof-of-concept, first-in-human, open-label trial was conducted between Sept 8th 2014 and Oct 5th 2015 at the phase I unit of CRS Clinical Research Services, Mönchengladbach, Germany according to GCP and Declaration of Helsinki guidelines.", "The protocol was registered with the European Medicine Agency (EudraCT No. 2013-004514-18) and approved by the relevant regulatory authorities and IRBs.", "An external independent Data Safety and Monitoring Board (DSMB) provided oversight.", "All subjects provided written informed consent at enrolment.", "The study was performed in two parts, a dosage escalation study (Part A) with two planned intramuscular injections of 50, 100, or 250 μg CV8102 given 21 days apart.", "A starting dosage of 50 μg CV8102 was selected based on preclinical dose toxicology studies that showed no adverse findings at 100 µg (mouse, rat) and 250 µg (mini-pig).", "The primary objective of part A was to determine the highest tolerable dosage and recommended dosage range of CV8102 for study part B.", "In part B the primary objective was to assess safety and reactogenicity of a bedside-mix of CV8102 with fractional doses of licensed rabies vaccine, Rabipur®; secondary objectives were to assess the antigen-sparing effect of CV8102 on Rabipur® based on immune responses measured as rabies virus neutralising antibody titres (VNTs).", "Control groups received full (B1), 1/5 (B2), 1/10 (B3) or 1/20 (B4) doses of Rabipur® alone.", "Based on the outcome of study part A two groups received two 1/10 (B5) or 1/20 (B6) doses of Rabipur® administered 21 days apart as bedside-mixes with 50 μg and 25 μg doses of CV8102, respectively.", "Eligible volunteers were healthy 18–40 year-old males.", "Major exclusion criteria were receipt of any other vaccination within 4 weeks of first dosing, use of immunosuppressive drugs or immunoglobulins, chronic immunodeficiency or autoimmunity, acute or chronic disease, serologic evidence of hepatitis B, C or HIV infection, current alcohol or drug use, or known allergy to vaccine components.", "Additional exclusions for part B were any known prior vaccination with a rabies vaccine or planned travel to regions with high rabies risk.", "Both study parts involved a screening visit in the two weeks before enrolment to assess basic health parameters, vital signs (blood pressure, heart rate, ECG), clinical chemistry and verify inclusion/exclusion criteria.", "Participants received study administrations on days 0 and 21 and enrolment was staggered with at least two days between subsequent subjects in a dose group.", "Participants recorded solicited local reactions (pain, redness, swelling, induration, ecchymosis, and itching) and systemic adverse events (AEs; fever, headache, myalgia, arthralgia, nausea, chills, and fatigue) on Days 0–7 after each administration, with severity determined according to WHO guidance [13].", "Unsolicited AEs from Days 1 to 28 after each administration were recorded using MedDRA terms together with any concomitant treatment.", "AEs were graded 1, 2 or 3 according to FDA recommendations and classified as related or unrelated to the treatment.", "Blood samples for safety assessments were drawn before administration on Day 0 and at eight (Part A), ten (Part B CV8102 groups) or five (Part B control groups) additional time points up to Day 49.", "Laboratory safety assessments included haematology/chemistry, abnormalities qualifying as AEs if the investigator considered they were clinically significant deviations from normal ranges.", "The principal investigator and sponsor medical representatives regularly reviewed AEs and SAEs, opening higher dose groups if there were no unexpected, treatment-related AEs in the previous group.", "Unexpected adverse reactions, severe AEs and SAEs were reviewed by the DSMB who advised on the enrolment of further subjects and opening of higher dose groups, and the enrolment of test subjects in part B (staggered vs parallel enrolment of subjects).", "An additional safety follow up call for documentation of any serious AE was done at 12 months.", "Rabies immune responses were measured in part B on Days 0, 14, 28, and 35 as VNTs using a standardised, WHO-recommended rapid fluorescent focus inhibition test (RFFIT) with the rabies virus strain CVS-11 (German rabies reference laboratory of the Friedrich-Loeffler-Institute, Greifswald, Germany) as challenge virus in an accredited reference laboratory (University Hospital Essen, Institute of Virology, Germany).", "The LD50 was calculated by the Spearman-Karber method and resulting titres converted to international units (IU)/mL using a WHO international standard (NIBSC, Potter Bar, UK), with median titres calculated per group.", "Measurements of extended serum dilutions were performed on samples with results outside the validated measurement range.", "Seroconversion was defined as having a neutralising titre ≥ 0.5 IU/mL after vaccination in the previously seronegative subjects (seropositivity for rabies was an exclusion criterion).", "In part A exploratory chemokine and cytokine measurements were performed in peripheral blood sera drawn before and 6 and 24 h after the first administration on Day 0, on Day 7, before and 6 and 24 h after the second treatment on Day 21, and finally on Day 28, using a cytometric bead array kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions.", "CV8102 is a purified, single-stranded, non-coding, long chain RNA molecule presented in a sterile aqueous, colourless solution containing the cationic peptide CR12C and trehalose, ready for mixing with an antigen.", "Lyophilized rabies vaccine, Rabipur® (GSK Biologicals, Wavre, Belgium) was reconstituted at the bedside in supplied diluent for Rabipur-only groups, or in CV8102 for combined groups, for intramuscular injection in volumes of 0.05–1 ml (using 1 ml BD Luer-Lok ™ syringes) in the deltoid.", "This was an exploratory trial with no confirmatory proof of hypothesis so statistical sample-size estimation was not performed.", "Six subjects per dose group were considered sufficient to down-select dosages which were not well tolerated.", "Safety analyses were performed on all subjects who received at least one study treatment and for whom any post-baseline safety data were available.", "Immunological evaluations were performed on all subjects who had at least one post-vaccination blood sample.", "Measured variables were summarised with descriptive statistics as appropriate.", "Between-group testing of VNTs was performed using the Mann-Whitney test or paired t tests as indicated, and correlation between CRP and VNTs was performed by Spearman’s rank correlation analysis.", "Fifty-six volunteers were enrolled into two groups in part A and six groups in part B and received at least one study treatment (Fig. 1).", "Demographic data including age, sex, body weight, and BMI were similar across groups.", "In part A, 6 of 7 subjects in group AL (50 μg CV8102) completed the study, with one drop-out after the first dose due to personal reasons.", "All 4 subjects enrolled in group AM (100 μg CV8102) completed after the first dose with further AM group enrolment being stopped due to transient marked CRP increases in two subjects after the first dose and no further dose escalation was done.", "In part B 45 volunteers were enrolled into six groups; 25 subjects in four control groups (B1–B4) received Rabipur alone (full, 1/5, 1/10, or 1/20 of the licensed dose), and 20 subjects in two groups received bedside-mixes of CV8102 and Rabipur®.", "Group B5 (50 µg CV8102 and 1/10 Rabipur®) was expanded from the 6 subjects originally planned to 14 subjects on the recommendation of the DSMB.", "Six subjects in Group B6 received 25 µg CV8102 and 1/20 Rabipur®.", "Four subjects discontinued after one vaccination, one from B3 (1/10 Rabipur® group) due to a positive drug screening test, and three from B5 (1/10 Rabipur® + 50 µg CV8102 group) due to a positive drug screening test, an unrelated SAE (dizziness) and personal reasons, respectively (Fig. 1).", "No SAEs or severe AEs were reported in study part A up to the end of the study at day 365.", "After one 50 µg CV8102 dose (group AL) six of seven subjects experienced mostly grade 1, self-resolving solicited AEs; five cases of local pain, two of fatigue, two of headache and one of fever.", "Incidence and severity did not increase after the second dose.", "All four subjects given one 100 µg CV8102 dose (group AM) reported grade 2 local pain on Days 0 and 1, three reported grade 1 or 2 fatigue, two grade 1 or 2 headache, two grade 1 myalgia/arthralgia, and one reported grade 1 nausea and another grade 1 fever.", "In groups AL and AM, 6 of 11 (54.5%) subjects reported grade 1 or 2 unsolicited AEs, all considered unrelated to study treatment, which resolved without sequelae and without study discontinuation.", "Laboratory assessment did not find any relevant changes in haematological or clinical chemistry parameters, other than transient mild changes in leukocyte populations, an expansion of neutrophils and monocytes and reduction of lymphocytes 1 day after treatment (not shown).", "Dosage-dependent increases in C-reactive protein (CRP) in the two days following CV8102 administration (Fig. 2A) was particularly marked in two subjects after 100 μg CV8102, peaking on Day 2 at 49.3 and 51.9 mg/L (ULN < 5.0 mg/L), respectively.", "All CRP values decreased to baseline within 7 days, and increases were not related to the severity of clinical adverse events.", "Transient increases in some inflammatory mediators, primarily chemokines, were also apparent at early time points after administration of CV8102 alone: CCL11 levels peaked at 6 h, CCL4 and IP-10 levels peaked at 24 h after treatment (Fig. 3).", "Mean CCL11 levels returned to baseline within 24 h and CCL4 and IP-10 essentially normalised by Day 7.", "A minor increase in pro-inflammatory IL-6 was observed in 2 subjects, but no correlation of increased inflammatory mediator levels with CRP increases were observed.", "Most other factors showed minor increases over detection limits or remained at undetectable levels (data not shown).", "In Part B 25 participants received 50 doses of Rabipur® alone as controls and 20 received 37 doses of mixed CV8102/Rabipur®.", "All were assessed for safety up to one year after the last dose.", "There were no SAEs or severe AEs in Rabipur® controls or 25 µg CV8102 and 1/20 Rabipur® recipients (group B6).", "One SAE after a dose of 50 µg CV8102 and 1/10 Rabipur® (group B5) was hospitalisation for diagnostic evaluation of grade 2 dizziness accompanied by grade 3 headache 7 days after the first vaccination, but symptoms resolved completely after symptomatic treatment.", "Although the SAE was considered to be unrelated to vaccination the second vaccination was not given.", "All solicited local reactions and systemic AEs after Rabipur® alone were grade 1, except for grade 2 injection site pain in two subjects after their second full dose (Table 1).", "In group B5 (50 µg CV8102 mixed with 1/10 Rabipur®) injection site pain occurred after 86% of first doses, including one grade 3 case, and after 64% of second doses.", "Solicited systemic AEs were less common; headache was reported by 4 of 14 (29%) and 5 of 11 (45%) subjects after first and second vaccinations, respectively, with two reports of grade 3 headache after each dose.", "Myalgia/arthralgia and fatigue were reported by 4 of 14 subjects (29%) of Group 5 after their first dose, with one grade 3 case of each, and 3 of 11 subjects (27%) after the second dose, with two grade 3 cases of each.", "In contrast, in Group B6 given 25 µg CV8102 and 1/20 Rabipur® 66% and 33% reported grade 1 or 2 injection site pain after the first and second doses respectively, and the only reports of systemic AEs were two cases of headache after the first dose, graded 1 and 2 respectively.", "Fifteen unsolicited AEs reported during the 49 day observation period by 13 of 45 (29%) subjects were mainly grade 1 (n = 9) or grade 2 (n = 4) general disorders such as back pain or nasopharyngitis; all were considered unrelated to vaccination.", "Two events, local rash at the injection site and increased CRP, were considered to be treatment-related.", "CV8102 dosage-dependent increases in CRP observed in Part A also occurred with CV8102/Rabipur® mixes (Fig. 2B).", "One subject in group B5 (50 μg CV8102 with 1/10 Rabipur®) had a peak CRP of 61.4 mg/L on Day 23, two days after the second dose, which was considered clinically significant and related to the treatment.", "This subject also experienced grade 2 headache and myalgia, and grade 1 influenza-like symptoms.", "All symptoms resolved spontaneously, CRP values returning to normal limits by Day 28.", "No CRP assessments were performed following Rabipur® alone.", "In part B, two fractional doses of Rabipur® in Groups B1–B4 elicited dose-dependent increases in rabies virus neutralising antibody titres (VNT) (Fig. 4A).", "CV8102 enhanced the kinetics of the VNT responses when expressed as the Day 14 seroconversion rate, i.e. the percentage achieving a VNT ≥ 0.5 IU/mL, the WHO protective threshold [12].", "After 1/20 and 1/10 Rabipur® doses 33.3% and 57.1% seroconverted at Day 14, respectively, increasing to 83.3% and 85.7% when mixed with CV8102 (Table 2, Fig. 4B, C).", "All subjects, except one in the B2 group (1/5 Rabipur® dose), had seroconverted one week after a second vaccination.", "Many subjects displayed VNTs above the upper limit of quantification (ULOQ), limiting our ability to compare the magnitude of the humoral immune responses across groups, so we performed a post-hoc exploratory analysis using an extended dilution range of available remaining sera from Groups B1, B3 and B5 (full and 1/10 Rabipur® ± 50 µg CV8102 groups).", "In this assay all 6 (100%) subjects given full-dose Rabipur® seroconverted by Day 14, compared with 4 of 7 (57.1%) given a 1/10 dose, and 13 of 14 (92.9%) subjects given the 1/10 dose mixed with 50 µg CV8102 (Table 2).", "All subjects in all three groups seroconverted one week after their second dose.", "Median VNTs after full and 1/10 Rabipur® doses were 2.9 and 1.1 IU/mL at Day 14, increasing to 9.8 and 2.1 IU/mL at Day 28 and to 23.2 and 2.9 IU/mL at Day 35, respectively (Fig. 4D).", "Mixing 1/10 dose with 50 µg CV8102 increased median titres although not to the full dose levels, at 2.7, 6.5 and 8.5 IU/mL at Days 14, 28 and 35, respectively.", "Due to the robust immunostimulatory nature of CV8102 observed in part A, we investigated whether early increases in CRP levels correlated with the VNTs in subjects receiving the combination of Rabipur® and CV8102 (cohorts B5 and B6).", "We identified a modest association between CRP levels and VNTs at Day 35 on Day 1, which was significant at Day 2 (Fig. 5).", "Highly purified or recombinant subunit vaccines generally have superior safety and tolerability but low intrinsic immunogenicity, so require adjuvantation to augment and shape immune responses, allowing for antigen- or dose-sparing, which may also be applicable to whole virus vaccines in resource-constrained situations.", "Furthermore, there is a important medical need for effective adjuvants for therapeutic vaccines, e.g. tumor immunotherapy, which will require novel adjuvants.", "We describe the first-in-human study of CV8102, an RNA-based adjuvant, using a rabies vaccine as model antigen.", "There was acceptable safety and tolerability with a 50 µg CV8102 given alone, but marked CRP increases in some subjects with higher dosages.", "The reactogenicity profile of 25 µg CV8102 with 1/20 Rabipur® was similar to that of full-dose Rabipur®, but reactogenicity to 50 µg CV8102 with 1/10 dose Rabipur® was higher.", "Consistent observations of transient increases in CRP with CV8102 with or without Rabipur® were only major in a few individuals and these all resolved rapidly.", "Although not directly associated with clinical symptoms or severe AEs the DSMB considered marked CRP elevations to be undesirable for a prophylactic vaccine adjuvant and recommended cessation of further dosage escalation past 50 µg.", "Early CRP increases have also been reported in infants after routine vaccinations [14,15] and adults after MF59 or TLR9 agonist adjuvanted vaccines [16,17].", "Influenza vaccine containing a TLR5 motif dose-dependently increased CRP in healthy adults associated with more severe reactogenicity in some subjects [18].", "Fractional doses of Rabipur® dose-dependently increased rabies immunity as shown by neutralising antibodies.", "Bedside-mixing lower doses of Rabipur® with CV8102 partially mitigated against lower immunogenicity, manifested as faster kinetics and increased magnitude of immune responses.", "With Rabipur, an inactivated viral vaccine that can induce innate immune activation by itself, the adjuvant effect was more pronounced with the 50 µg dose but associated with increased reactogenicity.", "In cohorts given Rabipur®/CV8102 mixes 85% (17 of 20) of subjects achieved seroconversion two weeks after one dose compared with 46% (6 of 13) of those given fractional doses of Rabipur® alone demonstrating the adjuvant role of CV8102.", "We used Rabipur as a model antigen with no plan to develop CV8102 as rabies vaccine adjuvant.", "Given the reactogenicity in combination with rabies vaccine CV8102 may be more suitable for combination with less reactogenic subunit or protein vaccines that are required for high medical need prophylaxis situations (epidemic outbreaks) or therapeutic vaccines where higher reactogenicity is acceptable.", "One limitation of this study is that we did not assess CRP responses to Rabipur® alone as the magnitude of these responses to CV8102 mixed with Rabipur® appeared higher than with CV8102 alone.", "This may be due to the TLR-activating motifs in viral vaccines like Rabipur® increasing inflammatory responses.", "Although excessively high levels of post-vaccination CRP are undesirable, we observed a modest correlation between transient CRP increases at Days 1 and 2 and VNTs at Day 35.", "Given the complexity of how the innate immune system activates and regulates the adaptive immune system there is a strong rationale for more in-depth analyses of early immune events [7–11,19].", "In light of this, another limitation was that samples were not taken in part B to examine the immediate effects of CV8102 and Rabipur® on the transient changes in cytokines and chemokines observed in part A.", "We cannot therefore fully conclude on the contribution of Rabipur® to the early inflammatory response.", "Another missing element was the inability to test 25 μg CV8102 mixed with a 1/10 dose of Rabipur® due to limitations in bedside-mixing with the available concentrations, but this combination may provide an optimal balance between tolerability and adjuvant enhancement of the response against the rabies antigen.", "Preclinical data supports the potential of CV8102 as an adjuvant for cancer vaccines [20].", "As transient severe reactogenicity and CRP increases are clinically more acceptable in therapeutic settings investigation of CV8102 including doses exceeding 50 µg is being pursued in combination with a peptide vaccine in patients with hepatocellular carcinoma in an EU-funded phase I trial by the HEPAVAC Consortium [21].", "Early observations in an ongoing phase I trial of CV8102 in patients with advanced solid tumours [NCT03291002] indicate that repeated intra-tumour injections ≥ 100 µg are tolerated without causing severe side effects [22].", "In conclusion, this first-in-human trial demonstrates that despite some transient severe systemic reactions, at dosages with acceptable reactogenicity the RNA-based adjuvant CV8102 is a potent immune stimulator of fractional doses of licensed rabies vaccine.", "At dosages up to 50 µg CV8102 is a promising adjuvant candidate and may have lower reactogenicity with inherently less reactogenic whole protein or subunit vaccines than the rabies vaccine we used.", "This study and editorial support for the preparation of this manuscript were funded by CureVac AG, Tübingen, Germany.", "All authors were involved in the design, performance and analysis of the study, and subsequently assisted in the drafting, review and approval of the manuscript.", "At the time of this study Fatma Doener, Henoch S. Hong, Angelika Daehling, Regina Heidenreich, Sven D. Koch, Mariola Fotin-Mleczek and Ulrike Gnad-Vogt were full-time salaried employees of the study sponsor, and Keyvan Tadjalli-Mehr was a contractor working for the study sponsor.", "Ingo Meyer has no conflicts of interest to declare." ]	[]
Fukuoka: Adapting to climate change through urban green space and the built environment?	This paper profiles Fukuoka City in Kyushu, Japan. We focus on the city's local climate change adaptation policies, and in particular the role of urban and greenspace planning in facilitating adaptation actions within Fukuoka. Fukuoka is a humid subtropical city which is currently experiencing significant population and economic growth. It has also made comparatively rigorous advances in climate adaptation, in a country context where local governments have been criticised for focusing more on mitigation. Fukuoka hence may yield lessons for other rapidly urbanising subtropical Asian cities. We illustrate that Fukuoka has a long tradition of science-policy connection towards the creation of a liveable urban environment. This creates a favourable research and policy infrastructure for adaptation, in particular mitigation of heat risk. This is evidenced in consideration of climate issues within the city's greenspace plans since the 1990s, and in an extensive body of underpinning applied research from local institutions into urban thermal environments in particular. Fukuoka's green terraced ACROS building has come to symbolise adaptation via the built environment, and has been followed by the emergence of further green roofs and through citizen and private sector involvement in smaller-scale greening actions. We caution that challenges remain around connecting different sections of local governments, and in maintaining climate and environmental imperatives in the face of ongoing development and expansion pressures.	[ "BUSI", "SOCI" ]	[ "Climate change adaptation", "Fukuoka", "Urban climate change governance", "Urban greenspace", "Urban planning" ]	[ "1409", "3303", "3312", "3322" ]	[ "Although Japanese policy on climate change has been criticised for focusing more on mitigation than adaptation at the local1 level (Baba et al., 2017; Kameyama, 2016), Fukuoka City has considered adaptation to the effects of environmental change within its greenspace and urban plans since the late 1990s (Fukuoka City, 1999).", "Following the adoption of the Kyoto Protocol in 1997, local governments in Japan have engaged enthusiastically with climate change mitigation via climate change plans geared towards emissions reduction and renewable energy deployment (Kameyama, 2016).", "However, progress on adaptation has been much slower.", "The 2018 Climate Change Adaptation Act now mandates municipal governments to form Local Climate Change Action Plans.", "Yet up until this point, few Japanese cities have had specific laws and plans to address adaptation action, and there is limited evidence of practical adaptation actions at the local level (Hijioka et al., 2016).", "In Fukuoka, by contrast, locally-based researchers have undertaken applied research on the relationship between urban greening, the built environment and urban thermal environments for several decades (e.g. Katayama et al., 1991; Nitta, Azuma, & Ishii, 1981; Yoda & Katayama, 1998).", "Fukuoka City Government also released in 2016 a climate change countermeasures action plan to integrate climate and environmental plans and link mitigation with adaptation, continuing actions which have developed since the city made its first local climate plan in 1994 (Fukuoka City, 2016a).", "This initial formalised climate action came several years ahead of comparable western Japanese cities such as Kyoto (1997); Kobe (2000); Osaka (2002); and Hiroshima (2003).", "Fukuoka is therefore significant for several reasons.", "Firstly, it can be considered a front-running city within Japan for local climate change policy, and also for actions targeted specifically towards adaptation to environmental change supported by the applied research history outlined above.", "Second, despite national trends, the population in Fukuoka is growing at the fastest rate of any Japanese city (Fukuoka Asian Urban Research Center, 2018), with resulting urban development and expansion (Fukuoka City, 2018a).", "Third, Fukuoka is among the southernmost designated cities in Japan in the humid subtropical climate zone.", "Assessing how Fukuoka has integrated climate adaptation-related issues into its local policies despite slow national progress, and clarifying remaining challenges, may hence yield lessons for other expanding Asian subtropical cities facing increasing climate-related risks such as urban heat island effects and flooding.", "In the wider international context, evaluation of how Fukuoka has managed urban heat in particular can contribute to a knowledge gap identified by the IPCC's Cities and Climate theme around knowledge of the relation between planning and the thermal environment in non-European or North American contexts (Prieur-Richard et al., 2018).", "Elaboration of how Fukuoka has drawn on techno-scientific expertise and evidence to support adaptation via urban planning can feed into debates – as per a recent Nature Sustainability expert panel - on how ‘the science of cities’ can inform urban climate responses (Acuto et al., 2018).", "For the purpose of this City Profile, we focus on Fukuoka City as an administrative unit.", "This is because (a) Fukuoka City Government is the municipal administrative unit governing the built-up area of Fukuoka City, and hence can make finer-scale recommendations than the higher-level prefectural government; and (b) Fukuoka Prefecture in any case is required to develop adaptation actions for a mixed rural and urban context.", "Indeed, it is noted in Fukuoka City's climate plan that the prefectural climate plan provides general overarching guidance for the city's actions, but that these are translated into practice via policies and spatial plans produced by the city government (Fukuoka City, 2016a).", "Unless otherwise stated, we therefore use Fukuoka City to describe specific actions undertaken by Fukuoka City Government, and Fukuoka to refer to more general social and cultural characteristics in the wider Fukuoka area.", "Actions and policies relevant to Fukuoka City but enacted by Fukuoka Prefecture are of course considered within the profile as and when appropriate.", "The information presented in this paper is derived from three sources.", "First is policy documentation produced by Fukuoka City and also Fukuoka Prefecture relating to climate change adaptation and the urban environment, including climate change plans, greenspace plans, and urban planning masterplans.", "Second is a review of scholarly research outputs pertaining to urban planning and environmental change with an empirical focus on Fukuoka.", "These were collated via ‘snowball’ sampling based on (a) searching Web of Science for the terms ‘Fukuoka’ and ‘climate’, ‘greenspace’ or ‘planning’; and (b) searching the Japan Society for the Promotion of Science's KAKEN database for additional Japanese-language projects and outputs, using Japanese equivalents of the same search terms.", "Third are eight in-depth interviews with people associated with local adaptation responses: one at Fukuoka City Environment Division; one at the Fukuoka City Green City Promotion Department; one at Fukuoka Prefecture Environment Division; one at the Fukuoka Center for Climate Change Actions; one at Kyushu Environmental Evaluation Association; one with an academic involved in the expert committee for Fukuoka City's Climate Change Countermeasures Action Plan; one with an academic involved in local and regional adaptation planning in Japan; and one at a private-sector consultancy providing public bodies with advice on climate change responses and environmental management actions.", "Whilst the profile is based largely on the review of policy and scholarly outputs, the interviews provided additional contextual information.", "This allowed observations from the policy analysis and review of academic research to be clarified, and also allowed respondents to provide a more critical and evaluative insight on the extent to which policy rhetoric translates into practice (see Section 6.5 in particular).", "Fukuoka is located in the north of Kyushu, the southern-most of Japan's four main islands (see Fig. 1).", "It is bordered by Hakata Bay (which opens into the Genkai Sea) to the north, and by the Sangun Mountain Range to the east and the Sefuri Mountain Range to the south-west.", "Fukuoka City is located within Fukuoka Prefecture, on the Fukuoka Plain.", "Fukuoka Prefecture is one of the 47 regional administrative units in Japan, and includes the urbanised area of Kitakyushu and the more rural areas of Chikuho and Chikugo over an area of 4584 km2.", "The population of Fukuoka Prefecture was approximately 5.1 million people in 2018, of whom around 3.8 million people were living in the urbanised areas of Fukuoka and Kitakyushu (Fukuoka Prefecture, 2019).", "The population of Fukuoka City was approximately 1.5 million people in 2018, over an area of 340 km2 (Fukuoka City, 2017).", "Fukuoka City has a humid subtropical climate, with the highest temperatures reaching around 37 °C in the months of July and August and an average of 1612 mm of rainfall annually (see Section 6 for a more comprehensive overview of weather and climate and its relation to climate change) (Fukuoka City, 2017).", "Fukuoka's population grew by 7.1% between 2010 and 2017, compared to 5.8% for Tokyo – giving it the biggest growth of any major city in Japan (Fukuoka Asian Urban Research Center, 2018).", "A large proportion of this population change is due to in-migration, with 6.8% of the population in-migrants from elsewhere in Japan or overseas (Fukuoka Asian Urban Research Center, 2018) Most recent data from 2016 shows that people over 65 – a group often considered vulnerable to climate risks – make up 21.4% of the city population (Fukuoka City, 2017) compared to 27.2% for Japan overall (Ministry of Internal Affairs and Communications, 2017).", "According to data from 2014, service industries (25.6%) made the biggest contribution to Fukuoka City's real gross regional product by economic activity, followed by wholesale (13.3%) and real estate activity (12.6%) (Fukuoka Asian Urban Research Center, 2018).", "Fukuoka developed as twin cities from the 17th Century onwards.", "Fukuoka, to the west of the Naka River, was a castle town of the samurai military class.", "Hakata, to the east of the river, was a city of merchants.", "The legacy of this historical structure is still visible today in the divide between the commercial centre of the city east of the Naka River, and the administrative and entertainment areas to its west.", "Settlement in the area where Fukuoka is now located began approximately 26,000 years ago, when people crossed from the Asian continent.", "Around 15,000 years ago, the region became separated from the rest of Asia by what is now the Genkai Sea.", "Rice farming superseded a hunter-gatherer lifestyle, and the people of the region started to run agricultural communities.", "The first state in the Japanese archipelago was established in the region, with records showing that in 57 CE, the Emperor of the Late Han Dynasty gave a gold seal to envoys sent by the king to request support for their kingdom (Fukuoka City Museum, 2013).", "After the centre of the nation moved east, the region where Fukuoka is located today played an important role for diplomacy, commerce and defence.", "The government of the time established a diplomatic facility called the Korokan, which in turn was succeeded as a centre of commerce by an area known as Hakata, which supported Chinese traders.", "Following attempted Mongolian invasions in the late 13th Century, trade with Ming (China), the Ryukyu Islands and the Korean Peninsula made Hakata a flourishing trading port (Fukuoka City Museum, 2013).", "The Hakata name is still visible today as one of the seven wards in Fukuoka City, and as the name for the main high-speed rail station in the city.", "In 1601, the House of Kuroda established a new castle and town near to Hakata.", "This new town was named Fukuoka after the feudal lord Nagamasa Kuroda's birthplace in Okayama Prefecture.", "In April 1889, the government issued an order for municipalisation, and the towns of Fukuoka and Hakata were merged.", "‘Fukuoka’ was chosen as the name for the new city.", "At this time, their populations were 20,410 and 25,677 respectively (Fukuoka City, 2009a).", "Fukuoka City continued to expand and developed a streetcar network in the early part of the 20th Century, but the city was heavily damaged by bombing raids in World War II.", "The city rebuilt and continued to develop along with Japan's post-War economic miracle, becoming Kyushu's core city by the mid-1970s.", "At this time, the Tenjin Underground Shopping Mall, Tenjin Station on the Fukuoka Municipal Subway Line, and several large department stores opened in the Tenjin area.", "The opening of the Tenjin Subway Line shifted in the flow of people in the central part of Fukuoka, changing the flow from along Meiji-Dori on an east-west axis, to Watanabe-Dori on a north-south axis (Fukuoka City, 2009b).", "In 1987, in response to citizen preferences for comfort and aesthetics over function and efficiency, Fukuoka City Government enacted legislation focusing on the urban landscape and initiated the Fukuoka City Urban Landscape Award to recognize those who contributed to the creation of an appealing urban landscape (Fukuoka City, 2009b).", "Whilst the different characters of east- and west Fukuoka are rooted in several centuries of history, the precise urban form of the city and its current development trajectory are informed by topographical constraints and more recent events (see Figs. 2 and 3).", "The current layout of the city's central area still largely reflects the form laid down during systematic post-World War II reconstruction planning.", "Meanwhile, mountains to the south and the sea to the north mean development entails either expansion in an east-west direction, or intensification.", "After Fukuoka was heavily damaged by saturation bombing during World War II, the current form of the city's central area was largely set by implementation of post-War reconstruction plans in the 1950s and 1960s (Fukuoka City, 1979).", "Hakata Station was moved 600 m to the north at this time, and became the meeting point for the Sumiyoshi and Taihaku roads.", "With development to the south constrained by mountain ranges, land reclamation has taken place into Hakata Bay to the north.", "Reclamation commenced in the 1930s, but intensified from the 1970s onwards to the north-east in particular (Ishibashi & Shibata, 2014).", "Island City Fukuoka, completed in 2005, is one such development with mixed residential, recreational and institutional land uses.", "As outlined in Section 3, Fukuoka has experienced notable population growth and in-migration.", "Yet in addition to geographical constraints, noise protection controls around Fukuoka Airport to the east, and strict municipal urbanisation control regulations to manage sprawl, further limit expansion and development.", "Current real estate development and expansion is thus focused in the direction of Itoshima City to the west (aided by the relocation of some parts of Kyushu University from the Ropponmatsu area of Jonan Ward to a new campus in the far west of Fukuoka City), and Kasyua County to the north-east.", "Development has also taken place into the 21st Century along the Japan Railways line to Kasuga City and Onojo City to the south-east, however this area is now reaching saturation.", "Given these geographical and land use constraints, the current Fukuoka City Urban Planning Masterplan states that consolidation and development of the Hakata-Tenjin core area is key to attaining the goal of making Fukuoka City an interchange and focal point for Asia, and also to extracting more economic value from Fukuoka's core area in line with the city's growth (Fukuoka City, 2014).", "To aid this process, Fukuoka City Government has initiated the ‘Tenjin Big Bang’ project, which has relaxed regulations on the height of buildings and on floor area ratio to encourage redevelopment of 30 old buildings in Tenjin by 2024 (Fukuoka City, 2018a).", "Administratively, Fukuoka City is divided into seven wards (see Fig. 4).", "The ward names loosely correspond to names of areas from the time of the Kuroda samurai clan in the 17th Century.", "However, the wards in their contemporary form are a superimposed political structure.", "The wards were created when Fukuoka was made a designated city by the Japanese government in 1972.", "Initially the city consisted of five wards, however the former Nishi Ward was subdivided into Jonan Ward, Nishi Ward and Sawara Ward in 1982.", "The main business and commerce district is Hakata Ward, whilst most entertainment and retail is located in the Tenjin area of Chuo Ward.", "Together, the Hakata and Tenjin areas make up the core of Fukuoka City.", "Most of the Fukuoka metropolitan area falls under the jurisdiction of Fukuoka City Government.", "This is the municipal government for Fukuoka City, and is located within Fukuoka Prefecture – the regional level of government.", "As far as climate change governance is concerned, it is Fukuoka City (i.e. the municipal level) which is mandated to produce a climate adaptation plan for the city.", "The municipal government is responsible for translating climate-related legislation, policies and directives from the national and prefectural governments into practice.", "Fukuoka City Government is able to respond to these directives through control of areas such as land use/greenspace planning and municipal climate action plans, as discussed in Section 6.", "Adaptation to climate change in Fukuoka City comes against a backdrop of an increasing population, with associated pressures on urban development and awareness of the need to preserve or increase greenspace.", "The population density of Fukuoka City grew from 3602 people per km2 in 1990, to 3907 people per km2 in 2000, and 4481 people per km2 in 2015 (Statistics Agency of Japan, 2015).", "Fukuoka City acknowledges a problem in that the ratio of green coverage in Fukuoka City declined from 60.2% in 1985, to 58.0% in 1996, and 55.4% in 2007 (data from the last time the city's greenspace plan was updated) (Fukuoka City, 2009c).", "Over the same time periods, the ratio of green coverage in the city's defined built-up areas reduced from 25.9% (1985) to 23.8% (1996), then down to 20.7% (2007).", "Nonetheless, through actions such as planting street trees, greening of public spaces, and greening of private residences, the area of ‘created greenery’ in Fukuoka City's defined built-up areas was increased by 9.1% from 1996 to 2008, and over the same time period the area of formal parks and greenspaces increased by 21.4% due to creation and incorporation of new parks (Fukuoka City, 2009c).", "Table 1 below shows how this greenery and greenspace is distributed by ward.", "Moreover, as actions such as rooftop gardens, green walls and proliferation of greenery across the city are stated as important heat island mitigation actions in the city's greenspace and urban plans, the number of community and private sector greening actions (e.g. roadside greenery, improvement within parks and at railway stations) supported by Fukuoka City in each ward under the Flower City Fukuoka project is listed.", "This illustrates the number and distribution of smaller-scale actions across the city.", "Given the focus of this profile on climate adaptation, it is worth noting recent experience with natural disasters, weather and climate in the city.", "Whilst not as seismically active as other areas in Japan, Fukuoka was struck by a powerful earthquake registering shindo 6 (the most powerful being shindo 7) on March 20,2005, causing damage to buildings, killing one person and forcing evacuation of much of Genkai Island at the edge of Hakata Bay.", "In 2009, heavy rainfall in northern Kyushu in late July caused increases in river water levels.", "This resulted in flooding in many parts of Fukuoka, forcing citizens to temporarily evacuate and causing five deaths in the Fukuoka metropolitan area (Hashimoto & Saito, 2012).", "The summer 2018 Japanese heatwave affected Fukuoka too.", "A temperature of 38.3 degrees Celsius was observed in Fukuoka City on 20 July 2018, the highest since records began in 1890 (Japan Meteorological Agency, 2019).", "A record 821 people in the city were hospitalised by heat stroke over the summer of 2018 (up to 30 September), an increase of 241 people over the same period in 2017 (Fukuoka City, 2018b).", "Based on observational research into Fukuoka's thermal environment, the Hakata-Tenjin area was identified as a ‘hot spot,’ and has subsequently been the focus of further research and experimental measures to mitigate heat island effects (Fukuoka City, 2016a; Tanaka, Hagishima, Kitayama, & Yoda, 2009).", "Table 1 shows that community and private sector greening activities, whilst not undertaken explicitly for heat mitigation, are also focused on the Hakata and Chuo Wards (where Hakata-Tenjin is located) where green coverage is lower.", "Flood risk and evacuation maps have been produced for each ward, identifying areas at risk in the event of inundation and illustrating evacuation routes and shelters (Fukuoka City, 2011).", "Compared to Japan as a whole, Fukuoka has experienced above-average warming to date and is likely to continue to feel the effects of climate change into the future.", "This is reflected in the risks identified in the city's climate change planning and the measures proposed in response.", "Average annual air temperatures in Fukuoka Prefecture increased by 2.54 °C between 1898 and 2017, compared to 1.69 °C for the wider Kyushu and Yamaguchi area and 1.19 °C for Japan over the same period (Fukuoka District Meteorological Observatory, 2017).", "Under the IPCC's A1B Emissions Scenario (very rapid economic growth, global population peaking in mid-century and declining thereafter, and rapid introduction of new and more efficient technologies with a balance of fossil- and non-fossil sources (IPCC, n.d.)), average air temperatures in Fukuoka Prefecture are predicted to increase a further 2.9 °C by 2100, with 18 more extremely hot days (over 35 °C) and 42 more hot days (over 30 °C) per year predicted by 2100 (Fukuoka District Meteorological Observatory, 2014).", "Fukuoka City's Climate Change Countermeasures Action Plan accordingly identifies five key climate risks requiring adaptation actions: (a) natural hazards from heavy rainfall and flooding; (b) pressure on water resources; (c) health risks from increased heat; (d) biodiversity loss; and (e) effects on agricultural produce (Fukuoka City, 2016a).", "Heavy rainfall leading to flooding in Fukuoka City in July 2009, and the significant increase in consecutive hot days observed in summer 2013 within Fukuoka are cited as examples of current climate effects (Fukuoka City, 2016a; Japan Meteorological Agency, 2013).", "Within the wider Japanese context, Fukuoka may be considered one of the relatively early adopters of climate change thinking in urban planning.", "The actions proposed in Fukuoka in response to climate risks are perhaps not unique to the city, but what is distinctive is the comparatively high degree of evidence and specificity provided by the city's greenspace and urban plans.", "Local governments in Japan generally started to form climate change plans from the mid-1990s onwards, to support national obligations under the Kyoto Protocol.", "These activities were also supported by the 1993 Basic Environment Law, which integrated wider environmental considerations into previous legislation focusing on anti-pollution measures.", "The production of climate change plans has been a legal obligation for larger local governments – including Fukuoka City – since 2008 due to the Act on Promotion of Global Warming Countermeasures.", "From 2018, local governments are obliged to develop plans specific to adaptation under the Climate Change Adaptation Act, and also to nominate a local adaptation centre for data collection and provision.", "As Table 2 shows, it is hence not unusual for larger local governments in Japan to have local climate plans preceding national legislation.", "It is however notable that Fukuoka's first climate change plan came earlier than other large cities in west Japan (in addition to those listed in Table 2, Kyoto's plan came in 1997 and Osaka's in 2002) and indeed pre-dated the Kyoto Protocol.", "Table 2 compares Fukuoka's actions on climate change within greenspace- and urban planning to two other western Japanese coastal cities of similar size and climatic characteristics – Hiroshima and Kobe.", "The intention is simply to provide some comparative context, and not to compare or criticise the cities in question.", "However, it is striking that Fukuoka's greenspace plan in particular contains a relatively high level of detail and specificity, especially for urban heat island mitigation.", "Table 3 summarises the main policies informing climate adaptation via planning in Fukuoka City.", "The core guiding policy is the Fukuoka City Climate Change Countermeasures Action Plan (2016a), known as the Cool and Adapt Project for short.", "This was developed to integrate the city's local climate change plan and its broader environmental plan.", "Previously, Fukuoka City Government produced its first local climate change plan – the Fukuoka City Local Climate Change Countermeasures Promotion Plan - in 1994, which was updated in 2001 and again in 2006.", "Climate issues are also considered within greenspace planning in Fukuoka, via the Fukuoka City Green New Basic Plan (2009c,d).", "This replaces the Fukuoka City Green Basic Plan (1999), and is due to be updated by 2020.", "The Fukuoka City Urban Planning Masterplan (2014) also discusses climate issues, albeit largely in relation to mitigation and with adaptation actions similar to those in the New Green Basic Plan.", "At regional level, the Fukuoka Prefecture Climate Change Countermeasures Action Plan (2017) provides climate change response guidance for all of Fukuoka Prefecture (including rural areas and Kitakyushu City as well as the Fukuoka urban area), setting guidelines which are enacted via Fukuoka City.", "As outlined previously, these actions take place under national-level legislation set by the Japanese Government: Act on Promotion of Global Warming Countermeasures (2008); National Plan for Adaptation to the Impacts of Climate Change (2015); and Climate Change Adaptation Act (2018).", "As outlined in Section 6.1 and Table 2, what is notable is that whilst Fukuoka is one of a group of cities which were early to engage with climate issues in urban governance, the city's plans appear grounded in a comparatively rich techno-scientific evidence base.", "Fukuoka has a history of environmental science research and planning considerations aimed at understanding how the built environment may be configured to create a comfortable urban climate (Mabon et al., 2019).", "This predates the city's current interest in climate change adaptation.", "Understanding of the urban climate in Fukuoka City – in particular the thermal environment - is underpinned by evidence from both national and local levels.", "Under the direction of the Ministry of Land, Transport and Infrastructure, Fukuoka was one of the first two municipalities in Japan – along with Tokyo – to produce an urban climate atlas, creating a climate function map focusing on effects of green space on urban heat islands with urban climate simulation system (Ichinose, Mikami, Niitsu, & Okada, 2003).", "Remote sensing-based studies aimed at understanding how planning can mitigate heat effects have been conducted in Fukuoka by researchers at local institutions since at least the early 1990s (e.g. Katayama et al., 1990), with Fukuoka City Government undertaking a neighbourhood-level study into thermal environments in 2006 in collaboration with locally-based researchers (e.g. Tanaka et al., 2009).", "Academic knowledge and research findings connect with local government policy through the participation of researchers from local institutions (e.g. Kyushu University/Kyushu Institute of Design, Fukuoka University, Kyushu Environmental Evaluation Association/Fukuoka Center for Climate Change Actions) on expert committees for the adaptation-related plans listed in Section 6.1.", "This provides a contrast to Baba et al. (2017), who identify a lack of expert knowledge and experience at the municipal level as a barrier to local climate adaptation in Japan.", "The role of Naohito Asano of Fukuoka University at the academia-policy interface is particularly noteworthy.", "Asano was an expert panel member for Fukuoka City's Cool and Adapt Project, and also chaired the expert committee for Fukuoka Prefecture's Climate Change Countermeasures Action Plan.", "What is significant is that Asano is an environmental lawyer whose career has been conducted largely in Fukuoka, with a professional interest in the use of local urban planning legislation and policy as a way to secure quality of living for citizens (e.g. Asano, 1988).", "As such, the connection of the research base with planning policy actions has been aided by a community of academics with an understanding of the technical and scientific principles of urban climate adaptation gained at many cases at local institutions, and also knowledge of the legislation and policy that can put these actions into practice.", "This rich evidence base may in part be explained by the longer interest in Fukuoka in local environmental quality and in managing the urban climate, both of which pre-date climate adaptation considerations.", "The incidents of Minamata Disease and air pollution in Kitakyushu both led to calls in the 1960s in Kyushu for independent environmental science undertaken in the public interest (e.g. Fujikura, 2001).", "The Kyushu Environmental Evaluation Association, which has been involved in monitoring urban thermal environments and in hosting the Fukuoka Center for Climate Change Actions, was specifically set up by academics in the early 1970s to monitor water quality as a result of events in Minamata.", "Asano too was engaged with the legal aspects of Minamata Disease earlier in his career (Asano, 1992).", "There is also a concurrent interest in Fukuoka in the provision of kaiteki kankyou – a liveable environment – through urban planning and environmental governance.", "This language was utilised in Fukuoka City's first Environmental Plan (Fukuoka City, 1986), and also in the writings of Fukuoka-based academics working at that time (e.g. Asano, 1988; Mitsuyoshi, 1985).", "A group of academics from Kyushu University of Design (now part of Kyushu University) in 1981 published Design of micro-weather in urban greening, which assessed how urban greenspace at a neighbourhood- and city-scale could be used to ensure thermal comfort (Nitta et al., 1981), tallying with Hebbert's (2014) observation that Fukuoka was one of the Japanese cities to engage early with ideas of urban climatological planning.", "Indeed, the Fukuoka City Green Basic Plan (1999) and subsequently the Fukuoka City New Green Basic Plan (2009c,d) consider such ideas of preserving wind corridors, creating a city-wide green network, and targeted strategic greening for cooling (see Fig. 5).", "Climate adaptation actions via planning in Fukuoka are thus supported by a number of local institutions with significant competence in understanding the urban environment, and willingness to engage with the policy-making process.", "However, this is arguably the result of a much longer concern in Fukuoka with a liveable local environment, which has helped these local institutions to develop the skills to create an environmental science evidence base towards maintaining quality of life (Mabon et al., 2019).", "This appears particularly true when it comes to mitigating heat effects.", "Whilst Hijioka et al. (2016) are right to claim that Japanese cities already have a number of robust local environmental and social policies which give a basis for climate change adaptation, it may hence also be the case that Fukuoka's distinctive environmental history has supported the comparatively early and rigorous consideration of climate adaptation concerns within Japan.", "As above, ‘success’ may come through Fukuoka's early adoption of climate change and adaptation-related policies, especially the specificity with which these are considered within greenspace and urban plans.", "In Japan, existing urban policies developed for areas such as disaster risk reduction and environmental management may mean that cities are already relatively well-prepared for climate change adaptation and may not require new areas of policy or planning to deal with weather and climate risks (Hijioka et al., 2016).", "Indeed, as per Table 2, Hiroshima City Government explicitly states that climate adaptation is considered within wider disaster risk reduction measures.", "What is perhaps distinctive about Fukuoka is therefore the explicit focus on climate change within greenspace- and urban planning measures.", "Fukuoka City produced its first climate change plan in 1994, placing it ahead of other local governments developing similar plans (e.g. Hiroshima, Kobe, Kyoto, Osaka) by several years.", "In comparison to other Japanese city contexts, Fukuoka's greenspace planning has also made rigorous attempts to consider climate change, the urban thermal environment and to work towards understanding the city's greenspace in terms of a city-wide green network, supported by research from within the government and by local institutions (Fukuoka City, 1999, 2009c).", "The city's support for research into localised heat island effects, and subsequent utilisation of these findings to inform decisions on development of a grass area outside Fukuoka City Hall (Tanaka et al., 2009), indicates there is a willingness within government to consider urban climate issues within their governance of the built environment.", "Moreover, although at an early stage, it is significant that Fukuoka Prefecture's Climate Change Countermeasures Action Plan (2017) identifies the need to understand green infrastructure and ecosystem services across the Fukuoka area from now into the future.", "The ACROS Fukuoka Prefectural International Hall is an example of a location where greening has delivered local cooling and also biodiversity benefits (see Figs. 6 and 7).", "Completed in 1995 and designed by green architecture pioneer Emilio Ambasz, ACROS features woodland on the stepped façade of a 15-storey building (Jim, 2017a) and is directly connected to the adjacent Tenjin Central Park.", "The building provides public good by containing a concert hall and government service points, as well as restaurants and office space.", "It is reported to host 40,000 plants and trees spanning 120 species.", "The ACROS terraces were not designed to provide climate or biodiversity benefits.", "Rather, the focus was on aesthetics, and on reducing the general environmental impact of the development (Jim, 2017a).", "However, the terraces have been demonstrated to provide rainfall runoff quality benefits (Berndtsson, Bengtsson, & Jinno, 2009); and to deliver cooling effects in the immediate area (Hagishima, 2018).", "Internationally, ACROS is a well-known example of vertical greening.", "ACROS was featured in an article on flood risk reduction from green roofs in academic blog site The Conversation (Howell, Drake, & Margolis, 2017), and included in an article on green roofs and heat island mitigation in UK newspaper The Guardian's Cities section (The Guardian, 2018).", "Both in academic literature and also public-facing material aimed at informed audiences, then, ACROS appears to be viewed internationally as a success story of climate change adaptation.", "Locally, signage on the terraces explains ACROS' biodiversity (authors' own visit, 2018), and the image of ACROS is frequently used in public-facing material relating to urban climate in Fukuoka (e.g. the New Green Basic Plan Summary Leaflet, (Fukuoka City, 2009d)).", "The site hence has a symbolic role within Fukuoka in motivating action towards climate change countermeasures and urban climate protection.", "Since ACROS was built, other large-scale green roofs have developed in Fukuoka.", "The Grin Grin active learning space and botanical garden in Island City Central Park has a green roof area of 1000 m2 over three connected buildings and opened in 2005 (Island City Central Park, 2019).", "The Tenjin Core shopping centre also installed a green roof, but is scheduled to close as part of the Tenjin Big Bang intensification works.", "Fukuoka City Government is responsible for the Green Curtain Project and Flower City Fukuoka initiatives, which collaborate with citizens and the private sector to encourage development of green walls and localised greenery respectively.", "Indeed, another aspect in which Fukuoka has made progress in climate adaptation through the built environment is in community and citizen engagement in greening.", "Engagement of communities in greening strategies has been encouraged to maximise climate benefit from greening in dense cities where greenspace may be at a premium and available space may lie outside formal greenspace plans (Mabon & Shih, 2018; Tan & Jim, 2017).", "Engagement on community-level greening can also help to build social relations (Tidball & Atkipis, 2018) and hence perhaps increase resilience to future climate events.", "As part of the Green Curtain Project, Fukuoka City offers cash prizes for growing vertical greenery (Fukuoka City, 2016a).", "The competition is framed in terms of delivering cooling and reducing energy consumption, and is supported with training sessions in growing and maintaining greenery.", "Fukuoka City Botanical Gardens also operates a ‘Green Advice Centre’ providing free technical advice to citizens and community groups wishing to develop greenery in their locale.", "Whilst not specifically targeted at climate adaptation or maximisation of ecosystem services, this nevertheless goes some way to addressing the lack of technical competence (Jim, 2017b) which may act as a barrier to enhancing or maintaining urban greenery within a city.", "Thus far, this profile has illustrated that Fukuoka has a sound policy base for climate adaptation action through competences in greenspace and urban planning in response to environmental change.", "However, the extent to which this basis extends to other urban policy areas, or to tangible action, may be more limited.", "The first difficulty lies in putting the visions laid out in the city's greenspace plan fully into action.", "Whilst there is very good knowledge stretching over three decades of the urban climate in Fukuoka and of how it may be controlled via greening (e.g. Hagishima, 2018; Nitta et al., 1981; Yoda & Katayama, 1998), interventions in the built environment are perhaps limited beyond the flagship examples laid out in Section 6.4.", "For more expansive measures such as the preservation or construction of wind corridors, it is also very difficult if not impossible to remove or alter existing buildings along the seafront (interview with KEEA, April 2018).", "The data presented in Section 5.2 indicates that Fukuoka's continuing growth is putting pressure on green spaces, with a declining trend in green coverage at the time of the last greenspace planning cycle.", "Nonetheless, this is recognised by the city, and the inclusion of more green and open spaces within the city's formal greenspace system – as well as city government-led initiatives such as the Green Curtain Project and Flower City Fukuoka – indicate efforts to re-establish greening as part of adaptation efforts.", "The second challenge represents the observations of Baba et al. (2017) that compartmentalisation of government bureaus may be a barrier to local climate adaptation progression in Japan.", "Whilst the Fukuoka City Urban Planning Masterplan and the Fukuoka City New Green Basic Plan both come under the remit of the Housing Division, the Climate Change Countermeasures Action Plan falls under the remit of the Environment Division.", "These local government divisions themselves report to different sections of national government.", "Housing is connected to the Ministry of Land, Infrastructure and Transport; and Environment to Ministry of Environment.", "Interviewed local government officials acknowledged there are initiatives to link different areas of local government through periodical face-to-face forums and also the connection of policies and plans to each other, but also that greenspace and climate adaptation plans do to an extent develop in silos (interview with Fukuoka City Environment Division, May 2017; interview with Fukuoka City Government Green City Promotion Department, April 2018).", "At the prefectural level, the Fukuoka Prefecture Climate Change Countermeasures Action Plan (2017) does however assign the identified tasks relating to climate change to specific prefectural government sections and departments.", "This may represent a move towards the “integrated adaptation strategy for the whole local government” which Baba et al. (2017: 10) believe is missing in Japan at present.", "Third, there is a potential tension in Fukuoka between economic development and environmental protection.", "Fukuoka has the highest growth rate of local tax revenue (4.2% between 2008 and 2015) of all major cities in Japan, and the fastest-growing population of major Japanese cities (7.1% between 2010 and 2017) (Fukuoka Asian Urban Research Center, 2018).", "Partly in response to this growth and associated demand for office and hotel space, Fukuoka City Government has initiated the ‘Tenjin Big Bang’ project described in Section 5.", "The Tenjin Big Bang regulations do incentivise pro-environmental actions in redevelopment by granting an increase in floor-area ratio for reduction of environmental impact (Fukuoka City Government, 2018c).", "This may help to encourage greening, given that Fukuoka City Government has tried, so far without success, to include greening actions within building codes due to perceived conflict with economic development (interview with Fukuoka City Government Green City Promotion Department, April 2018).", "However, although there is no clear opposition in Fukuoka yet, similar experiences in Taipei indicate that if not managed sensitively, such initiatives to engage developers in urban greening initiatives can backfire and/or be met with scepticism by pro-environmental and civil society stakeholders if they are perceived as giving developers too much power in planning processes (Mabon & Shih, 2018).", "There are also challenges in Fukuoka which reflect the general adaptation situation for Japanese cities.", "These include the still strong focus on mitigation compared to adaptation in the Cool and Adapt Project, which tallies with Kameyama's (2016) general observation on Japanese municipal climate policy.", "Respondents also commented (interview with KEEA, April 2018) that the conditions of government-funded projects sometimes made data sharing or re-use for further research into local climate adaptation difficult, an observation also made for Tokyo by Hijioka et al. (2016).", "This City Profile has illustrated that within Japan, Fukuoka has made relatively early progress on local governance of climate change adaptation, particular as regards consideration of specific implementation actions when compared to cities in western Japan of similar size and climate.", "What is perhaps most distinctive is that there is in Fukuoka a robust body of scientific evidence produced by local scholars and institutions on which municipal policymakers may draw to support their decisions.", "Our profile also supports the argument that whilst climate change adaptation may be a relatively new area for local governments in Japan, there are already comprehensive policy frameworks in place in areas such as environmental protection, disaster prevention, and appropriately-informed greenspace planning which can act as a foundation to facilitate actions necessary for climate adaptation (Hijioka et al., 2016; Morimoto, 2011).", "In Fukuoka, greenspace planning policies appear particularly well-suited to adapting to the effects of increased heat and rainfall, in that explicit consideration of climate adaptation may be considered only the latest iteration in the city of a much longer policy and research interest into how the urban green environment can be configured to create a ‘liveable environment’ for citizens.", "Nonetheless, there are limitations to the applicability of Fukuoka's experience with climate adaptation to other international cities.", "Whilst Fukuoka is indeed an expanding, subtropical, medium-sized city in Asia, the current and historical context makes it a distinctive and in some ways anomalous case.", "The strong technical and scientific competences in understanding the local environment held by local institutions and researchers in Fukuoka, and the associated drive to connect this with policy-making and undertake research in the public interest, may to an extent be the result of Kyushu's specific historical experiences with pollution.", "Fukuoka is also a large, well-resourced and still-growing city in a wealthy country context.", "This may afford the city access to funds to support the development of comprehensive climate and environmental policies, and also aid the recruitment and training of technically capable local government staff.", "Whether such actions would be possible to the same extent in other rapidly developing subtropical Asian cities without this level of resourcing and the specific social and environmental history is open to question." ]	[ "Fukuoka among early adopters of municipal climate change adaptation action in Japan", "Fukuoka demonstrates adaptation responses for an expanding sub-tropical Asian city.", "Understanding of wind corridors and cooling by greening informs Fukuoka city plans.", "Adaptation via planning continues much longer trend of liveable urban environment.", "Challenges remain in balancing economic development pressures with climate actions." ]
More than words: Judgments of politicians and the role of different communication channels	We collected short video clips of speakers and created five types of stimuli: (1) the original videos, (2) the audio tracks only, (3) single pictures only, (4) speech content, and (5) stick-figure animations displaying body motion. Participants rated these stimuli on a brief Big Five personality inventory. We then used ratings of the incomplete information conditions to predict ratings of the original video condition. Impressions in the audio track condition were strong predictors throughout all trait ratings. However, other cues were also non-negligible contributors to an overall impression. People even make sense of parsimonious cues, e.g., an animated stick-figure. Thus, presenters on a public stage are not only judged by what they say but also by how they move.	[ "PSYC" ]	[ "Body movements", "Impression formation", "Nonverbal communication", "Personality", "Snap judgments", "Thin slices" ]	[ "3200", "3207" ]	[ "When people form impressions about others, words often seem to affect them less than the observed outward appearance and nonverbal behavior.", "Visual and auditory cues affect people’s judgments of their interaction partners; judgments that are made spontaneously, effortlessly, and without conscious processing (Ambady, Bernieri, & Richeson, 2000; Sunnafrank & Ramirez, 2004).", "On the one hand, first impressions can be misleading and a source of prejudices and stereotyping (Zebrowitz & Montepare, 2005).", "On the other hand, they can pick up relevant information about one’s social environment.", "After being exposed to brief extracts of nonverbal or verbal information, people are able to assess other people’s actual personality, their job performances, or a CEO’s abilities to generate company profits (Albright, Kenny, & Malloy, 1988; Ambady et al., 2000; Borkenau & Liebler, 1992a,b; Hecht & LaFrance, 1995; Rule & Ambady, 2008; Scherer, 1978).", "Irrespective of whether they are the key to someone’s actual personality traits or abilities, snap judgments can have a strong impact on impression formation and decision making.", "This is greatly important for those who enter the public arena.", "Politicians and leaders who vie for media attention and try to win the approval of an audience have to be aware that they are not only judged by the content they present.", "Nonverbal and salient cues are assumed to be processed efficiently and easily remembered and for this reason they can dominate over verbal information (Clark & Paivio, 1991) Their impact may even be more prevailing nowadays because news reports have been undergoing a shift from political content to image bites (see Bucy & Grabe, 2007; Stewart, 2010).", "Moreover, the flood of information that people are confronted with daily imposes an additional cognitive load, which increases the propensity to take mental shortcuts when making decisions (Olivola & Todorov, 2010).", "Thus, people may tend to choose their leaders not after careful deliberation but on the basis of superficialities.", "Empirical studies underscore that appearance cues and nonverbal behaviors influence judgments of politicians and other leaders.", "Research on the perception of charisma showed that potential leaders who display expressive non-verbal behaviors (i.e., more body gestures, more variations in intonation, more eye-contact, etc.) are seen as more charismatic than persons who showed less non-verbal behaviors (Awamleh & Gardner, 1999; Gardner, 2003; Holladay & Coombs, 1993, 1994).", "Moreover, experiments using manipulated voices revealed that vocal cues affect people’s attributions of leadership qualities and their voting behavior (Klofstad, Anderson, & Peters, 2012; Tigue, Borak, O’Connor, Schandl, & Feinberg, 2012).", "People even read leadership qualities, such as, competence, trustworthiness, or dominance, into photographs of political candidates.", "Interestingly, the consensus among such ratings is strong enough to make them reliable predictors of hypothetical voting decisions and actual election outcomes (Antonakis & Dalgas, 2009; Ballew & Todorov, 2007; Banducci, Karp, Thrasher, & Rallings, 2008; Little, Roberts, Jones, & DeBruine, 2012; Olivola & Todorov, 2010).", "All these empirical findings point to nonverbal cues as the prevailing influence on the perception of leaders and politicians.", "However, some researchers who compared the relative impact of visual, vocal, and verbal information on judgments of politicians came to different conclusions (Krauss, Apple, Morency, Wenzel, & Winton, 1981; Nagel, Maurer, & Reinemann, 2012).", "They found that speech content dominates over nonverbal information.", "This does not undermine the role of nonverbal behaviors in human communication but indicates that their influence varies with the situation in which behaviors are performed as well as with audience motivation and involvement (Allwood, 2002; O’Sullivan, Ekman, Friesen, & Scherer, 1985; Petty, Cacioppo, & Schumann, 1983).", "In summary, regardless of on which communication channel (i.e., vocal, visual, or verbal) people’s first impressions are based when making inferences of social relevance, research clearly shows that “thin slices” of behavior or appearance cues can have a strong impact on which traits and abilities people read into their social environment.", "Body motion is a form of nonverbal channel that comprises hand gestures, movements of the head, or position shifts of the whole body.", "Although the current study provided data on the interplay of different communication channels in impression formation we mainly focus on the relative role of body motion.", "Empirical studies have found that people are very adept at extracting information from motion cues.", "Even abstract stimuli such as circles or triangles flitting around on a screen, are often interpreted as animal or human behavior and elicit attributions of intentionality and personality (Heider & Simmel, 1944; Koppensteiner, 2011; Scholl & Tremoulet, 2000).", "Research on human body motion was strongly influenced by the point light approach, which was introduced by Gunnar Johansson (1973).", "He and his colleagues attached point lights and reflective markers to persons’ major joints and filmed them so that only a set of dots were visible in the resulting movies.", "Supporting the role of motion in human perception, these kinds of stimuli only became human-like when the dots were moving, whereas observers who saw single pictures of the movies perceived nothing but a random distribution of dots.", "Other researchers were inspired by Johansson’s methodical approach and demonstrated that such point light displays contain enough information to make quite accurate guesses of other people’s age and sex (Dittrich, Troscianko, Lea, & Morgan, 1996; Kozlowski & Cutting, 1978; Montepare & Zebrowitz-McArthur, 1988; Troje, 2002).", "Research using more elaborated versions of this technique or alternative methods of motion capture revealed that motion cues convey information of social relevance (Blake & Shiffrar, 2007; Chouchourelou, Matsuka, Harber, & Shiffrar, 2006).", "People appear to be able to perceive affect in arm movements (Pollick, Paterson, Bruderlin, & Sanford, 2001), emotions in movements of the whole body (Atkinson, Dittrich, Gemmell, & Young, 2004; Clarke, Bradshaw, Field, Hampson, & Rose, 2005), and personality in patterns of human gait (Thoresen, Vuong, & Atkinson, 2012).", "The frequency and duration of motion and other kinematic features play a role in mating behavior (Bente, Donaghy, & Suwelack, 1998; Grammer, Honda, Juette, & Schmitt, 1999) and affect the way females judge the attractiveness of male dancers (Neave et al., 2011).", "Moreover, self-ratings and observer-ratings of personality on scales measuring sensation seeking or the Big Five personality dimensions are related to the motion behavior of dancers (Bechinie & Grammer, 2003; Hugill, Fink, Neave, Besson, & Bunse, 2011; Luck, Saarikallio, Burger, Thompson, & Toiviainen, 2010).", "In the domain of politics, variations in body motion influence how people judge the personality and health of politicians (Kempter, 1998; Koppensteiner, 2013; Koppensteiner & Grammer, 2010; Kramer, Arend, & Ward, 2010).", "Such results clearly show that motion cues are an equally important nonverbal communication channel as appearance cues (e.g., clothes), vocal cues (e.g., voice pitch) and facial expressions.", "The speed, the duration, and the flow of a gesture or variations in the movements of the whole body seem to have a non-negligible impact on how people form first impressions of their social environment.", "Human communication works on different levels ranging from symbolic information mostly conveyed by verbal content to information that has no definite signal character and is expressed by certain qualities of motion.", "The studies of Mehrabian (1972) and those on the perception of charisma (see above) provide evidence that under some experimental conditions, visual information exerts a dominant influence in impression formation followed by voice quality and speech content on the last position.", "In addition, comparison of trait judgments based on full channel information (i.e., video with speech) with trait judgments based on incomplete information only (e.g., silent videos or voice only) indicates that cues from different communication channels convey redundant information (Borkenau & Liebler, 1992a; Friedman, Oltmanns, Gleason, & Turkheimer, 2006).", "However, there are variations.", "Some traits appear to be preferably ascribed to visual cues, while other traits are preferably ascribed to cues from other modalities (Friedman et al., 2006; Gifford, 1994; Naumann, Vazire, Rentfrow, & Gosling, 2009; Zebrowitz-McArthur & Montepare, 1989).", "Moreover, when asked to identify emotional expressions or statements of agreement and disagreement in political debates people are more accurate when multimodal information is available (Bänziger, Mortillaro, & Scherer, 2012; Mehu & van der Maaten, 2014).", "In this study we examined to what degree body motion affects social judgments relative to information from other verbal and nonverbal communication channels.", "To accomplish this, we broke down short video clips of politicians making a speech into five different versions of stimuli.", "Independent samples of participants then judged either the full channel version of the speeches, sound only, single pictures taken out of the speeches, the content of the speeches read by a computer voice, or stick-figure animations displaying the body movements of the speakers.", "Measures of the participants’ first impressions were obtained using a brief questionnaire that builds on the five-factor model of personality (i.e. Big Five), because the five-factor model had already been successfully applied in numerous “thin slices” studies (e.g., Borkenau & Liebler, 1992a,b; Friedman et al., 2006).", "These measures allowed us to estimate to what extent information from different communication channels influences snap judgments of the speakers and how different “portions” of nonverbal and verbal information are related to body motion.", "Our study extends previous research in several aspects.", "First, other studies on nonverbal communication often instruct actors to display specific behaviors.", "In contrast to that, the stimuli we used were real politicians that had given their speeches in the German parliament.", "Hence, ecological validity of the displayed behaviors is high.", "Second, although our experimental design was inspired by previous studies on the role of different communication channels (Borkenau & Liebler, 1992a,b; Friedman et al., 2006), these studies did not examine the behavior of speakers in a public arena.", "Finally, we applied other tools for stimulus preparation and stimulus presentation than other studies in the field.", "By translating the body movements of the speakers into animated stick figures, we diminished the influence of confounding variables and were able to determine for which traits body motion was a strong predictor and to what degree it interacts with information from other nonverbal and verbal sources.", "In previous work we already used stick-figure animations as stimuli and related data-driven descriptors of body motion (e.g., measures of amplitude height) to judgments of personality.", "This revealed that people form first impressions on the basis of simple cues embedded in the behavioral stream.", "However, it was unclear whether and to what extent body motion affects judgements of speakers when vocal and other cues are also available to observers (Koppensteiner & Grammer, 2010).", "The work presented here, which is based on the same method of motion capturing but on a new set of stimuli, is a first step to overcome such limitations.", "It is suggested that rapidly evaluating other people’s intentions, assessing one’s social environment on broad trait categories and making fast decisions on whether to approach or avoid an individual has an adaptive function (e.g., Buss & Greiling, 1999; Oosterhof & Todorov, 2008).", "Misjudgments can have negative consequences and for this reason people may base their social evaluations on cues from different communication channels because this makes evaluations more reliable.", "Previous work has already shown that first impressions of extraversion, agreeableness and emotional stability are related to motion cues and gesturing (Borkenau & Liebler, 1992b; Gifford, 1994; Koppensteiner, 2013; Koppensteiner & Grammer, 2010).", "Hence, we hypothesized that for these personality dimensions judgments of the speakers’ body movements (i.e., stick-figure animations) can serve as predictors of people’s judgments in the full channel condition (i.e., video plus sound).", "Moreover, we expected pronounced links between ratings of the voice only condition and ratings of body motion, because it is well established that gesturing accompanies speech (e.g., McNeill, 1985).", "Appearance cues and vocal cues have been found to be related to all dimensions of the Big-Five (Borkenau & Liebler, 1992b; Friedman et al., 2006; Naumann et al., 2009).", "Consequently, we expected to replicate such findings in our study.", "Research on nonverbal communication revealed speech content to play a minor role when people form first impressions (e.g., Awamleh & Gardner, 1999).", "However, there seems to be a link between conscientiousness and speech content (e.g., Friedman et al., 2006), which we also expected to reveal in our data.", "Sixty speeches taking place in the German parliament (30 male and 30 female speakers) were randomly selected out of three parliamentary sessions (November 29 & 30 and December 14, 2012).", "The selected speakers were non-prominent members of the parliament and were unknown to our participants.", "At random, we extracted video segments with a length of 15 s from these speeches.", "Afterwards, the video segments were used to create five different stimulus types for the experimental conditions described below.", "For the full channel condition we merely removed the lower portion of the video clip, which gave information about the speaker’s name and party affiliation.", "Consequently, in this experimental condition the participants of a rating experiment assessed the original video clips, which included all visual and all auditory information.", "To eliminate information on body movement we chose one picture out of the frame series of each video segment.", "Each of these pictures showed the speakers adopting similar body postures.", "For more detail, we chose pictures with the speakers standing upright having their arms positioned at their sides.", "In accordance with the length of all other stimulus types pictures were shown for 15 s in the rating experiment.", "For this experimental condition, we extracted the audio track from each original video segment.", "Thus, visual information was removed and the participants assessing such stimuli only listened to what was being said.", "We extracted what said during the selected video segments from the transcripts of each parliamentary session.", "These transcripts were converted into audio files using the document reader software Ghostreader.", "Because all transcripts were read aloud by the same computer voice during the rating experiment, they served as the control condition for speech content and wording.", "The main focus of the study was on the analysis of body motion in relation to information presented in the other conditions.", "To this end we created stimuli, which display body motion without confounding information, e.g., appearance cues.", "To accomplish this, we used a program that allows running through the video clips step by step.", "In the first frame of each video clip, we positioned landmarks on the speaker’s forehead, the hollow of the throat between the collar bones, ears, shoulders, elbows, hands, a spot in the middle of the body near the navel, and the corners of the lectern.", "Motion behavior occurring between single frames of the video clips was traced by rearranging the landmarks with the computer-mouse and software routines that automatically tracks positions shifts (Koppensteiner, 2013).", "Based on this data, we created stick figure animations, which served as abstract representations of the speakers’ body movements.", "To reduce the workload during the encoding process, we only used every third frame and filled in missing frames by linear interpolation.", "A total of 308 Caucasian participants (i.e., students of the Faculty of Life Sciences) was recruited at the University of Vienna for taking part in the rating experiments.", "Sixty-five persons (37 females and 28 males; M-age = 23.3 years, SD = 5.8) participated in the full channel condition, 60 people (35 females and 25 males; M-age = 22.8 years, SD = 3.4) in the static visual condition, 63 (33 females and 30 males; M-age = 23.8 years, SD = 4.4) in the original voice condition, 60 participants (35 females and 25 males; M-age = 23.4 years, SD = 4.5) in the artificial voice condition, and 60 persons (33 females and 27 males; M-age = 22.5 years, SD = 3.7) in the stick figure condition.", "Participants received a financial compensation of €5.", "Participants were approached in person and asked to take part in a short rating experiment.", "After accompanying them to our laboratory, we informed the participants briefly about the experimental set-up and the task to be accomplished.", "Subsequently, participants performed the rating tasks on their own using a computer-controlled interface.", "They were instructed to rate the stimuli after they had watched or listened to them.", "Pictures in the static visual condition were shown for 15 s, but participants were told that they could start their ratings whenever they felt to be ready for them.", "In the full channel condition, the original voice condition, and the artificial voice condition, participants wore headphones (AKG K 272 HD) connected to the computer to optimize sound quality.", "Stimuli were presented on the left-hand side of the user interface, while rating scales were displayed on the right hand side.", "Participants completed their ratings by dragging a slider to the favored position between the right pole (i.e., named, strongly disagree) and the left pole (i.e., named, strongly agree) of the scale using a computer mouse.", "The slider position corresponded to a position on a scale divided into 200 subunits.", "All ratings started with the slider being in the neutral position (i.e., middle position on the slider bar).", "To examine personality ratings of the speakers, we used the German version (Muck, Hell, & Gosling, 2007) of Gosling, Rentfrow, & Swann’s (2003) Ten Item Personality Measure (TIPI).", "This questionnaire is based on the five-factor model of personality and covers personality dimensions openness, extraversion, conscientiousness, emotional stability, and agreeableness.", "In each experimental condition, each participant rated a subset of 20 stimuli that were randomly selected from the 60 stimuli available.", "Trait ratings were averaged for each condition and each stimulus.", "This yielded a dataset containing 60 speakers that were rated on ten items in five experimental conditions.", "Corresponding items of the TIPI questionnaire were turned into the Big Five personality dimensions according to the instructions by Gosling et al. (2003).", "Because each personality dimension only comprised two items, internal consistency of the questionnaire was determined by calculating the Spearman–Brown coefficient (Eisinga, Te Grotenhuis, & Pelzer, 2013).", "To estimate the relative influence of voice, speech content, appearance, and body motion on judgments of personality, we correlated ratings collected in the experimental conditions presenting incomplete information with ratings of the full channel condition (i.e., original video clips).", "We also performed multiple regression analyses using ratings in the full channel condition as criterion, ratings of the incomplete information conditions as predictors and the speakers’ sex as control variable.", "We expected these predictors to be intercorrelated and distorted due to multicollinearity.", "For this reason we also calculated so-called relative weights.", "Relative weights or relative importance weights range between 0 and 1, are unaffected by multicollinearity, and therefore very helpful when interpreting each predictors’ relative contribution in the regression model (Johnson, 2000; Kraha, Turner, Nimon, Zientek, & Henson, 2012; Lorenzo-Seva, Ferrando, & Chico, 2010).", "Moreover, bivariate correlations between predictor variables provided insight into the interrelations between ratings of the incomplete information conditions.", "Previous studies comparing different communication channels found correlations of .35 or higher between nonverbal cues and personality ratings (Borkenau & Liebler, 1992b; Friedman et al., 2006; Koppensteiner, 2013).", "We used different modalities as predictors in a regression analyses and assumed their effects to add up.", "Consequently, we expected the multiple regressions we performed to explain at least 20 percent of overall variance.", "On the basis of such an effect size, an alpha level of .05, a power level of .8, and five predictors an a priori power analyses suggested an optimal sample size of 51 stimuli.", "All statistical analyses were carried out in the program R (R Core Team, 2013).", "Descriptive Statistics are presented in Table 1 and internal consistencies between corresponding items of the questionnaire in Table 2.", "Coefficients ranged from .06 to .94 and thus were unacceptably low in some cases.", "In particular, the item pairs anxious–easily upset and calm–emotionally stable did not combine properly in the stick figure, in the original voice, nor in the full channel condition.", "This might have been due to a weakness of the questionnaire on this dimension or to the choice of stimuli.", "Politicians on a public stage may rarely show body movements or produce vocal cues in which people perceive qualities associated with the items anxious and easily upset.", "To sum up, internal consistencies we obtained for emotional stability were very low and for this reason we discuss results for this personality dimension on the basis of single items (Tables 3–5).", "Statistical analyses of the relationships between incomplete information conditions and the full channel conditions are presented as correlation coefficients, relative weights, and regression estimates (see Tables 3 and 4).", "Results for extraversion revealed a strong relationship of the ratings in the stick-figure condition and the original voice condition with the ratings in the full channel condition.", "In addition, there was also a noteworthy relationship between ratings in the static visual condition and the full channel condition.", "These relationships were reflected in all types of analyses we applied (i.e., see correlations in Table 3, and estimates and relative weights in Table 4).", "We therefore came to the conclusion that in our setting, judgments of extraversion were mainly affected by the speakers’ voice and by their body motion as well as—to minor degree—by the speakers’ appearances.", "The content of the speeches had no important effect on ratings of extraversion.", "Perceived conscientiousness in the full channel condition yielded high correlation coefficients with ratings of conscientiousness in the static visual condition, in the original voice condition, and the artificial voice condition (i.e., computer voice presenting speech content).", "Similar results were obtained for estimates of the multiple regression and the relative weights (Tables 3 and 4).", "Consequently, impressions of conscientiousness were mostly guided by vocal information.", "However, appearance cues and speech content also played a role.", "Body motion was revealed as the weakest predictor.", "Ratings of perceived openness in the full channel condition were strongly related to ratings in the original voice condition and the static visual condition (Tables 3 and 4).", "Correlation coefficients also provided a noteworthy effect size for speech content (Table 3).", "In conclusion, participants mainly tended to ascribe openness to the speaker’s appearances and their voices.", "Full channel ratings of agreeableness showed strong bivariate correlations with ratings in the original voice condition, ratings in the stick-figure condition, ratings in the artificial voice condition and a noteworthy one for ratings in the static visual condition (see Table 3).", "These patterns of relationships were not in accordance with the ß-weights of the multiple regression (Table 4).", "It only provided strong estimates for the static visual condition and the original voice condition.", "This was an indicator of multicollinearity, which distorts ß-weights and, therefore, undermines accurate interpretation of the data.", "Relative weights, which are unaffected by multicollinearity, replicated the patterns found by the bivariate correlations (Table 3).", "Thus, we concluded that ratings of agreeableness were guided by appearance cues, by body motion, by vocal cues and by the verbal content the speakers presented.", "Due to the low internal consistencies results for emotional stability were analyzed on the level of single items.", "The item pair calm–emotionally stable yielded notable correlation coefficients and relative weights for all conditions, whereas regression estimates labeled ratings of the original voice condition as strong predictors and ratings of body motion and appearance as predictors with a moderate influence (Tables 3 and 4).", "The item pair anxious–easily upset provided a strong correlation of full channel ratings with original voice ratings and noteworthy ones with ratings of static visual cues and with ratings of body motion (Table 3).", "A similar pattern was provided by the regression estimates and the relative importance weights (Table 4).", "Analyses of the interrelations between ratings in the incomplete information conditions revealed strong correlations between voice and the content the speakers presented for nearly all personality ratings (Table 5).", "This is not very surprising, because the voice only condition comprised nonverbal vocal cues as well as the content of the speeches.", "We also found strong relationships between body motion (i.e., ratings of stick-figure animations) and voice for the personality dimensions extraversion and agreeableness (Table 5).", "This complements results obtained in the regression analyses.", "It shows that body motion was not only a communicator of agreeableness and extraversion but also is accompanied by vocal cues that convey similar information.", "Thus, for judgments of some personality dimensions there appears to be a coupling between voice and body motion.", "Perceived extraversion and conscientiousness showed notable relationships between different communication channels (Table 5).", "For instance, we found a link between appearance cues and speech content for these personality dimensions.", "This hints that people are able to form expectations about what politicians will say on the basis of their appearance or form expectations about their appearance on the basis of what they say.", "The most prominent finding of our study was that all personality ratings of the original voice condition, in which the participants only listened to an audio track of the selected speeches, were strongly related to corresponding ratings of the full channel condition, in which participants watched the unaltered original video clips.", "This was mostly due to paralinguistic cues such as intonation and pitch because the strong impact of the speakers’ voice on personality judgments did not disappear when speech content was controlled.", "Other studies also revealed that vocal cues are an important influential factor in impression formation (Borkenau & Liebler, 1992a; Friedman et al., 2006).", "However, they did not play such a predominant role as in our study.", "Participants were, of course, aware that they were judging politicians.", "For this reason, they might have attended more to the speaker’s voices than to other information.", "The majority of studies that compared the impact of different communication channels did not use politicians for their experiments or instructed actors to display certain behaviors (e.g., Awamleh & Gardner, 1999; Borkenau & Liebler, 1992a; Friedman et al., 2006).", "This is different from our study design and might explain why vocal cues were a strong contributor.", "Actors may overdo their acting and display stereotypical behaviors thereby drawing attention to cues that are less salient when real politicians present themselves.", "On the other hand, judging politicians may raise different expectations than judging people drawn from an average population.", "Judgments in the original voice condition did not only convey speech content but also information about intonation, voice pitch, and other qualities of the voice.", "To separate such vocal cues from speech content, we also collected ratings of the speeches’ wording read by a computer voice and related these ratings to ratings of full channel condition.", "As expected full channel judgments of conscientiousness, but also judgments of agreeableness and one item pair of the personality dimension emotional stability (i.e., calm, emotionally stable) could be predicted by speech content to a certain degree.", "In other words, participants read some traits and qualities into what the speakers said and not only how they said it.", "Unlike some other studies we were unable to show that verbal content dominates over nonverbal cues in judgments of politicians (Krauss et al., 1981; Nagel et al., 2012).", "Nevertheless, the results obtained indicate that people integrate speech content when forming first impressions.", "Static visual information, which was presented as single pictures, yielded notable relationships with the full channel condition.", "Previous research has already shown that appearance cues alone—clothing styles, physiognomic features and facial expressions, just to give a few examples—affect people’s first impressions (e.g., Naumann et al., 2009).", "We have supported such findings by demonstrating that static visual cues contribute to overall impressions of a person.", "Consequently, people form an impression on the basis of static cues before any motion behavior occurs or a word is spoken.", "The documented effects were particularly strong for ratings of conscientiousness and openness.", "Participants had no information about party membership, but static visual cues may already reflect a conservative or a more progressive mindset and this may more strongly guide attributions of openness and conscientiousness than attributions of other personality dimensions.", "Future studies including facial expressions, physiognomic features, and clothing style as variables in the analyses could support such assumptions.", "The study’s main focus was to estimate the relative role of body motion when people judge politicians giving a speech.", "To accomplish this, we translated the speakers’ body motion into animated stick-figures.", "Full channel ratings of extraversion, agreeableness, and an item of emotional stability were strongly related to corresponding ratings of these abstract stimuli.", "Although other communication channels, in part, showed stronger effects over a wider range of ratings, it is still surprising that a parsimonious cue, as simple as a stick-figure, can guide social perceptions.", "In previous research, where we applied the same method of motion capturing, we revealed that people associate personality traits with salient and simple nonverbal cues that are embedded in the behavioral stream (Koppensteiner, 2013; Koppensteiner & Grammer, 2010).", "In accordance with the results obtained here we found strong relationships of certain motion descriptors with ratings of agreeableness and extraversion.", "For instance, speakers who produced expansive vertical movements (i.e., mostly up and down movements of the hands) were judged as highly extraverted while speakers who showed less such vertical movements and a greater overall variation in motion amplitudes tended to be judged as agreeable.", "Strong effects were also found for emotional stability.", "Speakers who displayed jerky movements were perceived as less emotionally stable than speakers who displayed smoother movements.", "This might explain why one item of emotional stability also produced pronounced effects in this study.", "We did not examine body movements on the level of motion cues in the work presented here.", "Consequently, the next step in this line of research would be to use motion descriptors such as those presented in our previous work and test in what way they are related to cues from other communication channels (e.g., auditory cues).", "Extraversion is often attributed to conspicuous and expressive behaviors (Kenny, Horner, Kashy, & Chu, 1992).", "For this reason, it is a trait that may be easily read from simple behavioral cues, including body motion.", "Similarly, appearing friendly is crucial when approaching or avoiding a stranger and therefore cues of agreeableness may also be easy to detect and even visible in body motion.", "Politicians often attack their opponents, make points with great vigor, and underline their arguments with expressive gestures.", "Trait ratings belonging to emotional stability—e.g., calmness—might be influenced by these expressive displays; this might explain why the participants of our experiments can use body motion for such attributions.", "Perceived agreeableness and extraversion of the stick-figure animations showed a strong coupling to perceived agreeableness and extraversion in the voice only condition.", "This underlines that speech is accompanied by body motion and gesturing (McNeill, 1985) and indicates that cues people associate with extraversion and agreeableness convey essential social information.", "To sum up, like other research in the field, our findings indicate that people perceive social meaning in a great variety of visual, auditory, and verbal information.", "In addition to this, we demonstrate that, for some important traits, abstract animations displaying the speaker’s body movements predict people’s first impressions.", "We randomly extracted short video clips from speeches that were given in the German parliament.", "These clips were then turned into five different types of stimuli, which were rated on the Big Five personality taxonomy.", "Stimuli were either (1) short extracts of the original videos, (2) audio sequences of the speeches, (3) speaker’s body movements turned into stick-figures, (4) single pictures of the speakers, or (5) the content of the speeches read by a computer voice.", "To estimate the influence of these different communication channels on impression formation we related ratings in the incomplete information conditions to ratings in the original full channel video clip condition.", "People make sense of brief displays of nonverbal and verbal information and although such snap judgments can be a source of false beliefs and prejudices, they often convey socially relevant information (e.g., Ambady et al., 2000).", "Studies examining the influence of different communication channels on impression formation found that people read information into and from a variety of cues.", "Most research suggests that visual cues have a prevailing influence on social judgments and that speech content only plays a minor role (e.g., Awamleh & Gardner, 1999; Mehrabian, 1972).", "Other researchers were unable to replicate such findings, which supports the idea that the influence of nonverbal and verbal cues varies according to context and audience involvement (e.g., O’Sullivan et al., 1985; Petty et al., 1983).", "In this study, we found a prevailing effect of vocal cues on impression formation but the relative role of different communication channels may, of course, depend on different factors that can hardly be tested and controlled in a single study.", "Future studies should thus extend the current experimental set-up by manipulating the salience of cues (e.g., manipulating vocal parameters), refine analyses by describing behaviors and appearance cues in more detail (e.g., clothing style), and include different contextual information (e.g., party affiliation, status).", "Also, politicians acting in the public arena may trigger differential judgments than the stimuli some other researchers used to investigate the impact of different communication channels (e.g., Borkenau & Liebler, 1992a; Friedman et al., 2006).", "As in our study, Krauss et al. (1981) as well as Nagel et al. (2012) collected ratings of politicians and found that people mainly judge them by verbal content.", "This is partly in accordance with our findings because we also revealed a non-negligible relationship between speech content and some trait ratings.", "However, the outcomes of the regression analyses suggest that, in our study, prosody and voice qualities had a marked impact on the participants’ judgments.", "Overall, the results obtained show that people’s first impressions are guided by cues from different communication channels.", "Put simply, in real life encounters there is no single cue that creates a first impression.", "Our analyses supports this by showing that in most cases single modalities (e.g., body motion, voice only) explained less variance than combinations of these modalities.", "Consequently, when making their judgments people seem to rely on a great variety of nonverbal and verbal cues.", "Also, some modalities such as voice and body motion are intertwined and communicate redundant information to a certain extent, while other modalities do not show such a redundancy.", "For instance, conscientiousness was more easily attributed to speech content than extraversion.", "Irrespective of that our results support the idea that people’s first impressions are guided by information from different communication channels (Bänziger et al., 2012; Mehu & van der Maaten, 2014).", "By translating the speakers’ nonverbal behavior into animated stick-figures, we created an abstract stimulus that served as representative of body motion.", "It has already been shown that parsimonious cues displaying motion behavior communicate socially relevant information (e.g., Blake & Shiffrar, 2007; Chouchourelou et al., 2006).", "In this study, we extended such research by comparing the influence of cues conveyed by a moving body with information from other communication channels.", "We were able to show that, for perceptions of extraversion and agreeableness, body motion is a major player.", "Given that our stick-figure animations reduce information to an abstract stimulus with an artificial appearance, the results obtained are impressive.", "They indicate people do not only ascribe intentions and personality traits to abstract representatives of body motion, but they also use motion cues to form impressions of their interaction partners.", "Rapidly categorizing another individual’s intentions, traits and behavioral tendencies may be an ability that has been formed during human evolution (Buss & Greiling, 1999; Fiske, Cuddy, & Glick, 2007).", "However, some traits are assumed to be of higher social relevance in first encounters because they inform the decision whether to approach or avoid an individual.", "For instance, Oosterhof and Todorov (2008) found that judgments of neutral faces are reducible to the independent dimensions of valence (i.e., represented by facial trustworthiness) and dominance.", "They further suggest that evaluations on these dimensions are overgeneralizations for inferring harmful intentions.", "Other research has shown that attributions of extraversion to a stick-figure animation show a strong relationship with perceived dominance, while perceived agreeableness is strongly related to perceived trustworthiness (Koppensteiner, Stephan, & Jäschke, 2015).", "This supports the conclusion that people have a higher sensitivity for cues of extraversion and agreeableness because these belong to basic categories of social evaluation with a direct link to survival.", "For this reason, overgeneralizations with regard to potential threats may not only occur when faces are assessed but also when people categorize body motion.", "Furthermore, in contrast to facial information, body motion is recognizable from greater distances.", "It is thus conceivable that motion cues are socially relevant because they allow decision making as another individuals approach, before these individuals come too close.", "In real life situations, nonverbal and verbal information does not disintegrate into pieces.", "Our results, and other research, suggest that some cues are intertwined.", "A well-known link, for instance, is between speech and gesturing (e.g., McNeill, 1985).", "Gestures are sometimes used to emphasize what is being said or go together with voice intonation.", "Hence, an overlap in the communicative value of motion cues, speech content, and voice quality is well established and the results obtained in the present study provide further support for this.", "Future research could investigate such an overlap in more detail by extracting certain motion cues as we have done in previous studies (see Koppensteiner & Grammer, 2010) and relating them to vocal cues.", "Follow-up studies with a more in depth analyses on the interrelations between different cues would also build a bridge to research that investigates how observers react to violations of such linkages (Weisbuch, Ambady, Clarke, Achor, & Weele, 2010).", "Presenting contradictory nonverbal and verbal information could show that there is indeed redundancy and that different cues might form one single cue with a common communicative value.", "This might also provide further support for work showing that judgments on the basis of multimodal information are more accurate (Bänziger et al., 2012; Mehu & van der Maaten, 2014).", "A next step in this kind of research would be to investigate how different nonverbal information influences decision making on important issues.", "Researchers have already shown that facial photographs from political candidates can be used to predict hypothetical or actual election outcomes (e.g., Antonakis & Dalgas, 2009; Little et al., 2012; Olivola & Todorov, 2010).", "Moreover, other studies reveal a link between vocal cues and leadership qualities (Klofstad et al., 2012; Tigue et al., 2012).", "Taking these as a starting point future research could extend into the domain of leadership research to examine how the interplay of different cues or multimodal cues affects decision-making.", "In summary, our findings indicate that people make use of verbal and different nonverbal cues when forming first impressions of politicians.", "Static visual cues, body motion, verbal content, and vocal cues were related to people’s impressions.", "Vocal cues were revealed as a strong predictor for all trait ratings in our study, yet other communication channels also proved important.", "The impact of body motion was particularly strong for ratings of agreeableness and extraversion were also linked to corresponding ratings based on the speakers’ voices.", "In other words, with regard to these personality dimensions body motion is coupled to information from the vocal channel.", "Cues communicating agreeableness and extraversion may reflect social abilities, which are helpful in social encounters and facilitate establishing interactions.", "Hence, people may be more sensitive to cues of extraversion and agreeableness; for this reason, they also read these traits into body motion." ]	[ "Speeches of politicians were divided into different portions of nonverbal information.", "First impressions were recorded on the basis of the Big-Five Personality Dimensions.", "Vocal information dominated impression formation throughout all conditions.", "Body motion strongly affected impressions of extraversion and agreeableness.", "People’s impressions are influenced by information from different nonverbal channels." ]
Investigating intergroup attitudes in Europe: Cross-national data on news media, attitudes towards newcomers, and socio-psychological indicators	This dataset is part of a multidisciplinary project that investigates the relationship between attitudes of the adult population towards ‘newcomers’, and news media consumption and trust in Belgium, France, the Netherlands, and Sweden in 2017 (N = 6000). These countries were chosen for their response to the 2015 refugee crisis in Europe, with each country having a different media context. We ran an online survey which included questions on socio-demographic characteristics, attitudinal indicators, and information on news media consumption and trust. The data are representative for age and gender in each country and can be of interest for researchers who wish to explore dynamics of attitude formation, particularly with regards to the role of news media, in the wake of the 2015 refugee crisis in Europe.	[ "MULT" ]	[ "Attitudes towards newcomers", "Europe", "News media consumption", "News media trust", "Refugees" ]	[ "1000" ]	[ "The data presented in this article were collected in the context of two projects: Image of Immigrants in the Media: Thought-Provoking Effects (IM2MEDIATE) and Focusing on Refugees and Inclusion in European News Diversity Studies (FRIENDS).", "The aim of these projects is to investigate the dynamic interplay between media representations of the current non-EU immigrant situation with a specific emphasis on the refugee situation on the one hand and the governmental and societal (re)actions on the other.", "With these data, we provide more insight into these societal reactions by investigating attitude formation.", "Through an online survey, we collected quantitative data on attitudes towards newcomers, news media consumption, frequency and valence of direct intergroup contact, threat frames, and socio-demographic characteristics for the adult population aged 18 to 65 in four European countries: Belgium, France, the Netherlands, and Sweden.", "We collected the data in cooperation with a Belgian polling agency and selected the methodology for its cost-effectiveness in cross-country research.", "This is also related to the country selection: the polling agency we worked with has a strong presence in the four countries, which facilitated fieldwork efforts.", "Adding additional countries was desirable, but the financial means for this were not available at the time.", "Respondents received an e-mail asking them to participate in a survey without specifying the subject matter, which was essential to avoid priming.", "Three weeks of fieldwork in September and October of 2017 resulted in a dataset of 6000 respondents (1500 per country).", "Sample weights were applied to ensure that the sample is representative for gender and age in each country.", "The response rate was about 35% [1,2].", "Table 1 shows the distribution of respondents by several socio-demographic characteristics, while Table 2 presents mean scores of selected indicators on attitudes, news media consumption and trust, threat, and intergroup contact.", "Although several public survey platforms exist that measure public opinion on newcomers, insurmountable shortcomings in these national and international surveys stimulate us to collect new data, which includes information on news media exposure and news media trust (among others) alongside variables measuring public opinion/attitudes towards immigrants and refugees.", "To recapitulate, we developed an online public opinion survey, to be carried out amongst a sample of Belgian, French, Dutch and Swedish residents aged 18 to 65, representative for gender and age.", "The survey was distributed by iVOX, a market research and online polling agency active in all countries included in the dataset.", "The fieldwork included the use of incentives to maximize the response rate.", "Participants who completed the survey were entered into a pool to win a tablet at the end of the fieldwork period.", "The survey we have developed consists of eight themes: socio-demographic characteristics, media- and news consumption, trust in media, attitudes towards immigrants, contact with and knowledge on immigrants, attitudes towards and knowledge on refugees, culture of fear and psychological indicators.", "In what follows, we highlight several measures.", "All data were processed and cleaned through SPSS.", "As to accurately measure the difference in public opinion on immigrants and refugees, we decided to adapt a scale previously used in a rotating module of the European Social Survey.", "This module was created to measure immigration attitudes and was included in Round 1 (2002) and Round 7 (2014).", "The adapted scale consists of seven items asking which groups of immigrants should be allowed to come and live in Belgium: ‘Immigrants of the same race or ethnicity as most of the country's population.’", "; ‘Immigrants of a different race or ethnicity as most of Belgium's population.’", "; ‘Immigrants of the richer countries in Europe.”", "; “Immigrants of the poorer countries in Europe.’", "; ‘Immigrants of the richer countries outside Europe.’", "; ‘Immigrants of the poorer countries outside Europe.’", "; and ‘Immigrants coming from Muslim countries who wish to work in Belgium.’", "Answer categories range from 1 (Allow none) to 4 (Allow many).", "To collect the data, we presented the scale in its original form and added an extra item concerning immigrants from Muslim countries.", "The reason for the inclusion of this item lies in the fact that a majority of immigrants and refugees entering Europe in the current refugee crisis originate from Syria, Iraq or Afghanistan – predominantly Muslim countries [3].", "Prior to completing each block of items, respondents were presented with a definition of immigrants and refugees from the United Nations with the request they keep this definition in mind during completion of the survey.", "The definition of immigrants was as follows:", "An immigrant should be understood as covering all cases where the decision to migrate is taken freely by the individual concerned, for reasons of ‘personal convenience’ and without intervention of an external compelling reason (e.g., war, natural disaster) [4].", "(UNESCO, 2017, para. 3)", "The definition of refugees was:", "A refugee is someone who has been forced to flee his or her country because of persecution, war, or violence.", "A refugee has a well-founded fear of persecution for reasons of race, religion, nationality, political opinion or membership in a particular social group [5].", "(United Nations, 1951, p. 14)", "We clearly highlighted these two definitions so that respondents would be able to distinguish between immigrants and refugees and provide a reliable measurement of attitudes for each group [5,6].", "As evidenced by Table 2, mean scores indicate there are clear differences across the countries, French residents being most negative and Swedish residents being most positive towards both groups of newcomers.", "There are also differences between groups of newcomers: respondents hold more positive attitudes towards refugees than towards immigrants and are more likely to accept newcomers from the same ethnicity or originating from rich (European) countries.", "Respondents were asked about their media consumption patterns during the past month, with answer categories ranging from 0 (Never) to 7 (Every day).", "Both television and radio consumption were split into two groups: public and commercial broadcasters.", "For the classic newspaper and online news consumption, the most commonly read newspapers and commonly visited webpages in each region were included separately.", "This selection of newspapers was based on information concerning the circulation of newspapers in each country.", "Online and social media were also included.", "For online media, most of the websites of the newspapers that were included were selected, with some additional online-only news outlets.", "As for social media, the most commonly used media in 2017 were included.", "We measured trust in the news media brands by means of a five-point scale with answer categories ranging from 1 (No trust at all) to 5 (A lot of trust).", "Like the methodology for news media consumption, trust was measured separately for public and commercial television and radio.", "Trust in newspapers was aggregated into two groups: trust in quality newspapers and trust in popular newspapers to avoid multicollinearity in the data.", "Trust in news websites/apps, and trust in social media were also measured on the same scale.", "In the measurement on threat perspectives, we included several types of threat.", "A first set of indicators assesses economic (or realistic) threat.", "This was measured by asking four questions: ‘Would you say that refugees who come to live here generally take jobs away from workers in [country], or generally help to create new jobs?’,", "‘Most refugees who come to live here work and pay taxes.", "They also use health and welfare services.", "On balance, do you think refugees who come here take out more than they put in or put in more than they take out?’,", "and ‘Would you say it is generally bad or good for [country]'s economy that refugees come to live here from other countries?’.", "One additional item on realistic threat is ‘Average wages and salaries are generally brought down by refugees coming to live and work here.’", "Another threat perspective is on cultural threat and was measured through the following question: ‘Would you say that [country]'s cultural life is generally undermined or enriched by refugees coming to live here from other countries?’.", "Both types of threat were measured on an 11-point scale, with the high end of the scale signifying a low degree of threat.", "The only exception is the item on wages and salaries being brought down by refugees coming to live in the country, as this is coded on a five-point scale with the high end of the scale indicating strong agreement with this statement.", "A third type of threat is on fear of terrorism, which was particularly relevant to include given the current events in Europe around the time of the survey.", "We adapted the fear of terrorism-scale developed by Nellis and Savage [6].", "The scale consists of several hypothetical scenarios (‘Someday I may witness a terror attack’, ‘Someday I may be the victim of terror attack’, …), with answer categories ranging from 0 (Not likely at all) to 10 (Very likely).", "Whereas the original scale measured this fear for several scenarios separately (‘I could be on a plane that is hijacked’, ‘I could be on a subway or bus that is hijacked’), we have aggregated these into a single item (‘Someday I may be on a plane/subway/bus that is bombed’).", "Some of the original wording was also adjusted.", "Direct intergroup contact was measured by asking whether respondents have any interethnic friendships (no, some, or many), and how often they have interethnic random contact on the street, at work, in shops… [seven categories, ranging from 0 (Never) to 6 (Every day)].", "The valence of direct contact was measured by an 11-point scale, with 0 indicating a negative evaluation of intergroup contact, and 10 indicating a positive evaluation.", "These indicators on economic and cultural threat, and on direct intergroup contact stem from the rotating module on immigration in Round 1 and Round 7 of the European Social Survey [7]." ]	[]
Three-year performance of in-situ mass stabilised contaminated site soils using MgO-bearing binders	This paper provides physical and chemical performances of mass stabilised organic and inorganic contaminated site soils using a new group of MgO-bearing binders over 3 years and evaluated the time-dependent performance during the 3 years. This study took place at a contaminated site in Castleford, UK in 2011, where MgO, ground granulated blastfurnace slag (GGBS) and Portland cement (PC) were mixed with the contaminated soils in a dry form using the ALLU mass mixing equipment. Soil cores were retrieved 40-day, 1-year and 3-year after the treatment. The core quality, strength, and the leaching properties were determined via physical observation, unconfined compressive strength (UCS) and batch leaching tests. After 3-year treatment, the UCS values of ALLU mixes were in the range of 50–250 kPa; the leachate concentrations of Cd, Pb, Cu and Zn (except Ni) in all mixes were lower than their drinking water standards; and the leachability of total organics was in the range of 10–105 mg/L. No apparent degradation of the mass stabilised materials after 3 years’ exposure to the field conditions was found. MgO-GGBS blends were found able to provide higher strength and less leachability of contaminants compared to PC and MgO-only mixes in mass stabilised soils.	[ "ENVI" ]	[ "GGBS", "In-situ mass stabilisation", "Leachability", "MgO", "Strength" ]	[ "2304", "2305", "2307", "2310", "2311" ]	[ "Several techniques were applied to treat contaminated site soils over the past few decades, as contaminants can bring great risk to humans and animals [1,2].", "Among these techniques cement-based soil mix technologies have been widely employed and investigated by a large number of studies [3–7].", "Mass stabilisation as one of these soil mix technologies is effective and environmentally friendly for combining contaminated soil remediation and ground improvement treatments on the same site.", "It has been implemented for a few brownfields in the Nordic countries during the past few decades [8].", "Portland cement (PC) is the most popular material in soil mix technologies.", "However, the production of PC is known to be highly energy consuming and releases ∼5–10% CO2 during its production process [9].", "Under the drive of sustainability, by-products and novel binders such as ground granulated blastfurnace slag (GGBS) and MgO are drawing people’s attention recently [9,10].", "It was argued that MgO-based binders have advantages over CaO-based binders and provides additional unique benefits due to their adjustable properties and improved pH buffering capacity [10–12].", "The immobilisation mechanism of MgO is mainly through precipitating the hydroxides and encapsulating contaminants into its hydration products (e.g. brucite).", "The main advantages of using MgO include: 1) the hydration of magnesia gives an equilibrium pH of ∼10.5, and hence has a better precipitating action compared with PC/lime as the solubility of many heavy metals is lowest at that pH range [13,14]; and 2) brucite is able to accommodate a wide variety of heavy metals and organics by hydrogen bonding or through ion exchange [15,16].", "In addition, the use of reactive MgO with GGBS for soil stabilisation has been reported as being able to offer a range of mechanical and chemical advantages over PC or lime-slag blends [1,9,10,17–19].", "This is because the hydration products of MgO-GGBS mixes are calcium silicate hydrate (CSH)/magnesium silicate hydrates (MSH) and hydrotalcite-like (Ht) phases.", "The higher MgO/CaO ratio in these mixes increases the homogeneity of CSH gel structure, leads to a decreased Ca/Si ratio of CSH gel and the increased amount of voluminous Ht phases, which can fill the pores in the matrix more effectively [19,20].", "However, limited studies about the effectiveness of mass stabilisation in treating organic and inorganic contaminated soils can be found, with even less field trials studies using MgO-bearing binders, hence assessment of site soils in real projects is essential for the validation of the efficiency of this type of binders in mass stabilisation system.", "Apart from the type of binders, time is a significant factor that determines the remediation efficiency of contaminated lands.", "In the studies of Kogbara et al. [21] and Perera [22], it was found that with the aid of carbonation, the hydration process of PC-bearing binders were accelerated, hence the unconfined compressive strength (UCS) values of all mixes were improved with time at various extents.", "In most short-term cases (<10years) of stabilisation/solidification (S/S), due to the in-progress hydration, the release of contaminants is more likely to decrease with time [23].", "However, Wang et al., [24] studied the leaching performance of S/S treated site soils at 0.2, 2.4, 5 and 17 years.", "It was reported that slightly higher concentrations of heavy metals were leached at 5 years after treatment compared to these at 0.2 and 17 years using a toxicity characteristic leaching procedure (TCLP) leaching test, and the hydration process of treated samples did not complete at 5 years but fully completed at 17 years.", "The leachate pH and the further carbonation are the cause of this fact.", "To date, very limited attention was given to real-life S/S materials, with even less to mass stabilised soils [22,25,26].", "Although ground improvement (GI) treatment has been widely applied in the world, the application of MgO-bearing binders in mass stabilisation is a recent development, with very limited studies in the brownfield.", "For these reasons, it is necessary to understand the time-related performance of mass stabilised contaminated soils using MgO-bearing binders.", "The objectives of this trial were to: (1) investigate the 3-year performance of mass stabilised contaminated soil samples at different depths, (2) compare the strength and leaching performance of the soil samples 40-day, 1-year and 3-year after the treatment, and (3) compare MgO-bearing binders and PC in mass stabilised contaminated soils.", "The contaminated site characterisation can be referred to [1,27,28].", "Individual GI ALLU mixes were installed in 2011 to form 31 square pits, with a plan area of 1.96 m2, and 3–4 m in depth.", "Soils were pre-wetted, and then a total of 31 different combinations of binders were added in a dry form to the made ground soils.", "The soil strata consisted of made ground (∼5% clay and silt, and ∼95% sand) and the groundwater level was reported to vary between 3.2 and 3.9 m below ground level.", "The water content of the made ground soils was ∼25%, while the liquid limit was ∼30% and the plastic limit was ∼24%.", "The main contaminants in the made ground tested in 2010 are Pb (38 mg/kg), Zn (99 mg/kg), As (128 mg/kg), Cr (495 mg/kg), Cu (823 mg/kg), Ni (806 mg/kg) and total organics (3605 mg/kg) [9].", "The detailed contents of heavy metals and organic contaminants in the original soils at different depths can be seen in Fig. S1 (See in the online version at DOI: 10.1016/j.jhazmat.2016.07.018).", "The contaminated site characterisation can be referred to [1,27,28].", "Individual GI ALLU mixes were installed in 2011 to form 31 square pits, with a plan area of 1.96 m2, and 3–4 m in depth.", "Soils were pre-wetted, and then a total of 31 different combinations of binders were added in a dry form to the made ground soils.", "The soil strata consisted of made ground (∼5% clay and silt, and ∼95% sand) and the groundwater level was reported to vary between 3.2 and 3.9 m below ground level.", "The water content of the made ground soils was ∼25%, while the liquid limit was ∼30% and the plastic limit was ∼24%.", "The main contaminants in the made ground tested in 2010 are Pb (38 mg/kg), Zn (99 mg/kg), As (128 mg/kg), Cr (495 mg/kg), Cu (823 mg/kg), Ni (806 mg/kg) and total organics (3605 mg/kg) [9].", "The detailed contents of heavy metals and organic contaminants in the original soils at different depths can be seen in Fig. S1.", "Spatial distribution of the heavy metal and organic contamination in the site soils as obtained from previous site investigations [33].", "Although 31 GI mixes in ALLU mass treatments were applied in the SMiRT project, only these treated by PC (C), MgO (M) and MgO-GGBS (MG) were selected for a detailed study in this paper.", "The compositions, by weight percentage, of those soil-binder mixes are presented in Table 1.", "The compositions of the binders used are shown in Table 2.", "The cores of decreasing diameters of 85, 75, 65 and 55 mm, were obtained and tested at around 1 month after treatment, they were then stored in their plastic cover until reopened 1 year after the treatment as part of this study for further testing.", "Those containing MgO, together with CEM I for comparison purpose, were sampled again after 3-year treatment from the site for detailed study.", "The core samples were sealed in Ø 0.1 × 1.5 m plastic tubes and sent to the laboratory for testing.", "Detailed testing procedure can be found in [1,27].", "After 3-year treatment, the undrained shear strength on the soil cores was determined by a handheld shear vane tester and converted to the unconfined compressive strength by a factor of 2 (ASTM D2166).", "The crushed samples were then subjected to batch leaching according to BS EN 12457-2 (2002).", "The liquid to solid ratio (L/S) used was 10:1, adding 500 mL of carbonated deionised water in a bottle (pH = 5.4) containing 50 g of core sample.", "After 24 ± 3 h of agitation on a bottle roller, ∼10 mL of the leachate solutions was filtered through a 0.45 μm filter and the pH of which was measured.", "Finally, a Perkin Elmer inductive couple plasma optical emission spectrometer (ICP-OES) was used to measure the leachate concentrations of heavy metals.", "The remaining extraction fluid from the batch leaching test was abstracted into a 1000 mL plug-contained conical flask for organic extraction, where 5 mL of 12 mol/L hydrochloric acid was added to speed up the extraction reaction and also acted as a pH buffer.", "Then, 90 mL of dichloromethane (DCM), serving as the solvent, was divided into 3 equal parts and each was added separately to the flask.", "After the extraction process, the entire extracted sample was then poured into a container, whose weight was pre-recorded, for DCM evaporation in a fume cupboard.", "The extracted volume and the waste volume were recorded.", "After about 48 h of evaporation time, the mass of the residual was recorded in order to determine the total organic content leached.", "The physical conditions and integrity of the GI ALLU cores were observed 3 years after the treatment, with both good quality intact cores (Fig. 1a) and cores with significant fragmentation and fracturing and with missing sections in Fig. 1b. Different from the results found in [9] that qualities of the S/S treated cores vary significantly depending on the binder compositions, there was no significant difference observed between ALLU mixes taken from different depths and different binder compositions.", "This may be due to different mixing methods applied in-situ.", "Fig. 2 shows that the UCS values of these GI ALLU mixes were in the range of 50–250 kPa 3 years after the treatment.", "Note that no value of MG10 (1:4) is shown due to the poor quality of the cores.", "The UCS values of mixes showed slightly continued hydration, as shown by the in-situ Dynamic Probe ‘Super Heavy’ (DPSH) testing results obtained from the ALLU mass stabilisation mixes within a couple of months after the installations.", "The results showed very low strength development of the treated heavily contaminated soils, with corresponding cu in the range of ∼20–50 kPa [29].", "Mixes blended with different contents of MgO-only (M2.5, M5 and M10) produced lower strength compared to other mixes, which is due to the weak binding nature of brucite (the hydration product of MgO).", "Many research studies observed that blending GGBS with MgO has many advantages such as improved long-term strength and durability [10,18,19,30–32].", "This is verified through our field study of S/S, with weak strength at the very beginning and an obviously improved strength after 3-year treatment for all the MgO-GGBS blends [9,33].", "A laboratory study of MgO-GGBS in stabilising uncontaminated soils was reported that covering the 7-day, 28-day and 90-day UCS values of the 5% optimum GGBS-MgO mixes were 2–3.9 times higher that of corresponding PC mixes [18].", "In this study, MgO-GGBS blends at ratios of 1:4 and 1:9 were able to produce similar strength compared to PC-only.", "The discrepancies among these two studies are due to the contaminants in the site soils, for example, Zn has a significant negative effect on the hydration of the cement-based binders, which has been extensively addressed by Du et al. [6,7], and the more complicated site conditions [34].", "In addition, values over the full depth cored were varied without any obvious trends, which could be due to the poor mixing and/or varying soil properties.", "The leaching characteristics of treated soils are presented in Fig. 3 in terms of the leachate pH, the concentrations of leached contaminants and depth 3 years after the treatment.", "The average values of pH and heavy metals were calculated from duplicate samples of mixes taken from different depths.", "The average leachate pH values of all these mixes were in general in the range of 6.5–10 (Fig. 3b).", "M2.5 produced the highest pH value after 3-year treatment, which may be due to the heterogeneity of the contaminated site soils.", "The metals considered in this research are Ni, Cu, Zn, Cd and Pb.", "However, the leachate concentrations of Cd and Pb are lower than their limit of detections (0.5 and 20 μg/L for Cd and Pb, respectively).", "Therefore, they are not shown in Fig. 3.", "In Fig. 3a, it is clear that the leachate pH of M2.5 was ∼10.", "Since leachate pH was reported as governing the solubility of Ni hydroxides, the value of ∼10 produces the lowest solubility of Ni(OH)2.", "Hence, M2.5 leached the lowest Ni and is one of the only two mixes which did not exceed Ni’s drinking water standard (0.02 mg/L) [35].", "At a pH of ∼7.5, M5 leached the lowest Ni as well, the uneven distribution of Ni in the made ground may be the cause of the fact.", "This is followed by C5, the average leachate concentration of Ni of which is ∼0.1 mg/L.", "The result agrees well with the findings in [4] that the leachate concentration of Ni at pH 6–9 was lower than its solubility limit.", "MgO-GGBS blends did not show any advantages over other blends in immobilising Ni.", "This is due to the pH values of these mixes (at ∼6.5) were lower than the pH values of C5, M5 and M2.5, which producing a higher solubility of Ni(OH)2 [27].", "Fig. 3b&c show that the leachate concentrations of Cu and Zn in all mixes are lower than their drinking water standard (2 mg/L and 3 mg/L respectively).", "This agrees well with the relationship of pH, Cu and Zn, and solubility of their hydroxides [27].", "The variation of metals’ concentrations leached from different depths is varied and up to more than 1 order of magnitude.", "In addition, it was found that all mixes (except few mixes in Fig. 3b) leached higher concentrations of these three metals from deeper soils, indicating that the mixing of the deeper soils is not as good as that of the surface soils on the site.", "In addition, it is noted that extensive testing is necessary and it is acknowledged that a large body of data is needed to arrive at more firm conclusions.", "Fig. 4 shows that the leachability of the total organics was in the range of 10–105 mg/L, suggesting big variability in the contaminant level and binder composition.", "As can be found from the distribution of the leachability of total organics along the depth, in general, the adsorption ability of binders for organics is in the order of MgO-GGBS (1:4) > MgO > PC.", "The leachability of the total organics stabilised by MgO-GGBS (1:9) were generally <50 mg/L, with two points at a depth of 2.6–3 m leached higher concentrations of total organics.", "This may due to the uneven distribution of organic compounds in-situ.", "The higher immobilisation degree of organics by the application of MgO and MgO-GGBS blends is due to the structure of brucite produced by MgO and voluminous hydrotalcite-like phases produced by MgO-GGBS blends.", "They are able to accommodate a wide variety of extraneous anions, cations as well as organic compounds by hydrogen bonding etc, thus providing an excellent host for contaminants [14].", "A few conclusions can be drawn from these figures: (a) In general, under different depths, the average strength (UCS) of binders obtained from Fig. 2 is in the order of MgO-GGBS ≈ PC > MgO; (b) the average leachability of binders for organic contaminants is in the order of MgO-GGBS (1:4) > MgO > PC; (c) the average leachability of binders for inorganics is in the order of MgO-GGBS (1:4 and 1:9) > MgO > PC; (d) no significant trend can be draw along the depth among all mixes.", "In this study, GI ALLU samples tested 40-day, 1-year and 3-year after the treatment were used to study the time-dependent performance of mass stabilised contaminated soils.", "It is meaningful to mention that the samples tested after 1-year treatment were different from these after 40-day and 3-year treatment (window sampled from the site directly), as they were stored outdoor covered with hessian cloth in the Engineering Department, University of Cambridge (window sampling is the process uses either drop weight or hydraulic hammer to drive 1 m or 2 m long sample tubes into the ground.", "The sample tubes have open slots or “windows” along their length to allow for logging and sampling).", "Table 3 shows average UCS results of GI ALLU mixes up to 3 years.", "The deviation of UCS for different binder mixes was in the range of 8–21 kPa after 40-day treatment, 12–93 kPa after 1-year treatment and 2–36 kPa after 3-year treatment.", "In Table 3, The UCS value of C5 is ∼263 kPa 40-day after the treatment, the value of which increased to 310 kPa after 1-year treatment and was ∼248 kPa after 3-year treatment.", "The higher strength of C5 after 1-year treatment is due to its lower water content (stored ex-situ).", "M10, M5, M2.5 and MG2.5 (1:4&1:9) were too weak to be tested at 40 days.", "For MgO-only mixes, this was due to the slow hydration of MgO and the weak binding nature of brucite.", "The low strength of mixes MG2.5 (1:4&1:9) at 40 days’ treatment is due to the very low binder dosage of 2.5% adopted in these two mixes, and the relatively long duration that needed for the strength development of MG2.5 (1:4&1:9) caused by the nature of MgO as a weak alkali activator of GGBS.", "After 1-year treatment, the strength of 10% MgO-only mixes was ∼300 kPa, which is ∼3 times higher than the 2.5% MgO-only mixes.", "Furthermore, in spite of the common hydration products such as CSH, Ht produced during the curing process assist to produce a finer microstructure [20].", "Hence, the strength development of MgO-GGBS blends after 40 days and 3 years of curing were found higher than MgO-only mixes.", "In general, the strength of all mixes did not change significantly with time.", "This may due to the high moisture content of raw soils (before mixing soils were pre-wetted) that the hydration degrees of most binders were very high after 40 days.", "High water to cement ratio also leads to a reduction of strength development of these mixes [36].", "The average leachate pH values and concentrations of Cu, Ni and Zn in the leachates as a function of time are displayed in Fig. 5.", "In addition, the leachability of heavy metals in MG-10(1:9), MG-5(1:9) and all MG(1:4) mixes after 1-year treatment did not display in Fig. 5 due to there were no data of these mixes available.", "As can be seen, the pH values for all mixes after 40-day treatment were in the range of ∼9.8–11.2, and these after 1-year treatment displayed lower average values but with a broader range at ∼7.7–10.9, while the average of these after 3-year treatment were even lower at ∼6.5–10.", "Comparing the pH values of 40-day with these 3-year, decreased pH values were found in all mixes after 3-year treatment.", "This is due to the in-situ atmospheric carbonation, which has been extensively explored by previous study reported in Du et al. [6].", "Comparing the leachate concentrations of heavy metals with their drinking water standard individually, it can be found that the majority mixes exceeded Ni’s standard limit during the whole testing period.", "Although the Ni concentration of most mixes declined slightly from 40 days to 1 year, they were found even higher in some mixes 3 years after the treatment.", "The decreased concentrations of leached Ni from 40 days to 1 year are due to the different curing environment.", "When comparing the samples cured in-situ, the higher leachability after 3-year treatment compared to 40 days agrees well with the relation of Ni, pH and solubility curve of Ni hydroxide studied in [1].", "The leachate concentrations of Cu and Zn in all mixes were found well below their drinking water standards at different time points.", "Although atmospheric carbonation and complex site environment can degrade treated soils and increase the release of heavy metals to the environment with time, the leachate concentrations of metals in most mixes were in the same magnitude at 3 time points.", "The leachability of total organics of GI ALLU samples 40-day, 1-year and 3-year after the treatment is shown in Fig. 6.", "It shows that the leachability of total organics of majority mixes increase with time.", "Binding mechanisms between binders and organic compounds are physical entrapment in the produced matrix and sorption onto the surface of hydration products rather than chemical reactions [37,38].", "Thus the leachability of organics was not improved with the hydration process during the past 3 years.", "In addition, the curing condition such as rain flush (destroy the structure of produced matrix), atmospheric CO2 (lower soils’ pH) may be the main cause of this fact.", "MgO-GGBS blends at a ratio of 1:4 were found to be the most effective mixes in immobilising organics 40-day and 3-year after the treatment.", "It was followed by MgO-GGBS blends at a ratio of 1:9.", "This is because the hydration products of MgO-GGBS mixes are CSH/MSH and Ht, which are more voluminous and can fill the pores in the matrix more effectively [20].", "Hence, it is considered that more organic compounds are supposed to be trapped in the reaction products of MgO-GGBS mixes.", "In addition, the results of MgO-bearing mixes show that higher binder content did not result in a lower leachability of total organics.", "Since the leachability of total organics was determined by a large number of factors such as the contaminants level, soil type etc., this result indicates that an increase of binder content among these mixes (from 2.5% to 10%) may not be as significant as factors mentioned above in improving the effectiveness of immobilising organics.", "According to the relationship studied between UCS and leachability of total organics in a previous study [1], when the leachate concentrations of organic pollutants are >30 mg/L, high strengths of S/S treated samples cannot be achieved.", "High organic contaminants are able to inhibit the hydration of binders [37] and produce significant micro- and macrostructural changes to their products may be the cause of this result.", "The results of UCS tests and the leachability of heavy metals and organics at 3 time points indicate that there is no degradation of the GI ALLU mixes up to 3 years.", "This agrees well with the finding of Jin et al. [9] that no degradation of S/S materials was found after 3 years’ exposure to the field conditions.", "The time-related performance of mass stabilised soils was evaluated in terms of their physical and leaching properties.", "It also investigates the application of the innovative MgO-bearing binders in mass stabilisation for the first time.", "The results showed that MgO-bearing binders are very promising binders which can be used as a replacement of PC in this field.", "The main findings of this study are summarized as follows: 1) No degradation of mass stabilised materials was found after 3 years’ exposure to the field conditions, while the full range of high to poor quality cores were observed at both sampling time points (40 days and 3 years); 2) The overall range of strength and leachability results at different depths was varied throughout, suggesting a complicated distribution of contaminants in soils; 3) After 3-year treatment, regardless of the depth, the strength of binders is in the order of MgO-GGBS ≈ PC > MgO, while the leachability of binders for contaminants is in the order of MgO-GGBS > MgO > PC; 4) MgO is found to be very effective in immobilising both heavy metals and organics, however, it showed weak mechanical performance.", "MgO-GGBS blends at a ratio of 1:4 were found to be the most effective GI ALLU mixes in immobilising organics; 5) High moisture content in-situ has adverse effect on the efficiency of mass stabilisation system; 6) The leachate concentrations of Cu, Pb, Cd and Zn meet their drinking water standard, but the concentrations of Ni in some mixes are above its drinking water standard regardless of time.", "And it was found that the leachability of organics was not improved with the hydration process during the past 3 years." ]	[ "No apparent degradation of the mass stabilised materials after 3 years’ exposure to the field conditions was found.", "The overall range of strength and leachability results at different depth was varied throughout.", "MgO-GGBS blends provided better performances compared to PC and MgO-only mixes in mass stabilised soils." ]
Cell diameter doesn't affect lipid productivity of Chlorococcum littorale	We hypothesized that cells with different diameter have different division rates, which could affect lipid productivity (lipid content × biomass productivity). In the present work we assessed the influence of cell diameter, as a sorting parameter, on both biomass and lipid productivity of Chlorococcum littorale. Prior to sorting, cells were grown in a batch-wise nitrogen run-out including a long nitrogen depleted phase (N −) to stop cell division, thus only having vegetative cells (Pre-sorting). Cell sorting was done at the end of this N − phase using FACS (fluorescence assisted cell sorting) based on forward scatter as a proxy for diameter (size ranges (μm): 5–6 (small), 8–9 (medium), 11–14 (large) and 5–14 (control)). The sorting was done in 2 pools: multiple-cell (100 cells) and single-cell. After sorting, cells were recovered under low-light for 2 weeks, and used to start the Post-sorting experiment (analogous to Pre-sorting). The populations derived from different sorted pools, single-cell and multiple-cell, showed similar size distributions after re-growth. No difference was observed in biomass and lipid productivities among Post-sorting cells and when compared to Pre-sorting cells under nitrogen depletion. We concluded that cellular size had no effect on both biomass and lipid productivity of C. littorale.	[ "AGRI" ]	[ "Biomass productivity", "Cell diameter", "Cell sorting (FACS)", "Chlorococcum littorale", "Lipid productivity", "Microalgae" ]	[ "1102" ]	[ "Microalgae have shown potential to replace current feedstock for bulk commodities [11,36].", "The positive aspects of microalgae bulk production include sustainability and versatility of the production chain when compared to land crops [10].", "Nevertheless, reported biomass and lipid productivities need to be improved to make bulk production economically feasible for commodity products, such as plastics or fuels [26,28].", "One approach to increase productivity is to develop more productive strains.", "This can be done by taking advantage of the natural genetic variability of a parental population to screen and sort cells with different features (e.g. high lipid producers) [22,37].", "Another possibility is to increase genetic variability by inducing mutations in a parental population and screen and sort for mutants with abnormal improved features [7,13,32,35].", "Fluorescence assisted cell sorting (FACS) is a method which allows rapid screening and sorting of cells with desired characteristics.", "Sorted cells can be regrown, possibly leading to a new improved culture [13,32,35,37].", "FACS can sort cells based on multiple fluorescence and light scattering parameters, which makes it a versatile technology.", "Chlorococcum littorale has a high lipid content [29], high photosynthetic efficiency under nitrogen stress [3], and has been used in FACS before [9] and for this reason has been chosen in this study.", "C. littorale is a unicellular microalga whose cell size can range from 5 to 14 μm in the vegetative stage [25].", "C. littorale can reproduce sexually and asexually by multiple-fission of the mother cell, into 2 to 16 daughter cells (in the shape of spores that can be haploid or diploid, in case of asexual or sexual reproduction, although there is no morphological differences between the two kinds of spores) [25].", "The formation of spores is relevant for the sorting, since dividing cells could be mistaken for a large vegetative cell.", "Up to now the regulation of cell size in microalgae is not completely understood [4,27,33].", "Studies with Chlamydomonas, a genus with multiple-fission cell division, showed a positive correlation between mother cell's sizes and the number of generated daughters [27].", "Other authors have shown that cells might even continue their division without doubling their cell volume.", "Hence, such cells would produce bigger daughter cells, that would generate more and smaller cells in the next division [4].", "Both studies point to a possible influence of cell diameter on the growth patterns of following generations.", "Up to date, no report has focused on the possible effect of size of vegetative cells on the biomass productivity of bulk cultures.", "We hypothesized that cells with different diameter have different division rates, which could affect biomass productivity.", "Hence, differences in biomass productivity could consequentially affect lipid productivity (lipid content × biomass productivity).", "In the present work we assessed the influence of cell diameter, as sorting criterion, on both biomass and lipid productivity of vegetative cells of C. littorale.", "C. littorale was grown in a batch-wise nitrogen run-out followed by a long nitrogen-depleted phase to stop cell division and to induce lipid accumulation, thus having only vegetative, lipid-rich cells.", "At the end of the nitrogen-depleted phase (15 days, after cell size was stable) cells were sorted in four groups of different diameters: small (5–6 μm), medium (8–9 μm), large (11–14 μm) and a control (5–14 μm).", "We compared both biomass and lipid productivities of sorted cells of C. littorale with the growth of the parental population.", "Additionally, we compared the daily differences in cell size distribution, photosystem II quantum yield, autofluorescence and intra-cellular lipid fluorescence.", "Inoculum of C. littorale (NBRC 102761) was prepared from samples preserved under low light conditions (16 μmol m− 2 s− 1) in borosilicate tubes containing growth medium and agar (12 g l− 1).", "Small samples were transferred from agar to 200 ml sterile borosilicate Erlenmeyer flasks, containing 100 ml of sterile growth medium.", "C. littorale was grown in salt water-like medium with the following composition (g l− 1): NaCl 24.55, MgSO4·7H2O 6.60, MgCl2·6H2O 5.60, CaCl2·2H2O 1.50, NaNO3 1.70, HEPES 11.92, NaHCO3 0.84, EDTA-Fe(III) 4.28, K2HPO4 0.13, KH2PO4 0.04.", "The medium also contained the following trace elements (mg l− 1): Na2EDTA·2H2O 0.19, ZnSO4·7H2O 0.022, CoCl·6H2O 0.01, MnCl2·2H2O 0.148, Na2MoO4·2H2O 0.06, CuSO4·5H2O 0.01.", "The sequence of experiments performed in this work is represented in Fig. 1.", "After inoculum preparation we set-up a preliminary test to determine the boundaries for the sorting gates based on cell size using fluorescence assisted cell sorting (FACS).", "The preliminary test was performed before but analogous to the experiment explained in the next section.", "The experimental set-up was the same for both Pre-sorting and Post-sorting experiments (Fig. 1).", "Experiments were inoculated with an initial OD750 value of 0.5 (~ 0.7 g DW l− 1).", "The algae were cultivated in 200 ml borosilicate Erlenmeyer flasks (100 ml medium per flask) and kept in an Infors Multitron Pro orbital shaker incubator.", "Growth conditions were: 25 ± 0.2 °C, 120 rpm, 60% humidity, 2% CO2 enrichment on air, and 120 ± 2 μmol m− 2 s− 1 continuous light (24 h).", "Both experiments had 2 phases: a growth phase (N +) and a nitrogen depleted (N −) phase.", "During the growth phase nitrogen (N–NO3) was allowed to run-out progressively (at day 5 NO3 was completely consumed by the cells).", "After reaching stationary phase, the cultures were diluted 5 × to a biomass concentration of 1.4 ± 0.1 DW g l− 1, in sterile medium without NO3−, to allow further light penetration in the culture and enhance lipid production.", "After dilution, cells were monitored until maximum size was achieved, and was constant for 3 days (after 15 days from start experiment).", "All daily measurements were done at the same time every day and are described in the following section.", "Multiple-cell sorting (3 × 100 cells) was done based on cell size in four groups, at the end of the stationary phase (Fig. 3).", "After sorting, cells were re-suspended in 50 ml sterile falcon tubes containing fresh and sterile nitrogen-replete medium into.", "Cells grew for 2 weeks in the tubes under low light conditions (16 μmol m− 2 s− 1).", "After this, cells were transferred to sterile borosilicate Erlenmeyer's, where Post-sorting experiments were conducted under similar conditions as the Pre-sorting experiments and with the same daily measurements.", "Additionally single-cells were sorted from the same parental population on the same day (Fig. 1).", "Fifteen replicates per group of single-sorted cells were taken to increase sample size and to assure reproducibility of Post-sorting results.", "Single cell sorting was done analogous to and immediately after the multiple-cell sorting.", "After 18 days of growth under low-light conditions (16 μmol m− 2 s− 1) the sorted cells were centrifuged at 1224 × g for 10 min, washed with sterile medium and readily analyzed for size and autofluorescence using the FlowCAM® fluid imaging system (settings are presented in the next section).", "All daily measurements were done at the same time every day.", "Optical densities were measured on a daily basis using a spectrophotometer (HACH, DR5000) on two different wavelengths: 680 and 750 nm.", "Samples were diluted within the range of the detection limit (0.1–1 units of OD).", "OD750 was used as a proxy for biomass concentration and OD680 was relative to the chlorophyll fluorescence [16].", "FlowCAM® analyses were performed daily to follow-up the cell's diameter, autofluorescence (chlorophyll fluorescence) and lipid-dependent fluorescence (BODIPY505/515 fluorescence).", "For FlowCAM® analyses samples were first diluted 100 to 1000 times and then stained for neutral lipids to achieve a final concentration of 0.4 μg/ml of BODIPY 505/515 and 0.35% of ethanol, following the protocol of [9].", "After 5 min the samples were run in the FlowCAM® (Fluid Imaging Technologies, Yarmouth, Maine) using the following settings: 20 × optical magnification and Trigger-mode-on with channels 1 and 2, autofluorescence (650 nm long pass filter) and BODIPY505/515 (525/30 filter), respectively.", "The trigger mode activates the system to take a camera image when a particle produces a fluorescence signal.", "Diameter (μm), autofluorescence (Ch1) and BODIPY505/515 fluorescence (Ch2) of each sample were taken for further analyses.", "Daily samples were taken for measurement of nitrogen content (N–NO3 mg l− 1) in the medium.", "1 ml of culture was centrifuged at 13,000 × g and the supernatant was used for analysis.", "Nitrogen content was measured daily with the Sulphanilamide N-1-naphthyl method (APHA 4500-NO3-F) using the SEAL AQ2 automatic analyzer.", "At the end of the nitrogen-depleted phase of the Pre-sorting experiment, a sample was collected and diluted to an OD750 of approximately 0.3, to keep cell concentration in the flow cytometer at 200 cells/min.", "The sorting was carried out with a FACScalibur® flow cytometer (BD Biosciences, San Jose, California).", "FACScalibur® is equipped with an argon ion laser with excitation at 488 nm and in our work the emission at 670LP (FL3 channel) was used, relative to the cells autofluorescence (Chlorophyll-a emission).", "FL3 channel was read in logarithmic scale and the sensitivity was set at 300 mV while FSC channel was read on linear scale.", "Cells were collected in sterile 50 ml falcon tubes and sterile PBS was used as sheath fluid (phosphate saline buffer; composition: NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10.0 mM, KH2PO4 1.8 mM).", "The sorting was done in two steps: multiple-cell sorting and single-cell sorting.", "For multiple-cell sorting 100 cells were collected per tube to produce inoculum for the Post-sorting experiments (in triplicate).", "Subsequently, single-cell sorting was carried-out using the same sample and same machine settings as the multiple-cell sorting (with 15 replicates for each size).", "After sorting, cells were centrifuged at 1224 × g for 5 min and re-suspended in sterile growth medium (both multiple and single-cell sorting).", "The tubes with sorted cells were placed under constant light at 16 μmol m− 2 s− 1, allowing cells to recover for 2 weeks.", "After the recovery time, triplicates containing 100 cells each were transferred to Erlenmeyer flasks to produce inocula for the Post-sorting experiments.", "The cultures originating from the single-cell sorting experiments were readily analyzed with the FlowCAM® after 18 days of growth in the tubes.", "Biomass samples were collected at the end of both nitrogen replete (N +) and nitrogen depleted (N −) phases of Pre- and Post-sorting experiments.", "Biomass was centrifuged twice and washed with MilliQ water (3134 × g for 10 min at 4 °C) and followed by freezing at − 20 °C and freeze-drying for 24 h.", "Fatty acid and triacylglycerol's (TAGs) extraction and quantification were performed as described by Breuer et al. [8], using GC/MS column chromatography of the chloroform fraction of lipids.", "Data from FlowCAM and cytometer were exported to Microsoft Excel to be edited and analyzed.", "For statistical analysis, one-way analysis of variance (ANOVA) was used to access the significance of differences between and among sorted groups (SigmaPlot, v. 12.5).", "The premises of ANOVA, i.e. the homogeneity of variances and the normality of the data, were also measured with SigmaPlot.", "A p-value lower than 0.05 was considered significant.", "The increase in BODIPY cellular fluorescence was used to calculate the BODIPY accumulation rate (BPr), relating to the increase of cellular neutral lipids in time.", "The BPr was estimated with a similar approach as used for Eq. (2), only that the values of DW where replaced with the values of BP fluorescence (relative fluorescence units, RFU).", "The period of time used for calculation was the period between the start (d = 5) and the end (d = 10) of the nitrogen run-out (being d = 0 the inoculation day).", "This period was chosen because it shows the first increase in intracellular lipid content; hence it is an estimate of the first metabolic response of the intracellular lipid accumulation.", "The decrease in photosystem II quantum yield (QYr) was calculated using an identical approach to the one used in Eq. (2), but replacing DW values by QY measurements.", "Two QYr were calculated: at the end of the nitrogen run-out (between d = 5 and d = 10), and at the end of the nitrogen depleted phase (between d = 10 and d = 25).", "A preliminary test was done to check 2 assumptions: 1] that the intracellular lipid content is not correlated with the cell diameter and 2] that FACS could be used to sort cells based on cell size.", "The first assumption is relevant not to include the lipid content as a sorting criteria.", "As stated in the introduction, we hypothesized that cells with different diameters can have different division rates and we want to evaluate the effect of such differences on lipid productivity.", "We found no correlation between cell diameter and intracellular lipid-fluorescence, as it can be seen from the R2 value in Fig. 2.", "The preliminary test was done using the same experimental set-up as it was used for the actual experiments (Fig. 1 and Section 2.2 of methods).", "Once the first assumption was checked we used the same sample to do a preliminary sorting test to check the second assumption: can FACS be used to sort cells based on diameter?", "Cell diameter cannot be directly measured in FACS.", "However, measurements of the forward light scattering (FSC) of every cell can be used as a proxy for cell size [2,37].", "Although calibration beads are available to convert FSC into diameter [23], we decided not to use calibration beads due to intrinsic differences in e.g. surface smoothness between the calibration beads and microalgae cells.", "Furthermore, we worked with size intervals that differed from each other in only 2 μm.", "Hence we decided not to use calibration beads to convert FSC to cell size, instead we decided to use FSC as a proxy for cell size and to perform a test to confirm the results of the set gates boundaries by measuring the cell size of sorted cells immediately after the sorting (using FlowCAM fluid imaging).", "A histogram of FSC was firstly established to show the distribution of size within the population and to identify the different size groups (Fig. 3B).", "The information from the histogram (Fig. 3B) was crosschecked with simultaneous plotting of autofluorescence (AF) versus FSC (Fig. 3A) as a positive control for algae cells (to avoid gating non-fluorescent particles).", "Autofluorescence was used as a proxy for cellular chlorophyll content to confirm the identity of the particles as microalgae cells.", "We kept the ratio AF/FSC the same for all gates to avoid AF as a second sorting criteria (Small: 1.2, Medium: 1.1, and Large: 1.1).", "The gates used for sorting were established on the dot-plot shown on Fig. 3A.", "This approach was important to avoid the inclusion of cell debris, especially for the group containing small cells.", "Once the gates were established, as shown in Fig. 3A, the cells were sorted (100 cells per group, in triplicate) and subsequently analyzed for size.", "The cell size was assessed with the FlowCam imaging, which provides direct measurement of cell diameter (μm) and autofluorescence, supplementing the analysis from FACS.", "The results from the preliminary test (Fig. 3) confirmed the suitable gate boundaries to sort cells based on size: small (5–6 μm), medium (8–9 μm), large (11–14 μm) and a control including all previous sizes (5–14 μm).", "Detailed pictures of the cells from all different groups can be found in the supplementary materials (SM1).", "C. littorale was cultivated phototrophically, batch wise in nitrogen replete medium (Pre-sorting experiment).", "At day 5 the concentration of nitrogen in the medium was zero (Fig. 4A, light gray area marks the 5th day).", "We kept the cultivation for 5 additional days after nitrogen run-out, until biomass concentration reached its maximum, meaning that the cells had consumed their internal nitrogen pools while finishing to divide (which can also be noticed on the size evolution, Fig. 4C).", "At this point, the cultures were diluted in nitrogen depleted medium to increase the light intensity inside the culture necessary to further stimulate lipid accumulation in microalgae [18,21,30,34] (Fig. 4A), and a consequent increase in size (Fig. 4C).", "Table 1 presents the growth rate, biomass and TAG volumetric productivities and fatty-acid and TAG content (%DW biomass).", "The growth rates and productivities calculated in the present work were used to compare the Pre-sorting with the Post-sorting populations.", "In the growth phase we observe that the quantum yield (QY) of photosystem II remained above 0.5, supporting the hypothesis that the cells were not under photochemical stress (Fig. 4A).", "The QY is an indicator of stress at values below 0.4 [19,38].", "We can detect a drop in the QY after the nitrogen run-out (light gray area, Fig. 4A) and a further drop to half of the initial value during the nitrogen depleted phase (dark gray area, Fig. 4A).", "Low values of QY are an indirect indication of lipid formation, because under nitrogen stress no biomass can be produced and the photochemical energy is used to produce storage lipids [1].", "The experiment was kept until values of QY close to 0 were achieved (Fig. 4A).", "Both autofluorescence and lipid-related fluorescence (BODIPY) were monitored during the experiment.", "The autofluorescence was stable throughout the experiment, showing only a reduction of 6.8% in autofluorescence from the beginning to the end of the experiment (Fig. 4B).", "The autofluorescence is related to the fluorescence of both photosystems in the chloroplast [14], hence it can be used as a proxy for the chlorophyll content per cell.", "We can infer that almost no chlorophyll degradation took place, since only a small fraction of autofluorescence was reduced.", "Fluorescence of the BODIPY dye was used as a proxy for cellular lipid content [2,9,15,24].", "BODIPY505/515 is a dye used to stain non-polar lipid bodies in microalgae cells [9,12,24].", "The average BODIPY505/515 fluorescence per cell is presented in Fig. 4B, where it is possible to see an accumulation of neutral lipids from the moment of nitrogen run-out onward (Day 5).", "The increase in cellular lipid content is a survival mechanism in most microalgae, which directs the energy converted from photosynthesis to the synthesis of storage lipids in the absence of substrate (nitrogen) to grow [1,17,20].", "Sorting based on cell size took place at the end of the nitrogen-depleted phase (d = 25), after vegetative cells had reached their maximum size (As showed in 4C).", "Size measurements were plotted in a histogram after the regrowth phase (Fig. 5).", "The overlap of the curves indicates that, after sorting, cells showed the same behavior as before the sorting.", "We also measured the distribution of cell diameter in the population originated from sorted single-cells to compare the 4 sorted populations among each other.", "The comparison of the average cell diameter among the sorted cells showed no statistical difference (Fig. 6, p > 0.05, one-way ANOVA).", "These results were the first evidence that cell size might be an intrinsically regulated phenotypical distribution of C. littorale.", "Cell cycle studies with Chlamydomonas, a microalgae strain with multiple-fission division as C. littorale, showed that there is a positive correlation between mother cell's sizes and the number of generated daughters [27].", "Other authors, however, have shown that cells might divide without doubling their cell volume.", "Hence, such cells would produce bigger daughter cells, that would generate more and smaller cells in the next division, which is an evolutionary advantage for natural unstable conditions [4].", "All sorted populations were grown batch wise in similarity to the Pre-sorting experiment (Fig. 4) in order to be able to compare the Pre- with the Post-sorting culture on biomass and lipid productivity.", "All populations, including the parental population, showed similar growth (Fig. 7 and Table 1).", "All four groups showed similar biomass productivities during the nitrogen run-out phase, ranging from 0.32 to 0.35 g l− 1 d− 1 (Px Nout, Table 1).", "The lack of statistical difference among the groups confirms the previous results from the size distribution of single cell and multiple-cell sorting's (Figs. 5 and 6).", "The Post-sorting populations showed, however, a higher value of Px at Nout when compared with the Pre-sorting population (Px Nout, Table 1, p < 0.05).", "The higher Px values from Post-sorting could be a result of the low light acclimation that the cells experienced during recovery (Fig. 1), before being again cultivated, which is known to increase photosynthetic efficiency, hence, the biomass production [5].", "Another observation that could corroborate this effect is the evolution of the photosystem II quantum yield (QY) (Fig. 7).", "The QY values dropped at a faster rate in the Pre-sorting experiment when compared with the Post-sorting experiment (Figs. 4 and 7).", "The evolution of the QY, however, showed no difference when Post-sorting populations were compared among each other (Fig. 7).", "This effect can also be seen with the similar QY decrease rates (QYrate) between experiments at the end of both N + and N − phases (from 0.3 to 0.4, Table 1).", "Despite an apparent physiological change possibly due to photo-acclimation during nitrogen replete conditions, no difference was observed in the Px among the sorted groups and in comparison with the Pre-sorting population at nitrogen depleted conditions (Px N −, Table 1).", "These results are evidence that cell size of vegetative cells has no effect, as sorting criteria, on biomass productivity.", "The evolution of BP fluorescence over time (Fig. 7B) is related to the values of lipid productivity (Table 1).", "All sorted populations showed similar trends among each other (Fig. 7) and when compared to the Pre-sorting experiment (Fig. 4B) in the evolution of lipid/cell accumulation.", "However, all sorted populations reached lower values of BP fluorescence at the end of the Nout phase when compared with the Pre-sorting experiments (Figs. 4 and 7).", "Such lower values of BP fluorescence match the results of lipid content in biomass: both TAG/total fatty-acids content and TAG productivity (PTAG) were similar among Post-sorting populations at the end of the Nout, but statistically different when compared to the Pre-sorting experiment (Table 1, ANOVA p < 0.05).", "An exception was for both TAG/total fatty-acids content and TAG productivity (PTAG) of large cells (Table 1), which showed values statistically lower than the other Post-sorted populations and the parental population.", "However, such differences were not found in the N − phase, indicating that the delay in lipid accumulation was temporary during the Nout.", "These differences may be an effect of the low-light acclimation period the sorted cells went through for 2 weeks before growing under the same conditions as the Pre-sorting experiment (as shown in Fig. 1).", "The photo-acclimation effect is corroborated by the above-mentioned slower decrease in the QY values (Fig. 7A and Table 1).", "Due to photo-acclimation cells produced more biomass in comparison to the Pre-sorting experiment, which explains the different TAG fluorescence per cell (Fig. 7B) and the lower TAG content/productivity in biomass (Table 1) in the Post sorting experiment.", "These results point to a temporary change in the physiological response of sorted cells, seen in the lower values of BP at the beginning of the nitrogen run-out in comparison with the Pre-sorting experiment (Figs. 4 and 7).", "However, the accumulation rate of BP fluorescence (BPr d− 1) shows that the biological mechanism still responded at the same rates.", "Likewise, the PTAG at the end of the nitrogen depletion phase (N −) showed no differences among the Post-sorting populations and no difference when compared with the Pre-sorting population (Table 1).", "We conclude that sorting vegetative cells of C. littorale according to cellular diameter does not result in differences in biomass and lipid productivity.", "We established the gates for cell sorting based on size using the FSC as a proxy for diameter in combination with autofluorescence.", "A Pre-sorting experiment was performed including a growth phase (N +), a nitrogen run-out (Nout) and a long nitrogen depleted phase (N −).", "The N − phase assured that cells stopped dividing, thus resulting only in vegetative cells, and assured that cells only increased their size due to lipid accumulation.", "Vegetative cells of C. littorale were sorted at the end of the Pre-sorting experiment based on cell size.", "Both single-cell and multiple-cell sorted populations showed similar size distributions after re-growth.", "The growth rates and productivities (biomass and lipid) were calculated to compare Pre- and Post-sorting populations.", "No difference among Post-sorting populations was detected, however, Post-sorting cells showed a higher value of biomass productivity (Px) when compared with Pre-sorting cells.", "This result could be due to the low light acclimation that the cells experienced during recovery after sorting.", "Nevertheless, no difference was observed in the TAG's productivity (PTAG) among Post-sorting cells and when compared to Pre-sorting cells at the nitrogen depletion phase.", "We conclude that cellular size of vegetative cells has no effect on both biomass and lipid productivities of C. littorale." ]	[ "Successful cell sorting based on light forward scatter as proxy for cell diameter.", "Single and multiple-cell sorting lead to populations with similar size distribution.", "Cultures started with different diameters had similar final lipid productivities.", "Cell size, as sorting criterion, had no effect on biomass and lipid productivity." ]
Notch Activity Modulates the Responsiveness of Neural Progenitors to Sonic Hedgehog Signaling	Throughout the developing nervous system, neural stem and progenitor cells give rise to diverse classes of neurons and glia in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges through the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh gradient depends on both the amount of ligand and duration of exposure, but the mechanisms permitting prolonged responses to Shh are not well understood. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to attain sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates subcellular localization of the Shh receptor Patched1, gating the translocation of the key effector Smoothened to primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch shaping the interpretation of the Shh morphogen gradient and influencing cell fate determination.	[ "BIOC" ]	[]	[ "1300", "1307", "1309", "1312" ]	[ "Neuronal and glial diversity in the CNS emerges in large part through the concomitant and combinatorial actions of morphogen signals such as Sonic hedgehog (Shh), Bone Morphogenetic Proteins (BMPs), Wnts, and retinoids that organize neural progenitor cells (NPCs) into discrete domains along the dorsoventral and rostrocaudal axes (Briscoe and Novitch, 2008; Le Dréau and Martí, 2013; Butler and Bronner, 2015).", "Each of these domains is defined by its expression of unique combinations of transcription factors and ability to generate specific classes of neurons and glia (Briscoe and Novitch, 2008; Rowitch and Kriegstein, 2010; Le Dréau and Martí, 2013; Butler and Bronner, 2015).", "The prevailing model for morphogen signaling posits that differential cellular responses arise due to the signal concentrations that cells encounter (Rogers and Schier, 2011), yet the duration of exposure to a fixed amount of signal can also elicit graded domain responses and influence fate decisions (Kutejova et al., 2009).", "These results suggest that an important aspect of morphogen interpretation is the ability of cells to maintain their responsiveness to these cues as development proceeds.", "However, the mechanisms that permit this competence over time are not well understood.", "One of the best studied examples of morphogen signaling is the patterning response of NPCs in the ventral spinal cord to Shh.", "Shh acts on NPCs in a dose-dependent manner, binding to its primary receptors Patched1 and 2 (Ptch1/2) to initiate a cascade of intracellular signaling events centered on the translocation of the G-protein-coupled receptor Smoothened (Smo) to primary cilia (Eggenschwiler and Anderson, 2007; Dessaud et al., 2008; Ribes and Briscoe, 2009).", "The presence of Smo in cilia modulates the proteolysis and activity of the Gli family of Zn-finger transcription factors, which in turn regulate the expression of many NPC fate determinants that subdivide the ventral spinal cord into three distinct ventral NPC domains: p3, pMN, and p2 (Briscoe and Novitch, 2008; Dessaud et al., 2008; Ribes and Briscoe, 2009).", "These domains are distinguished by their shared expression of the transcription factor Nkx6.1 and differential expression of Nkx2.2, Olig2, and Irx3, respectively (Mizuguchi et al., 2001; Novitch et al., 2001; Briscoe and Novitch, 2008; Dessaud et al., 2008).", "The pMN gives rise to motor neurons (MNs), while the p3 and p2 domains produce distinct classes of spinal interneurons that modulate MN activities.", "Later in development, Olig2+ NPCs form a domain of oligodendrocyte precursors (pOLs) that disperse and migrate throughout the spinal cord before differentiating into myelinating oligodendrocytes (Rowitch and Kriegstein, 2010).", "The p3 and p2 domains similarly transform into astroglial progenitor groups (pVA3 and pVA2), producing astrocytes that colonize distinct regions of the ventral spinal cord (Muroyama et al., 2005; Hochstim et al., 2008).", "While these fates can be specified through the administration of different concentrations of Shh ligand in vitro (Dessaud et al., 2008; Ribes and Briscoe, 2009), NPCs also acquire their ventral identities through time-dependent mechanisms.", "NPCs treated with moderate doses of Shh initially express the pMN determinant Olig2; however, if Shh/Gli signaling is sustained, they subsequently express Nkx2.2 and adopt the more ventral p3 fate (Dessaud et al., 2007, 2010; Balaskas et al., 2012).", "Recent studies in the zebrafish spinal cord have further demonstrated that progenitor maintenance mediated by the Notch signaling pathway plays an important role enabling later born Shh-induced cell types to emerge (Huang et al., 2012).", "Together, these findings indicate that cells must remain in an undifferentiated state to properly interpret the Shh morphogen gradient, but do not resolve the mechanism by which the maintenance of NPC characteristics influences Shh responsiveness and whether retaining cells in a progenitor state influences spatial patterning.", "The Notch signaling pathway serves as a major regulator of NPC maintenance and both neuronal and glial development (Gaiano and Fishell, 2002; Pierfelice et al., 2011).", "Notch receptors are broadly expressed by NPCs and are activated by the Delta-like and Jagged families of transmembrane ligands presented by neighboring cells (Kageyama et al., 2009; Pierfelice et al., 2011).", "Activated Notch receptors are cleaved by the Presenilin γ-secretase complex, liberating Notch intracellular domain (NICD) fragments.", "NICD subsequently forms transcriptional activating complexes with the DNA binding protein Rbpj and members of the mastermind-like (MAML) coactivator family (Kageyama et al., 2009; Pierfelice et al., 2011).", "Rbpj-NICD-MAML complexes regulate a number of targets most notably Hes genes, bHLH transcription factors that repress proneural genes, inhibit neuronal differentiation, and promote NPC maintenance (Kageyama et al., 2007, 2009; Pierfelice et al., 2011).", "Through these actions, Notch signaling suppresses neuronal differentiation and endows cells with gliogenic potential.", "NICD misexpression can further promote specific glial cell fates, such as radial glia in the forebrain, Müller glia in the retina, and astrocytes in neural stem cell cultures (Furukawa et al., 2000; Gaiano et al., 2000; Scheer et al., 2001; Ge et al., 2002) while inhibiting oligodendrocyte differentiation (Wang et al., 1998).", "These data implicate a role for Notch in glial fate selection, although the mechanisms underlying these effects remain unclear.", "Here, we test the contributions of Notch signaling on both the establishment of NPC identities and glial fate determination.", "We show that activation and inactivation of the Notch pathway modify the responses of NPCs to Shh, altering both their dorsoventral register and ability to generate distinct classes of neurons and glial cells.", "Notch activity strikingly acts at the most proximal steps in the Shh transduction pathway, affecting the trafficking of Smo and Ptch1 to primary cilia.", "Together, these findings reveal a role for Notch signaling shaping the interpretation of the Shh morphogen gradient and assignment of cell fates.", "We first used Olig2Cre mice (Dessaud et al., 2007) to selectively activate or inactive Notch signaling in the p3 and pMN domains between embryonic days (E) 9.5 and 10.5 (Figures S1A–S1W).", "This strategy was accomplished by crossing Olig2Cre to mice harboring (1) a Cre-inducible R26RGFP transgenic reporter (Mao et al., 2001) (control condition), (2) a R26RNICD-GFP transgene and reporter (Murtaugh et al., 2003) (“Notch-On” condition), or (3) a Cre-inactivatable Rbpj allele (Han et al., 2002), along with the R26RGFP transgenic reporter (“Notch-Off” condition) (Figures 1A).", "The impact of these Notch pathway manipulations was evident by E11.5, as Notch-On mice displayed elevated expression of the Notch effectors Hes1 and Hes5, which are normally very low in the pMN and reduced expression of proneural transcription factors, including Neurog2, Ascl1, and Neurog3 (Figures S2A–S2N).", "Conversely, Notch-Off mice displayed reductions in Hes1 and Hes5 expression and increased levels of Neurog2, Ascl1, and Neurog3 (Figures S2O–S2U).", "While the ventral ventricular zone (VZ) narrowed in Notch-Off mice, a contiguous band of Sox2+ NPCs was maintained throughout development, and both the neuroepithelial architecture and apicobasal polarity of progenitors were preserved (Figures S2V–S2AI).", "This phenotype contrasts with mutations in other members of the Notch pathway such as Hes1 and Hes5 whose combined loss disrupts the neuroepithelium (Hatakeyama et al., 2004).", "The persistence of NPCs and neuroepithelial organization in Olig2Cre;Notch-Off mutants may be explained by the lasting presence of Hes1 in ventral progenitors despite the loss of Rbpj (Figures S2Q and S2R), most likely due to Notch-independent activation of Hes1 by Shh, as has been described in other tissues (Ingram et al., 2008; Wall et al., 2009).", "We next examined the impact of these Notch manipulations on dorsoventral patterning.", "Remarkably, activating Notch signaling led to a notable reduction in Olig2+ pMN cells by ∼E11.5 and a nearly complete loss of Olig2+ NPCs throughout the rest of embryonic development (Figures 1B–1K and 1Q).", "Notch-Off mice exhibited the reciprocal phenotype, with an ∼1.5 to ∼2.5-fold increase in the number of Olig2+ progenitors from E11.5 to postnatal day (P) 0.5 (Figures 1L–1Q).", "While Olig2+ cells were reduced in Notch-On mice, the overall number of ventral NPCs expressing Nkx6.1 increased by ∼50% (Figure 2M).", "The loss of Olig2 from Nkx6.1+ NPCs coincided with the increased expression of the p3 determinant Nkx2.2 (Figures 2A–2H and 2N).", "Given that Nkx2.2 can repress Olig2 (Mizuguchi et al., 2001; Novitch et al., 2001; Sun et al., 2003), the loss of pMN cells in Notch-On mice is likely due to their transformation toward the more ventral p3 fate.", "This conclusion was supported by the reduced percentage of Nkx6.1+ progenitors expressing Nkx2.2 and corresponding increase in Olig2+ cells seen in Notch-Off spinal cords (Figures 2I–2L and 2N).", "Collectively, these data demonstrate that Notch signaling plays a critical role enhancing the ventral character of NPCs and influencing their partitioning between pMN and p3 identities.", "We next used R26RGFP lineage tracing to assess the fate of the Notch-manipulated cells.", "Consistent with the loss of Olig2, Notch-On spinal cords exhibited an ∼35% reduction in MN formation (Figures S3A–S3F and S3J–S3L).", "Most of this deficit resulted from the selective loss of Foxp1+ lateral motor column (LMC) MNs at limb levels and preganglionic column (PGC) MNs at thoracic levels, with little change to Foxp1− medial and hypaxial motor column (MMC and HMC) MNs (Figure S3K) (Rousso et al., 2008).", "LMC and PGC MNs are among the last MN subtypes to be formed (Tsuchida et al., 1994), suggesting that Notch activity must be silenced for the generation of these later-born cell types.", "Nevertheless, Notch-Off spinal cords did not exhibit any obvious defects in either MN formation or segregation into different columnar subgroups (Figures S3G–S3L).", "Olig2Cre-mediated Notch manipulations produced more striking changes in glial fate selection.", "In E18.5 control embryos, Olig2Cre derivatives include both Sox10+ Pdgfrα+ oligodendrocyte progenitors scattered throughout the spinal cord and BLBP+ Nf1A+ Nkx6.1+ Fgfr3+ Slit1+ VA3 astrocyte precursors and differentiated astrocytes located in the ventral-most white matter (Figures 3 and S3M–S3U) (Hochstim et al., 2008).", "Notch-On spinal cords exhibited a nearly complete loss of pOLs and corresponding increase in VA3-like astrocyte precursors (Figures 3A–3H, 3M–3O, and S3M–S3R) (Hochstim et al., 2008).", "Conversely, Notch-Off spinal cords produced more pOLs and fewer astrocyte precursors and differentiated VA3 astrocytes (Figures 3I–3O and S3S–S3U).", "Together, these data show that early changes in NPC fates following Notch pathway manipulation lead to corresponding alterations in neuronal and, more strikingly, glial identities.", "Previous studies observed that glial fates could be altered by deleting Rbpj function from all spinal NPCs (Taylor et al., 2007), raising the question of whether our results stemmed from direct effects of Notch activity on glial fate selection or were a secondary consequence of altered dorsoventral patterning.", "To distinguish between these possibilities, we examined the consequences of manipulating Notch activity in the p0 domain of the intermediate spinal cord using a Dbx1Cre driver (Bielle et al., 2005; Dessaud et al., 2010).", "Dbx1Cre-mediated Notch activation expanded the numbers of Dbx1+ and Dbx2+ progenitors (Figures S4A–S4D and S4G), while Notch inactivation disrupted neuroepithelial organization and depleted these cells (Figures S4E–S4S).", "Despite these effects, we observed no changes in the dorsoventral register of NPCs or shifts in glial identities as seen with Olig2Cre-based manipulations (Figures S4T–S4AI).", "Thus, while manipulation of the Notch pathway can change the balance between NPC maintenance and differentiation within the intermediate spinal cord, it appears insufficient to evoke changes in dorsoventral patterning and associated shifts in neuronal and glial fates.", "The selective effects of Notch activity on cell fate assignment in the ventral versus intermediate spinal cord suggests that Notch modulates the responsiveness of NPCs to Shh ligand produced at the ventral midline.", "To test this possibility, we used a chick intermediate [i] neural plate explant system to examine the fates of NPCs exposed to moderate (1 nM) or high (4 nM) amounts of Shh and varying amounts of the γ-secretase inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) to reduce Notch receptor cleavage and downstream signaling (Dovey et al., 2001; Geling et al., 2002; Dessaud et al., 2007).", "High amounts of Shh produced numerous Nkx2.2+ p3 cells and a small number of Olig2+ pMN cells (Figure 4D), as previously described (Dessaud et al., 2007).", "However, when Notch activity was reduced using DAPT, the number of Nkx2.2+ progenitors was reduced while Olig2+ cells increased (Figures 4E and 4F), recapitulating the phenotype seen in Notch-Off mice (Figures 2I, 2J, and 2N).", "Interestingly, the effects of DAPT up to 25 μM appeared selective, as it blunted the Nkx2.2-inducing activity of high doses of Shh but did not block the Olig2-inducing activity of lower doses of Shh (Figures 4A–4C).", "These results suggest Notch is required for NPCs to experience high but not low levels of Shh signaling.", "To verify that these NPC identity shifts were due to effects of Notch on Shh pathway activity, [i] explants were isolated from chick embryos electroporated with a Gli binding site-luciferase (GBS-luciferase) reporter to measure Gli function after Shh administration (Stamataki et al., 2005; Dessaud et al., 2007).", "DAPT addition led to a >50% decrease in GBS-luciferase activity over that seen with Shh alone (Figure 4G).", "Similar results were obtained with measurement of GBS-luciferase activity in ventral neural plate plus floor plate [vf] explants, in which Gli activity is driven by the endogenous Shh produced by floor plate cells (Figure 4H).", "Collectively, these data demonstrate that Notch signaling is required for NPCs to attain the highest levels of Gli activity and assume the ventral-most fates.", "We next sought to determine a mechanism that could explain the modulatory effects of Notch signaling on Shh responsiveness.", "Given that the requirement of Notch for Shh responses appears to be conserved in NPCs across species, we tested whether it was also conserved across cell types.", "NIH 3T3 mouse fibroblasts are a cell line shown to be Notch responsive (Small et al., 2003) and in which the cellular and molecular details of Shh signaling are well established (Taipale et al., 2000; Rohatgi et al., 2007; Tukachinsky et al., 2010).", "We first validated the system by exposing Shh-Light2 cells, a NIH 3T3 derivative stably transfected with a GBS-luciferase reporter, to increasing concentrations of Shh and observed dose-dependent increases in luciferase activity (Figure 5A).", "The addition of DAPT to these cultures strikingly reduced Shh-induced GBS-Luciferase activity (Figure 5B), recapitulating the effects seen with neural plate explants (Figures 4D–4H).", "Quantitative PCR (qPCR) analysis showed that DAPT similarly impacted endogenous Shh response genes such as Gli1 and Ptch1 (Figure 5C).", "We then used the NIH 3T3 fibroblast system to pinpoint where Notch activity acts in the Shh transduction cascade.", "One of the first steps is the translocation of Smo to primary cilia, which initiates the conversion of Gli proteins into transcriptional activators (Corbit et al., 2005; Rohatgi et al., 2007).", "DAPT dramatically reduced Shh-induced Smo accumulation within primary cilia, acting in a dose-dependent manner (Figures 5D–5F, 5I–5K, and S5A).", "This change occurred without any obvious impact on Smo mRNA, alterations in cell polarity, or presence of primary cilia, although DAPT addition alone reduced average cilia length by 12.6% ± 1.3%, p < 0.001 (Figures 5C and S5B–S5I).", "To confirm that reductions in ciliary Smo were due to changes in Notch pathway activity, we repeated these experiments using two additional small molecule inhibitors: SAHM1, a peptide that prevents assembly of the NICD-Rbpj-MAML1 transcriptional activator complex (Moellering et al., 2009), and JLK6 (7-Amino-4-chloro-3-methoxyisocoumarin, also referred to as γ-secretase inhibitor XI), a molecule that blocks activation of some γ-secretase targets such as beta-amyloid precursor proteins while sparing others, including the Notch receptors (Petit et al., 2001).", "Verifying these activities, we found that both DAPT and SAHM1 reduced Hes1 gene expression in NIH 3T3 cells by ∼65%–75%, whereas JLK6 had no discernible effect (Figure 5I).", "Importantly, SAHM1 reduced Shh-induced ciliary accumulation of Smo in a manner similar to DAPT (Figures 5G and 5J).", "JLK6 in contrast had no effect on Smo localization (Figures 5H and 5K).", "We further tested whether the impact of Notch activity on Shh-induced Smo localization was limited to NIH 3T3 cells or more broadly applicable to other cell types including human NPCs, primary mouse embryonic fibroblasts (MEFs), and C2C12 mouse myoblasts.", "In all cases, DAPT reduced Shh-induced Smo accumulation within primary cilia (Figures S6A–S6M), suggesting that the cross-talk between the Notch and Shh pathways is conserved across germ layers and species.", "Since Notch inhibition reduced both the presence of Smo within primary cilia and Shh pathway activity, we tested whether the converse was also true.", "NIH 3T3 cells were transiently transfected with a vector expressing NICD and an IRES-nEGFP reporter cassette to activate Notch signaling, and both Smo localization and the expression of Shh-target genes evaluated.", "NICD-transfected cells exhibited an ∼40-fold increase in Hes1 expression irrespective of Shh stimulation (Figure 5L).", "Primary cilia were also slightly longer (17.5% ± 3.9%, p < 0.001) in NICD-transfected cells compared with nEGFP-only transfection controls, consistent with the reduced cilia lengths seen with DAPT addition.", "Upon Shh treatment, NICD-transfected cells exhibited an increase in the level of Smo within primary cilia and ∼2- to 3-fold higher levels of Gli1 expression (Figures 5M and 5N).", "These effects were only seen after the addition of Shh.", "Together, these results illustrate that Notch activity is not only required for Shh responsiveness, but can also potentiate its signaling function.", "Given that Hes1 was notably changed in all of our Notch manipulations, we tested whether direct elevation of Hes1 could similarly increase cellular responses to Shh ligand.", "Interestingly, Hes1 misexpression was sufficient to increase Shh-evoked activation of Gli1 ∼1.8-fold (Figures S5J–S5K).", "Collectively, these results suggest that the potentiating effects of Notch on Shh signaling result from activation of Hes genes and likely other downstream effectors.", "Given the ability of Notch signaling to promote localization of Smo to cilia in cultured cells, we examined whether this effect could also be seen in the developing spinal cord.", "In E10.5 control embryos, high amounts of Smo were present in the cilia of both floor plate and Nkx2.2+ p3 cells and lower levels present in Olig2+ pMN cells (Figures 6A–6B′′).", "In Notch-Off spinal cords, most Olig2Cre-derived NPCs exhibited lower levels of ciliary Smo, and this change preceded shifts in Olig2 and Nkx2.2 expression (Figures 6C–6D′′ and 6K).", "By E11.5, the extent of Smo localization within cilia along the dorsoventral axis of Notch-Off mutants was reduced by ∼60% compared with littermate controls (Figures 6E–6F′ and 6I–6L).", "Notch-On mutants by contrast showed a dorsal expansion in the extent of Smo localization within primary cilia (Figures 6G–6H′ and 6L).", "Changes in the ciliary accumulation of Smo following Notch manipulations could stem from either direct effects of Notch on Smo trafficking or indirect effects related to Notch having altered NPC identities.", "To distinguish between these possibilities, we examined Smo staining in the spinal cords of Nkx2.2, Olig2, and Pax6 mutant mice, where dorsoventral patterning is known to be severely disrupted (Dessaud et al., 2008).", "Remarkably, the dorsal limits of ciliary Smo in all mutants were similar to control littermates, despite clear changes in NPC fates (Figures S7A–S7R).", "In Nkx2.2 mutants, this alteration permitted the unusual presence of Olig2 in cells exhibiting high amounts of Smo in their cilia (Figures S7J and S7N), a phenotype that was never seen in control embryos or those in which Notch activity had been manipulated (Figures 6E–6L).", "Collectively, these data show that Notch activity influences Smo accumulation within primary cilia in multiple cell types in vitro and spinal cord NPCs in vivo and acts upstream of the transcription factor network controlling dorsoventral fates.", "We next considered the mechanism by which Notch might impact Smo localization.", "Our observations that Notch activation only promoted the accumulation of Smo within cilia following Shh addition suggested that it most likely acts upstream of Smo in the Shh transduction cascade.", "Consistent with this model, we found that DAPT was unable to block Smo accumulation when cells were treated with either Purmorphamine (Pur) or Smoothened Agonist (SAG), small molecules that directly stimulate Smo activity in a Shh ligand-independent manner (Chen et al., 2002; Sinha and Chen, 2006) (Figures 7A–7E).", "We thus focused our attention on the actions of Notch on the Shh receptor Ptch1.", "In the absence of ligand, Ptch1 localizes around the base and within primary cilia, where it inhibits Smo entry and Gli activation (Rohatgi et al., 2007).", "Shh binding to Ptch1 promotes its exit from primary cilia and concomitant Smo accumulation (Rohatgi et al., 2007).", "Since endogenous Ptch1 protein was difficult to detect in NIH 3T3 cells by antibody staining, we utilized Ptch1-YFP MEFs generated by infection of Ptch1LacZ/LacZ mutant cells with a retrovirus expressing a Ptch1-YFP fusion protein (Rohatgi et al., 2007).", "In the absence of Shh, ∼75% of primary cilia contained Ptch1 (Figures 7F and 7J).", "When DAPT was added for 12 hr, the number of Ptch1+ primary cilia increased to ∼90% (Figures 7G and 7J).", "DAPT was also able to impede the clearance of Ptch1 from primary cilia upon Shh stimulation (Figure 7H–7J).", "Remarkably, the effects of DAPT on Ptch1 localization occurred without any change in either Ptch1 mRNA or protein levels in both Ptch1-YFP MEFs and NIH 3T3 cells (Figures S8G–S8I).", "These results prompted us to examine whether the effects of DAPT on Smo trafficking to primary cilia occur immediately after its addition or rather require more time to enable Ptch1 to increase and thereby block Smo entry.", "Smo normally accumulates in primary cilia within 4 hr of Shh addition (Rohatgi et al., 2007) (Figures S8A and S8B).", "When Shh and DAPT were coadministered, there was no decrease in Smo presence within primary cilia at either the 4- or 6-hr time points; rather, Smo reduction only became evident after ∼12 hr (Figures S8A and S8B).", "In contrast, when cells were pretreated with DAPT for 8 hr and then exposed to Shh plus DAPT for an additional 4 hr, significant reductions in Smo ciliary accumulation were observed (Figures S8C and S8D).", "These data indicate that the suppressive actions of DAPT on Smo localization follow the time course of Ptch1 accumulation within primary cilia.", "We further found that the actions of DAPT required new transcription, as changes in Smo localization were partially blocked by coadministration of DAPT and the RNA polymerase inhibitor α-amanitin (Figures S8E and S8F).", "These results suggest that Notch modulates Ptch1 and Smo levels in and around primary cilia through a transcriptional mechanism.", "To test whether Ptch1 mediates the inhibitory effects of DAPT on Smo, we measured the impact of DAPT addition to Ptch1LacZ/LacZ mutant MEFs.", "Whereas DAPT potently inhibited Smo accumulation in the cilia of Shh-treated control MEFs, it was unable to do so in Ptch1 null cells (Figures 7K–7O and S8J).", "Collectively, these data show that Notch signaling influences Smo accumulation by regulating the ciliary presence of Ptch1.", "Finally, we tested whether altered localization or abundance of Ptch1 protein was observed after manipulations of the Notch pathway in the ventral spinal cord.", "In Notch-On mutants, Ptch1 protein staining in and around the primary cilia was notably reduced, fitting with the observed increase in Smo presence (Figures 6E–6H′, 7P, 7Q, and S7).", "In contrast, Notch-Off mutants showed elevated Ptch1 at the apical membrane and cilia in accordance with the reductions in Smo staining (Figures 6I–6J′, 7R, and 7S).", "Together, these in vitro and in vivo experiments demonstrate that Notch signaling plays an integral role modulating Ptch1 localization to gate Smo entry into primary cilia.", "Through these actions, Notch can regulate the downstream activation of the Shh transduction pathway and assignment of NPC fates.", "It is well established that the dorsoventral identity of NPCs in the spinal cord and other regions of the CNS is influenced by the concentration of Shh ligand they are exposed to (Fuccillo et al., 2006; Dessaud et al., 2008; Ribes and Briscoe, 2009).", "However, Shh concentration is only part of the means though which graded signaling responses are achieved.", "Other important factors include (1) the duration of time over which cells are exposed to Shh, (2) the ability of cells to modulate their responsiveness to Shh through changes in the expression and/or subcellular distribution of key signal transduction components such as Ptch1 and Smo, (3) changes in the expression of proteins that modulate Shh-Ptch1 interactions or modify Shh itself, and (4) cross-regulatory interactions between Shh-regulated transcription factors that assign specific cell fates (Dessaud et al., 2008; Ribes and Briscoe, 2009; Briscoe and Thérond, 2013).", "Our studies show that Notch signaling plays a crucial role in these first two processes, serving to sustain NPCs in an undifferentiated, Shh-responsive state while also influencing the ciliary trafficking of Ptch1 and Smo and the downstream activation of Gli transcription factors (Figure 8).", "Together, these data provide important insights into the mechanisms through which NPCs interpret the Shh gradient and reveal a potentially general mechanism by which the Notch and Shh signaling pathways collaborate to direct cell fate decisions.", "Our data show that manipulating the Notch pathway modulates the dorsoventral register of NPCs, with Notch activation and inactivation respectively increasing or decreasing the formation of the ventral-most cell types reflected by alterations in Nkx2.2 and Olig2 expression and shifts in specific classes of neurons and glia.", "Importantly, multiple lines of evidence indicate that these changes are due to the ability of Notch to modulate how NPCs interpret the endogenous Shh signaling gradient rather than more direct effects on cell fate determination.", "First, all changes in NPC fates occurred within the context of Nkx6.1+ progenitors, which reflect the limit of endogenous Shh signaling in the spinal cord (Briscoe et al., 2000).", "Second, Notch manipulation in the intermediate spinal cord impacted NPC maintenance without any change in dorsoventral patterning or shift in glial cell types.", "Third, in fibroblasts, Notch activation and inactivation were unable to modulate Smo trafficking to primary cilia or Gli transcriptional activity without the coadministration of Shh ligand.", "Collectively, these data indicate that Notch plays a supporting role in tissue patterning by tuning the response of cells to Shh present in the developing embryo or culture media.", "It has long been appreciated that the influences of Shh on neural fate selection are generally restricted to dividing cells (Ericson et al., 1996).", "Recent studies have provided molecular explanations for this relationship showing that most Shh/Gli-regulated genes are coregulated by SoxB1 transcription factors such as Sox2 that are broadly expressed by NPCs (Oosterveen et al., 2012; Peterson et al., 2012; Oosterveen et al., 2013).", "Some of the positive effects of Notch on Shh signaling could thus be accounted for by its ability to elevate SoxB1 levels as it maintains NPCs in an undifferentiated state.", "However, our data indicate that Notch can also act at a more proximal level, regulating the ciliary localization of at least two key components of the Shh transduction pathway, Ptch1 and Smo.", "Ptch1 appears to be the most directly impacted by Notch, as the addition of DAPT alone to fibroblasts promotes Ptch1 accumulation within primary cilia (Figures 7F, 7G, and 7J), and Ptch1 is known to block Smo entry and downstream signaling events (Rohatgi et al., 2007).", "Moreover, DAPT was unable to reduce Smo accumulation within cilia in the absence of Ptch1 or in the presence of Pur and SAG, small molecules that bypass Ptch1 function (Figures 7A–7E and 7K–7O).", "These observations in fibroblasts also hold true for spinal cord NPCs, as Rbpj deletion increased Ptch1 protein in and around primary cilia, whereas NICD misexpression reduced it.", "These changes correspondingly impacted Smo presence in cilia and, ultimately, the expression of specific NPC fate determinants (Figures 7P–7S).", "How might Notch signaling alter Ptch1 and Smo trafficking?", "In epidermal cells, Notch receptors and processing enzymes are located in and adjacent to primary cilia, and ciliary transport is required for Notch pathway activity (Ezratty et al., 2011).", "Based on this proximity, Notch signaling components could conceivably impact the interactions of ciliary transport proteins with Shh signaling components.", "However, our results point to Notch acting through a transcriptional mechanism.", "First, changes in NPC fates and Gli transcriptional activity were seen with either removal of Rbpj function or increased expression of NICD, components whose main sites of action are known to be in the nucleus.", "Second, the Shh-potentiating activities seen with NICD misexpression were recapitulated by the forced expression of Hes1, one of the best known downstream transcriptional effectors of the Notch pathway.", "Third, the effects of DAPT administration on Ptch1 and Smo trafficking were not immediate, but rather required at least 12 hr of exposure—more than sufficient time for a transcriptionally mediated response.", "Finally, DAPT effects on Smo trafficking were blocked by the addition of the transcriptional inhibitor α-amanitin.", "Together, these results lead us to propose that Notch and Hes genes modulate Shh signaling by regulating the expression of genes whose products impact the trafficking of Ptch1, Smo, and potentially other Shh signaling components to primary cilia, designated as “X” for direct Notch effectors and “Y” for Hes-suppressed effectors (Figure 8).", "While a great deal is known about the transcriptional control of Ptch1 in response to Shh pathway activation, relatively little is known about the regulation of Ptch1 protein trafficking.", "Some insights into this process have been recently made by observations that Ptch1 exit from primary cilia requires the function of the intraflagellar transport (IFT) protein Ift25 (Keady et al., 2012), and endocytic turnover mediated by the ubiquitin E3 ligases Smurf1 and Smurf2 (Yue et al., 2014).", "Loss of these components results in Ptch1 accumulation within primary cilia and reduced cellular responses to Shh (Keady et al., 2012; Yue et al., 2014), reminiscent of the effects seen with the loss of Notch signaling.", "However, none of these genes were changed by our Notch manipulations (J.H.K. and B.G.N., unpublished data).", "A better understanding of the downstream targets of Notch and Hes1 should yield important new insights into how the localization and function of Ptch1 and other Shh signaling components may be controlled.", "The primary cilium is a nonmotile organelle that is present on almost all vertebrate cells (Pazour and Witman, 2003).", "Although primary cilia were first observed over a century ago (Zimmermann, 1898), their function as an antenna-like organelle that allows cells to detect extracellular environmental stimuli and modulate an appropriate intracellular response has only recently been realized.", "In addition to Shh signaling, primary cilia are thought to be essential for Hippo, mTor, Notch, Pdgfrα, and Wnt signaling (Schneider et al., 2005; Boehlke et al., 2010; Ezratty et al., 2011; Habbig et al., 2011; Lancaster et al., 2011).", "The importance of primary cilia is perhaps best illustrated through ciliopathies, a group of genetic disorders that are due to defects in the generation or function of cilia, which collectively affect nearly every major organ in the human body (Novarino et al., 2011).", "As no protein synthesis occurs within the cilium, the formation of the cilium and the accumulation of signaling pathway components within the cilium are entirely dependent on the IFT system to shuttle proteins to their proper areas (Pedersen and Rosenbaum, 2008).", "While our study focused on the impact of Notch on Shh signaling by altering the localization of Ptch1 and Smo, the mechanisms used to achieve this result are likely to have a broader impact on other signaling pathways that depend upon the IFT system.", "Consistent with this hypothesis, we have carried out a series of preliminary expression profiling experiments in NIH 3T3 cells, which indicate that DAPT addition reduces the expression of several proteins known to be associated with primary cilia (Ishikawa et al., 2012), including components of the Pdgfrα and Wnt signaling pathways, and various extracellular matrix proteins (J.H.K. and B.G.N., unpublished data).", "In this regard, the mechanism through which Notch gates the responsiveness of cells to Shh might signify a more general role for Notch modulating ciliary transport that could impact multiple signaling pathways involved in both development and disease.", "Olig2Cre and Dbx1Cre mice were generated as previously described (Bielle et al., 2005; Dessaud et al., 2007).", "Cre mice were crossed with R26RGFP transgenic reporter mice (B6;129-Gt(ROSA)26Sortm2Sho/J; Jackson Labs Stock #004077) (Mao et al., 2001); R26RNICD-nGFP transgenic floxed mice (Gt(ROSA)26Sortm1(Notch1)Dam/J; Jackson Labs Stock #008159) (Murtaugh et al., 2003), or RbpjCKO mice (Han et al., 2002).", "Olig2−/−, Nkx2.2−/−, and Pax6Sey/Sey mutant mice were generated as previously described (Novitch et al., 2001; Rousso et al., 2012).", "All mice were maintained and tissue collected in accordance with guidelines set forth by the UCLA Institutional Animal Care and Use Committee.", "Chick neural plate explants were generated as previously described (Dessaud et al., 2007).", "All spinal cord tissues were fixed, cryoprotected, sectioned, and processed for immunohistochemistry or in situ hybridization as previously described (Novitch et al., 2001; Gaber et al., 2013).", "Antibodies and probes used are listed in the Supplemental Experimental Procedures.", "NIH 3T3 fibroblasts (CRL-1658) and C2C12 myoblasts (CRL-1772) were purchased from ATCC.", "Shh-LIGHT2 cells were used as previously described (Taipale et al., 2000).", "Ptch1−/− and Ptch1−/−;Ptch1-YFP MEFs were generated as previously described (Rohatgi et al., 2007, 2009).", "Primitive human neuroepithelial progenitors were generated from embryonic stem cells as previously described (Hu et al., 2009).", "For cilia analysis in fibroblasts, cells were plated onto glass coverslips, grown to 80%–100% confluency in DMEM containing 10% bovine calf serum (BCS) and then changed to low serum media (0.5% BCS) at the beginning of experiments.", "Cells were fixed in 4% paraformaldehyde, incubated with indicated primary and secondary antibodies, and mounted in Prolong Gold (Invitrogen).", "See also Supplemental Experimental Procedures.", "Unless otherwise stated, cell counts, luciferase assays, and qPCR analyses are presented as mean values ± SEM.", "For Figures 1Q, 2M, 2N, 3M, 3N, 5I, 6K, 6L, 7S, S3J, S3K, S4G, S4AF–S4AI, S7Q, S7R, and S8I, experimental conditions were compared with the control, and an ANOVA with a Dunnett’s post hoc test was performed.", "For the data shown in Figures 4G, 4H, 5C, 5L, 5N, S5D, S5I–S5K, and S8H–S8J, unpaired, two-tailed t test were performed.", "All ciliary Smo fluorescence data sets did not pass the Shapiro-Wilk normality test.", "Thus, for all ciliary Smo analyses between two groups (Figures 5M, 7E, 7J, S8B, S8D, and S8F) two-tailed nonparametric Mann-Whitney tests were performed.", "For analyses between three or more groups (Figures 5J, 5K, 7O, S5A, S6D, S6H, and S6L), nonparametric Kruskal-Wallis tests were used along with Dunnett’s post hoc tests.", "All statistical analyses were calculated using Graphpad Prism 6 software.", "Significance was assumed when p < 0.05.", "J.H.K., L.Y., E.D., K.C., and D.M.M. performed the experiments.", "R.R. contributed vital reagents and insights.", "J.H.K., L.Y., J.B., and B.G.N. designed the experiments and wrote the paper." ]	[ "Changes in Notch signaling alter the dorsoventral identity of neural progenitors", "Activation and inactivation of Notch signaling alter cellular responses to Shh", "Notch activity is required for efficient trafficking of Smo to primary cilia", "Notch activity regulates the subcellular distribution of the Shh receptor Ptch1" ]
Argonaute2 mediates compensatory expansion of the pancreatic β cell	Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity. © 2014 Elsevier Inc.	[ "BIOC" ]	[]	[ "1307", "1312", "1314" ]	[ "Adaptation to environmental stress is a fundamental cellular process that promotes the maintenance of the physiologic steady state (Spriggs et al., 2010).", "Stress responses have been shown to induce numerous changes, such as activation of gene expression programs, which have evolved to allow for the cell to promote its own survival (Kültz, 2005; Ebert and Sharp, 2012).", "For example, in response to insulin resistance, the pancreatic β cell undertakes measures to proliferate and increase its output of secreted insulin.", "A coordinated increase in both β cell mass and secretory function constitutes the compensatory response to maintain normoglycemia (Muoio and Newgard, 2008).", "Although the underlying mechanisms directing these processes are still not completely understood, several studies have illustrated a role for metabolic changes in catalyzing β cell expansion (Steil et al., 2001).", "Furthermore, cellular pathways enabling the β cells to proliferate and adapt to increases in metabolic load may act by ultimately promoting signaling cascades essential to increasing both secretion and islet mass (Rhodes, 2005).", "Recent evidence has shown the microRNA (miRNA) pathway as an important regulator of gene expression in response to metabolic stress (Leung and Sharp, 2010).", "Central to this mechanism are the Argonaute (Ago) proteins, which mediate this pathway by facilitating the interaction between miRNAs and their target mRNAs (Höck and Meister, 2008; Bartel, 2009).", "In addition, Ago proteins have been shown to accumulate in stress granules upon exposure to oxidative stress; however, their role in this compartment is not understood (Leung et al., 2006).", "Although loss of Argonaute2 (Ago2) expression in the MIN6 β cell line model resulted in enhanced secretion, its role in the stress response of the β cell has not been described (Tattikota et al., 2013).", "We have previously shown that loss of miR-375 expression, among the most abundant miRNA in the pancreatic islet, inhibited the compensatory β cell proliferation in leptin-deficient ob/ob mice and resulted in severe hyperglycemia and diabetes (Poy et al., 2009).", "The absence of any dramatic effect on the development or specification of the different cell populations in the miR-375 knockout mouse may indicate a larger role for this miRNA in stress responses (Mendell and Olson, 2012).", "Furthermore, these observations suggest that many of the targets of miR-375 are also relevant to the adaptive response of the β cell and likely play a role in proliferation during metabolic stress.", "Although extensive sequencing efforts have identified ∼2,000 mature miRNA sequences in human tissues, relatively little is understood regarding how small RNAs coordinately function in these cellular processes (Kozomara and Griffiths-Jones, 2011).", "Here, we show that miR-184 is silenced during insulin resistance to promote the expression of Ago2 in the pancreatic β cell.", "Deletion of Ago2 in ob/ob mice reduced compensatory proliferation of this cell type, thereby underlining an integral role for the miRNA pathway in this process.", "Moreover, we observed that Ago2 mediates the function of miR-375 in regulating the growth suppressor Cadm1.", "Taken together, our results show that several components of the miRNA pathway contribute in a concerted effort to facilitate proliferation of the β cell to meet metabolic demand during insulin resistance.", "In light of the essential role of miR-375 in adaptive growth of the pancreatic β cell, we first sought to identify the additional components of the miRNA pathway that coordinately mediate this mechanism.", "We performed small RNA sequencing on total RNA from islets of 12-week-old ob/ob mice (Table S1 available online).", "Consistent with results by Zhao et al. (2009), expression of miR-184 was the most reduced miRNA identified (Figure 1A; Table S1).", "We then measured miR-184 in the islets of ob/ob mice from age 4–16 weeks and observed the decrease in expression starting at 8 weeks of age with the onset of resistance (Figures 1B, S1A, and S1B).", "Similarly, the pri-miR-184 transcript in the islets of ob/ob mice by quantitative real-time PCR was also silenced, indicating that this miRNA is regulated on a transcriptional level (Figure 1C).", "As recently described, miR-184 is enriched in pancreatic β cells as shown by quantitative real-time PCR from fluorescence-activated cell sorting (FACS)-sorted, GFP-positive β cells (MIP-GFP) (Figure 1D) (Hara et al., 2003; van de Bunt et al., 2013).", "We also observed a similar loss of expression of mature miR-184 in the islets of 12-week-old leptin receptor-deficient db/db mice and in mice on a high-fat diet (HFD; 60% calories from fat), all of which showed that this observation is not limited to one mouse model of obesity and insulin resistance (Figures 1E and 1F).", "In contrast to miR-184, miR-375 was modestly increased in HFD-fed animals as previously observed in ob/ob mice (Figure 1G) (Poy et al., 2009).", "In addition, the suppression of miR-184 was not observed in the eye of ob/ob mice, the highest site at which expression has been measured (Figures S1C and S1D).", "In order to identify direct targets of miR-184, we induced its expression in the MIN6 line with doxycycline after transfection with plasmids expressing the rtTA transactivator and miR-184 under control of the operator sequence of the Escherichia coli tetracycline-resistance operon (184-tetO).", "Illumina WG6v2 arrays measured changes in gene expression after 16 hr of treatment with doxycycline in triplicate (Figure S1E).", "Several computationally predicted genes for miR-184 were downregulated in both the array and luciferase-based experiments, including Ago2 (Figure 1H; Figures S1F–S1H) (Friedman et al., 2009).", "We cloned a portion of the mouse 3′ UTR of Ago2 (912 nt) and the complete human UTR of Ago2 (899 nt) into a luciferase reporter construct and observed decreased activity in the presence of the miR-184 mimic.", "In addition, mutating six nucleotides in the binding site within the mouse or human UTR (at position 152) from UCCGUCC to CGGGCGG abolished the inhibitory effect of miR-184 (Figure 1H).", "Conversely, transfecting a mutant miR-184 mimic had no effect on reporter activity from constructs bearing either the mouse or human Ago2 3′ UTR sequences (Figure 1I).", "Western blotting after overexpression of miR-184 resulted in an approximately 60% decrease in Ago2 protein levels, whereas quantitative real-time PCR revealed a 40% decrease in Ago2 mRNA in MIN6 cells (Figures 1J and 1K).", "To address the relevance of this microRNA:target interaction in the islets of human subjects, we measured the expression of miR-184 and Ago2 in the pancreatic islets from 15 nondiabetic and 12 type 2 diabetic (T2D) donors.", "miR-184 was significantly decreased in T2D islets, indicating that the expression of this specific miRNA is linked to changes in metabolic status in both mice and humans (Figure 1L; Table S2).", "Whereas levels of Ago2 were not statistically significantly increased in T2D donor islets compared to the nondiabetic cohort, the inverse correlation between Ago2 and miR-184 expression was significant across the entire cohort (Figure 1M).", "We next measured Ago2 expression in the islets of insulin-resistant ob/ob mice and observed increased levels at 8 weeks of age, whereas Ago1 was not altered (Figure 2A).", "Similarly, Ago2 levels were increased in islets isolated from 12-week-old db/db mice and C57BL/6 mice on a HFD for 16 weeks (Figures 2B).", "As shown in other tissues, Ago2 is the most abundant of the Ago family in GFP-positive β cells as measured by quantitative real-time PCR (Figures 2C and S2A) (Wang et al., 2012).", "To understand the contribution of Ago2 to growth and function of the β cell, we generated transgenic mice bearing the mouse Ago2 cDNA under the regulatory control of a tetracycline-responsive promoter element and crossed them with mice expressing the reverse tetracycline-controlled transactivator (rtTA) protein under the control of the rat insulin2 (Ins-rtTA) promoter as described (dox-Ago2) (Nir et al., 2007).", "Administration of doxycycline via drinking water for 30 days to four different lines (14, 30, 61, and 69) resulted in ∼1.5- to 25-fold increase of Ago2 protein levels in isolated islets (Figures 2D and S2B).", "Characterization of all lines showed no changes in body weight, random glucose, or insulin levels.", "After a glucose challenge, dox-Ago2 mice (line 30) exhibited transiently elevated glucose levels compared to littermates; however, no difference was observed in insulin sensitivity (Figures S2C and S2D).", "Moreover, acute-phase plasma insulin levels were diminished after glucose challenge, indicating reduced insulin release (Figure S2E).", "Morphometric analysis in dox-Ago2 (line 30) animals showed increased β cell mass in addition to increased insulin-positive cell number and BrdU incorporation compared to littermates (Figures 2E–2H).", "We then established the conditional deletion in β cells by crossing mice bearing the floxed allele of the Ago2 gene with animals expressing Cre-recombinase under control of the rat insulin promoter (βAgo2KO) (O’Carroll et al., 2007; Herrera, 2000).", "Quantitative RT-PCR and western blotting analysis confirmed an efficient reduction in Ago2 expression in islets from βAgo2KO mice compared to littermate controls (Figures 2I and 2J).", "In contrast, Ago1 was upregulated in islets isolated from βAgo2KO mice, suggesting a degree of compensation by this family member on the protein level (Figure 2J).", "Both random-fed and fasted glucose and insulin levels were unchanged; however, βAgo2KO mice exhibited improved glucose tolerance without any alteration in insulin sensitivity at 10 weeks of age compared to littermates (Figures S2F and S2G).", "As previously observed in the MIN6 model, loss of Ago2 expression in vivo resulted in enhanced secretion of insulin after glucose challenge (Figure S2H) (Tattikota et al., 2013).", "In contrast to dox-Ago2 mice, we observed decreased β cell mass and number, BrdU incorporation, and pancreatic insulin content in βAgo2KO animals without any change in islet architecture, α cell number, or total pancreatic mass (Figures 2K–2N, S2I, and S2J).", "Moreover, no change was detected in TUNEL-positive β cells, indicating cell death was not a primary mechanism for the effect on β cell mass (Figure 2O).", "Lastly, we measured an increase in both β cell size and the total number of granules (Nv, measured as LDCV volume density), whereas the number of docked granules remained unchanged (Ns, LDCV surface density) in βAgo2KO mice compared to their littermates using electron micrographs (Figure S2K).", "To determine the functional role of miR-184 in the β cell, we characterized the constitutive miR-184-knockout mouse (184KO) for changes in β cell proliferation, insulin release, and glucose homeostasis.", "The loss of miR-184 expression resulted in decreased fasted glucose levels and increased fasted plasma insulin levels (Figures 3A–3C).", "Consistent with loss of miR-184 expression during insulin resistance, morphometric analysis of pancreatic sections from 184KO mice showed an increase in β cell mass and number and BrdU incorporation rate (Figures 3D–3F).", "Moreover, in line with islet expression analysis in insulin-resistant models, Ago2 expression was increased in miR-184-deficient islets (Figures 3G and 3H).", "Knockout mice exhibited transiently elevated insulin release and improved tolerance after glucose challenge, indicating additional targets, such as Slc25a22, an established regulator of insulin release, may mediate the effect of miR-184 on secretion (Figures S1H, S3A, and S3B) (Casimir et al., 2009).", "Lastly, insulin sensitivity was unchanged in 184KO mice, indicating that loss of this miRNA in the β cell does not contribute to insulin resistance (Figure 3I).", "Two gain-of-function models for miR-184 were then generated in order to restore its expression in the β cell and validate the regulation of Ago2 by this miRNA during insulin resistance.", "Similar to dox-Ago2 mice, Ins2-rtTA mice were crossed to newly generated mice bearing the mouse miR-184 precursor under regulatory control of a tetracycline-responsive promoter element (dox-184).", "Administration of doxycycline for 15 days via drinking water (1 mg/ml) resulted in ∼100-fold increase in expression (Figure 3J).", "No change was observed in body weight; however, dox-184 animals exhibited decreased plasma insulin levels, β cell mass and number, and BrdU incorporation (Figures 3K–3N and S3C).", "The number of apoptotic cells was unchanged, indicating the hyperglycemia (>250 mg/dl) and glucose intolerance in dox-184 mice were due to a decrease in β cell mass and rate of proliferation (Figures 3O and S3D–S3F).", "Isolated islets from dox-184 mice treated with doxycycline for 48 hr ex vivo exhibited decreased Ago2 expression (Figure 3P).", "Moreover, we crossed dox-184 mice onto the ob/ob background (dox-184ob) and observed a 50% reduction in Ago2 expression after doxycycline treatment and restoring expression of miR-184 (Figure 3P).", "Furthermore, whereas islet levels of miR-375 and Ago1 were unchanged, random blood glucose levels increased and plasma insulin levels were significantly reduced in dox-184ob mice (Figures S3G–S3J).", "An additional transgenic model of constitutive overexpression was developed to further confirm the role of miR-184 in β cell growth and function.", "We focused on three independent mouse lines (Tg-96, Tg-04, and Tg-32), which showed by Southern analysis varying numbers of the transgene containing the miR-184 precursor sequence under control of the rat insulin promoter (Figure S3K).", "All lines exhibited a mild decrease in body weight, but the highest overexpression of miR-184 (∼100-fold in Tg-04 and 32) resulted in severe hyperglycemia and reduced systemic insulin levels, whereas a 5-fold increase (Tg-96) induced glucose intolerance and impaired insulin release (Figures S3L–S3Q).", "Similar to dox-184 animals, Tg-04 and Tg-32 animals exhibited hyperglycemia and reduced circulating insulin due to loss of β cell mass and pancreatic insulin content (Figures 4A–4G and S4A–S4G).", "Crossing Tg-04 animals onto the ob/ob background (04ob) resulted in sustained expression of miR-184 and did not compromise the most abundant miRNAs, such as miR-375 (Figures 4A and 4B).", "As in dox-184ob mice, 04ob mice exhibited severe hyperglycemia as a result of diminished insulin levels and β cell mass and ultimately contributed to weight loss (Figures 4C–4I).", "Lastly, restoring expression of miR-184 in β cells of 04ob mice suppressed Ago2 expression compared to ob/ob littermates (Figure 4J).", "We next measured the expression of miR-375-targeted genes in islets from βAgo2KO mice and confirmed increased protein expression in vivo (Figure S5A) (Tattikota et al., 2013).", "Conversely, expression analysis of these miR-375 targets in islets from dox-Ago2 mice showed decreased levels by quantitative real-time PCR (Figure S5B).", "While immunoprecipitation of Ago2 from untransfected MIN6 cells efficiently recovered miR-375, we observed an enrichment of Gephyrin, Elavl4/HuD, and Rasd1 in the Ago2-containing complexes compared to control pull-downs (Figures S5C and S5D).", "Moreover, overexpression (OE) of Ago2 recovered higher levels of these specific mRNAs, whereas transfection of inhibitory antisense oligonucleotides against miR-375 (AS 375) depleted their presence in the protein complex (Figure S5D).", "We also observed that expression of the growth suppressor Cadm1 was increased in islets of βAgo2KO mice, further indicating that the regulation of several targets of miR-375 was dependent on Ago2 (Figures 5A and 5B) (Poy et al., 2009).", "The higher levels of miR-375 target genes were accompanied by no change in miR-375 expression, indicating that the loss of Ago2 can nullify the impact of abundant miRNAs on their respective targets (Figure 5B).", "We next measured Cadm1 expression after siRNA-mediated knockdown of Ago2 in MIN6 cells and observed a similar increase, but not after knockdown of Ago1 (Figures 5C and 5D).", "In addition, increasing Ago2 levels in both islets of dox-Ago2 mice and in MIN6 cells (Ago2OE) decreased Cadm1 expression (Figures 5E and 5F).", "As observed with the other miR-375 targets, we detected enrichment of Cadm1 in Ago2-containing complexes, indicating its regulation was also dependent upon this member of the Ago family (Figure 5G).", "In line with its established role as a growth suppressor, morphometric analysis in cadm1-deficient mice (cadm1KO) showed increased β cell mass and number and BrdU incorporation without any change in total pancreatic mass (van der Weyden et al., 2006, 2012) (Figures 5H and S5E).", "Lastly, consistent with observations in dox-Ago2 animals, quantitative real-time PCR analysis in the islets of ob/ob mice and littermate controls showed the miR-375 targets Cadm1, Gephyrin, Rasd1, and Elavl4/HuD to be decreased, whereas Ago2 expression was significantly upregulated (Figure 5I).", "To address whether Ago2 mediates the compensatory expansion of β cells during insulin resistance, we next crossed mice carrying the Ago2-floxed allele onto the ob/ob background to generate mice deficient in both Ago2 and leptin (Ago2ob).", "In line with βAgo2KO mice, β cell mass and the number of insulin-positive cells were significantly reduced in Ago2ob animals compared to ob/ob littermates without any alterations in the number of glucagon-positive cells or total pancreatic mass (Figures 6A–6D).", "Furthermore, random glucose levels and the corresponding insulin levels were decreased, thereby underlining an important functional role for Ago2 in the growth and function of the β cell (Figures 6E and 6F).", "In spite of the decrease in β cell mass, Ago2ob mice exhibited higher circulating insulin levels after challenge and improved glucose tolerance, confirming again that loss of Ago2 increases secretion in vivo (Figures 6G and 6H).", "Insulin sensitivity, body weight, or food intake (5.2 ± 0.4 in ob/ob and 5.0 ± 0.3 g/day in Ago2ob mice) were not significantly altered, but the decrease in both circulating glucose and insulin suggests insulin resistance has been partially attenuated (Figures 6I and 6J).", "In addition, western blotting analysis of isolated islets showed that loss of Ago2 expression resulted in increased levels of the miR-375 target Cadm1 (Figure 6K).", "Similar results in islet morphometry and glucose tolerance were observed in βAgo2KO mice placed on a HFD, indicating that the effect on β cell mass was consistent in multiple forms of insulin resistance and, although extrapancreatic recombination has been shown with this Cre line, including in the hypothalamus, energy expenditure or food intake was not affected (Figures 6L–6O and S6A–S6D).", "Published studies have shown that the administration of the ketogenic diet improves insulin sensitivity and glycemic status (Badman et al., 2009).", "To further support the functional relevance of miR-184 and Ago2 in the β cell, we next tested whether miR-184 is regulated after administering the ketogenic diet to C57BL/6 mice (Keto mice).", "After 24 days on the diet, we observed ∼4-fold increase of miR-184 in islets from Keto mice (Figure 7A).", "As previously shown, Keto mice exhibited decreased body weight and reduced glucose levels at steady state and during an ITT in line with improved insulin sensitivity (Figures 7B, S7A, and S7B).", "Consistent with mild ∼4-fold, overexpression of miR-184, Keto mice exhibited reduced insulin levels and impaired tolerance compared to control mice after a glucose challenge; however, no change was observed in β cell mass (Figures 7C, 7D, and S7C).", "Moreover, administration of the ketogenic diet to ob/ob mice (Keto/ob) for 15 days was sufficient to restore normal expression of miR-184; however, after 45 days, we observed a ∼18-fold increase in the expression of this miRNA in islets from Keto/ob mice compared to chow-fed, nonobese littermate controls (Ch-WT) (Figure 7E).", "Keto/ob mice also exhibited lower glucose levels during an ITT, reduced random glucose and plasma insulin levels, and no change in body weight (Figures 7F–7H, S7D, and S7E).", "Similar to Keto mice, Keto/ob mice were relatively more glucose intolerant as a result of decreased insulin release compared to Chow/ob littermates (Figures 7I and 7J).", "These results may reflect a significant reduction in the demand for insulin because Keto/ob mice are normoglycemic.", "Moreover, after 45 days on the ketogenic diet, we observed a decrease in β cell mass and Ki-67+ β cells in Keto/ob animals, and quantitative real-time PCR analysis at this time point showed Ago2 and Slc25a22 expression to be significantly reduced, whereas measurements produced by western blotting showed Cadm1 to be increased (Figures 7K–7M, S7F, and S7G).", "Taken together, these data further show that both miR-184 and Ago2 expression are inversely regulated in accordance with changes in the requirement for insulin.", "Here, we show that the onset of insulin resistance induces the silencing of miR-184 in order to promote the function of Ago2 in the pancreatic β cell.", "Our data suggest that miR-184 acts as a “brake” on Ago2 that becomes released to increase proliferation and accommodate the elevated demand for insulin.", "Moreover, we show that the increased expression of Ago2 will facilitate the function of miR-375 in suppressing genes, including Cadm1 in vivo.", "Our observations functionally associating miR-184 together with Ago2 and miR-375 now identify a network within the miRNA pathway that coordinately regulates the compensatory proliferation of the β cell.", "Interestingly, miR-184 is unique as the most downregulated islet miRNA during insulin resistance, and additionally, administration of the ketogenic diet in leptin-deficient ob/ob mice rescued its expression.", "Furthermore, the increase of miR-184 expression in the islets of ob/ob mice after restoring insulin sensitivity further underlines a dynamic role for the miRNA pathway in regulating β cell growth and function according to the demand for insulin.", "Published work identified the regulation of miRNAs in the retina according to light levels, thereby linking turnover of small RNAs with physiologic stimuli in neuronal cells; together, our results may indicate another common property between these cell types (Krol et al., 2010).", "Likewise, after treatment with serotonin in Aplysia sea snails, miR-184 was significantly downregulated in the nervous system, the site of orthologous insulin expression (Rajasethupathy et al., 2009; Floyd et al., 1999).", "Moreover, serotonin has been previously shown to mediate compensatory expansion of the β cell during pregnancy (Kim et al., 2010).", "In line with these studies, we observed that miR-184 was transiently decreased at day 18.5 of gestation in C57BL/6 mice and that the expression of this miRNA had been restored by day 5 in the postpartum phase.", "Neither tryptophan hydroxlase 1 nor 2 (Tph1/2) were regulated in the pancreatic islets of ob/ob mice, further suggesting that multiple independent pathways promote proliferation in these models (Rieck et al., 2009).", "However, the robust consistency of the silencing of miR-184 in both models of pregnancy and insulin resistance suggests that additional genes in the miRNA pathway will be identified as mediators of β cell proliferation (Jacovetti et al., 2012).", "Importantly, our results continue to build on the numerous studies now supporting a role for miRNAs in mediating cellular stress responses (Leung and Sharp, 2010; Mendell and Olson, 2012).", "The observations made in Ago2ob animals would continue to indicate that several abundant miRNAs in the β cell coordinately contribute to this process.", "In addition, our current investigation continues to reinforce the role of miRNAs as inhibitors of insulin release (Poy et al., 2004; Tattikota et al., 2013; Bolmeson et al., 2011).", "Although the precise role of miRNAs remains unclear, the accumulating evidence may suggest that “brakes” on gene expression are essential in managing the dynamic nature of both first- and second-phase insulin release (Wang and Thurmond, 2009).", "Of note, the increased release of insulin after loss of Ago2 expression in the β cells of ob/ob mice improved many aspects of the diabetic phenotype, including circulating glucose and insulin levels, and glucose tolerance; however, this came at the expense of β cell mass and may suggest that the miRNA pathway mediates a delicate balance between both growth and secretion.", "Many of the genes being targeted by small RNAs in the β cell may regulate this balance via independent mechanisms.", "Several studies indicate F-actin and the microtubule networks are actively engaged in managing insulin release and many of the targets of miR-375, including Cadm1 and Gephyrin, may contribute to the remodeling of the cytoskeleton (Seino et al., 2011; Charrier et al., 2010; Masuda et al., 2010).", "In addition, published studies have already shown that during pregnancy, Rasd1 acts as a growth suppressor and is inhibited during islet expansion and then is increased in the postpartum phase when islet mass is restored (Lellis-Santos et al., 2012).", "Also, inhibition of the RNA-binding protein HuD/Elavl4 was shown to promote expression of insulin (Lee et al., 2012).", "Lastly, as evidenced in the differences in the phenotypes of dox-184 and βAgo2KO mice, our observations supporting Slc25a22, a regulator of glutamate transport and secretion in the β cell, as an additional target of miR-184 indicates that this miRNA may also regulate both proliferation and exocytosis through the targeting of different genes (Casimir et al., 2009).", "Future studies will presumably continue to establish that numerous targets of all miRNAs conspire to orchestrate the demands placed on the cell during insulin resistance, including growth and the continual recruitment of insulin-containing granules.", "It will also remain to be seen whether specific miRNA-mediated gene regulations are unique to their metabolic context, including insulin resistance, fasting, hypoxia, and cold exposure (Dumortier et al., 2013).", "Published studies have illustrated reduced expression of miR-375 in the pancreatic islets of db/db mice and Goto-Kakizaki rats, suggesting that differential roles may exist for miRNAs and their targets according to alterations in the metabolic environment (Zhu et al., 2013; El Ouaamari et al., 2008).", "Therefore, it will be important to understand the factors that dictate the kinetics of miRNA regulation in the β cell.", "It also remains to be determined whether the same genes that expedite the decay of miR-184 during insulin resistance are the same factors that promote its expression as sensitivity improves after administration of the ketogenic diet.", "Moreover, the increase in miR-184 that results from this diet and the resulting suppression of insulin release indicate that miRNAs play a significant role in the maintenance of steady-state circulating glucose and insulin levels.", "Furthermore, the identification of increased miR-184 expression can explain the simultaneous occurrence of both improved insulin sensitivity and glucose intolerance observed in mice on ketogenic diet.", "Although the results appear paradoxical at first, this may be explained by the drastic reduction in both circulating glucose and the demand for insulin.", "Another apparent conflict appears in the mouse models exhibiting hypersecretion and decreased β cell mass.", "However, in spite of the reduction in β cell mass in Ago2ob mice (35% lower compared to ob/ob littermates), the decrease is significantly below the threshold that would induce hyperglycemia (Orland et al., 1985).", "Ago2-deficient models still maintain the capacity to transiently secrete higher amounts of insulin because β cells release a relatively small percentage of its stored granule pool in response to glucose (Seino et al., 2011).", "Conversely, although increased expression of Ago2 causes transient glucose intolerance in dox-Ago2 mice, these mice have normal circulating glucose and insulin levels and insulin sensitivity, and therefore it is unlikely that the increase in Ago2 levels in the islets of ob/ob mice plays a causal role in the hyperglycemia or hyperinsulinemia.", "As shown in dox-184ob animals, restoring miR-184 in the β cell and reducing Ago2 levels in an insulin-resistant model exacerbated the diabetic phenotype.", "In contrast, reducing glucose levels and the demand for insulin in ob/ob mice by administration of ketogenic diet to ultimately suppress Ago2 and restore β cell mass underlines an important function this gene plays in the adaptation of the β cell according to metabolic need.", "Many aspects of β cell physiology remain largely unexplored in terms of nutrient sensing, cellular metabolism, and intercellular signaling pathways.", "Our observations on the effects of the ketogenic diet on miRNA function in the β cell unify several poorly understood mechanisms and reinforce the potential in studying the role of small RNAs in physiologic stresses.", "Continued emphasis on characterizing the functional role of miRNA targets in this cell type should bring clarity in understanding how the network of small RNAs contribute to maintaining essential metabolic processes and how their failure ultimately leads to disease.", "Mice were maintained on a 12 hr light/dark cycle with ad libitum access to regular chow food, a HFD (containing 60% kcal fat, cat.", "no. E15741-347, ssniff Spezialdiäten GmbH), or a ketogenic diet (cat.", "No. E15149-30, ssniff Spezialdiäten GmbH) in accordance with requirements established by Landesamt für Gesundheit und Soziales (Lageso).", "All experimental procedures were approved under protocols G 0357/10, O 0405/09, and T 0436/08.", "Results were consistent in both genders; however, data from female mice are not shown.", "Total Cadm1 knockouts and floxed Ago2 mice were generated as described (van der Weyden et al., 2006; O’Carroll et al., 2007).", "The total miR-184 knockout was generated by D. Aberdam and R. Shalom-Feuerstein (Institut Clinique de la Souris) and genotyped with forward and reverse primers 5′ ACTGAACATTATTTCATGGGCCGGG and 5′ AACTACAACTGTTTGGCTAGCAGGGTG (knockout allele) and the substitute reverse primer 5′ CGCTGAGACCTTGTGATAAACCGTT (wild-type allele).", "To generate dox-184 mice, a 292 bp genome fragment encompassing miR-184 precursor was amplified using the primers 5′ TGCTGAAGAGTGGCCTGCTAGG and 5′ CTCCTCCTCACGTCCTGTGGTA, cloned into a pTRE-2 vector (Clontech), and the resulting construct was used for microinjections.", "The Ago2-tetO mice were generated from a mouse Ago2 cDNA construct (a kind gift of G. Meister, University of Regensburg) (Nir et al., 2007).", "Small RNA sequencing and expression analysis from cell lines and mouse islets was performed as described; primers used with FastStart SYBR Green PCR Master Mix (Roche) are available upon request (Tattikota et al., 2013).", "Relative quantification of Ago genes was determined after normalization with standard curves established with mouse Ago1-4 cDNA constructs (G. Meister).", "RIP experiments using Ago1 and Ago2 antibodies were performed as described (Keene et al., 2006).", "Human islets were obtained with research consent and selected based on islet cell purity and isolated at the University of Virginia, Oxford DRWF Human Islet Isolation Facility and Prodo Laboratories (USA) (Morán et al., 2012).", "Information for nondiabetic and diabetic donors is provided in Table S2.", "Insulin measurements from plasma and pancreatic extracts were measured by radioimmunoassay (RIA) (Millipore), and blood glucose and luciferase assays were measured as described previously (Poy et al., 2009).", "Islet morphometric analysis after intraperitoneal injections of BrdU on four consecutive days (50 μg/g BW, Sigma B5002) was performed on 8 μm sections of paraffin-embedded pancreas approximately 150–200 μm apart.", "Sections were dewaxed, washed, and stained for insulin (Dako A0564), glucagon (Millipore AB932), BrdU (Abcam ab6326), Ki-67 (Dako), or TUNEL (Roche cat.", "no.12156792910).", "Cell numbers from all islets in 3–7 sections were counted with ImageJ software from 20X images obtained using a Zeiss LSM700 (Schneider et al., 2012).", "β cell mass was measured as the ratio of insulin-positive cell area to the total tissue area, multiplied by the weight of the pancreas using Imaris software (Bitplane).", "In vivo insulin release and glucose (GTT) or insulin (ITT) tolerance tests were performed following a 6 hr fast and injected intraperitoneally with either glucose (2 g/kg BW) or insulin (0.75 U/kg BW).", "MIN6 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 4.5 g/l glucose supplemented with 15% v/v heat-inactivated FCS, 50 μM β-mercaptoethanol, and 50 mg/ml penicillin, and 100 mg/ml streptomycin and insulin release was performed as described (Poy et al., 2004).", "Antibodies were as described: Gephyrin (BD 610584), Rasd1 (Millipore AB15794), Ago2 (for immunoprecipitation, Wako 018-22021), Ago2 (for western blotting, Cell Signaling C34C6), Ago1 (MBL RN028PW), Slc25a22 (Sigma AV44041), HuD/Elavl4 (Santa Cruz sc-48421), γ-tubulin (Sigma T6557), Cadm1 (Sigma S4945), and β-actin (Sigma).", "Guinea pig anti-insulin and rabbit anti-glucagon antibodies (Millipore) were used on paraffin-embedded pancreata fixed in 4% paraformaldehyde for 3 hr.", "Pearson’s R coefficient and independent two-group Mann-Whitney tests were implemented using the R statistical package (http://www.r-project.org).", "Comparisons between data sets with two groups were evaluated using an unpaired Student’s t test.", "ANOVA analysis was performed for comparisons of three or more groups.", "A p value of less than or equal to 0.05 was considered statistically significant.", "Results presented as mean ± SEM.", "∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001." ]	[ "Silencing of miR-184 during insulin resistance promotes its target Ago2", "Loss of Ago2 during insulin resistance blocks pancreatic β cell proliferation", "Ago2 mediates the suppression of Cadm1 by miR-375 in the β cell", "Administration of the ketogenic diet to ob/ob mice rescues miR-184 in islets" ]
Why we must question the militarisation of conservation	Concerns about poaching and trafficking have led conservationists to seek urgent responses to tackle the impact on wildlife. One possible solution is the militarisation of conservation, which holds potentially far-reaching consequences. It is important to engage critically with the militarisation of conservation, including identifying and reflecting on the problems it produces for wildlife, for people living with wildlife and for those tasked with implementing militarised strategies. This Perspectives piece is a first step towards synthesising the main themes in emerging critiques of militarised conservation. We identify five major themes: first, the importance of understanding how poaching is defined; second, understanding the ways that local communities experience militarised conservation; third, the experiences of rangers; fourth, how the militarisation of conservation can contribute to violence where conservation operates in the context of armed conflict; and finally how it fits in with and reflects wider political economic dynamics. Ultimately, we suggest that failure to engage more critically with militarisation risks making things worse for the people involved and lead to poor conservation outcomes in the long run.	[ "AGRI", "ENVI" ]	[ "Anti-poaching", "Human-wildlife conflict", "Militarisation", "Poaching", "Political economy", "Protected areas", "Rangers", "Trafficking", "Violence", "Wildlife trade" ]	[ "1105", "2309" ]	[ "The rapid rise in poaching of elephants, rhinos and many other species over the last decade has prompted conservationists to think about how to respond effectively.", "One proposed response is a series of measures that include: more forceful or armed forms of conservation (Asiyanbi, 2016; Barbora, 2017; Massé and Lunstrum, 2016; Verweijen and Marijnen, 2018); the development and application of military style approaches (Annecke and Masubele, 2016; Büscher, 2018; Duffy et al., 2015); such as the development of informant networks, and counterinsurgency-like strategies (Adams, 2017; Büscher, 2018; Dunlap and Fairhead, 2014; Pimm et al., 2015); and the use and applications of technologies originally developed by the military (Lunstrum, 2018; Shresthra and Lapeyre, 2018).", "In this analysis, we term these developments the ‘militarisation of conservation’, because of the military origins and models that inform and guide these interventions.", "Proponents of militarisation present the integration of military approaches with conservation practice as a necessary development (Shaw and Rademeyer, 2016; Runhovde, 2017; Henk, 2005, 2006; Hübschle and Jooste, 2017; Mogomotsi and Madigele, 2017).", "However, the militarisation of conservation holds potentially far-reaching consequences for practices, rhetoric, policy, and interactions between conservation actors and other stakeholders.", "It therefore merits attention and so here we provide an overview of an emerging body of scholarship in conservation social science that provides evidence-based critiques of the militarisation of conservation.", "We seek to inform and advance debates amongst academics, policy makers and practitioners.", "Further, while there are historical antecedents in the use of violence and militarisation to sustain conservation (Brockington, 2004; Lunstrum, 2015), we argue that in the new urgent rush to save species from extinction, many practitioners, policy makers, and proponents of current militarisation have not paid adequate attention to the potential disadvantages and long term implications of relying on such a strategy.", "The body of literature we build on is firmly anchored in qualitative social sciences, and often draws on extensive fieldwork in places such as Nigeria (Asiyanbi, 2016), India (Barbora, 2017), Laos (Dwyer et al., 2015), Guatemala (Devine, 2014; Ybarra, 2016), Colombia (Bocarejo and Ojeda, 2016), Democratic Republic of the Congo (DRC) (Marijnen and Verweijen, 2016), South Africa (Büscher and Ramutsindela, 2016; Hübschle, 2017; Lunstrum, 2014), Mozambique (Massé and Lunstrum, 2016; Massé et al., 2017b; Witter and Satterfield, 2018), Tanzania (Mabele, 2016) and Central African Republic (CAR) (Lombard, 2016).", "Many of these studies are in-depth explorations of a single case, but there are few overviews (Duffy et al., 2015; Duffy et al., 2016; Büscher and Fletcher, 2018) or comparative studies (Lunstrum and Ybarra, 2018).", "Our aim in this Perspective Piece is to draw out the major themes, patterns and concerns about militarisation from the critical literature in order to inform more thorough dialogue in conservation; this synthesis of critical perspectives is intended to provide reflections in order to inform conservation policy making via the development of a clear counterpoint to the more commonly articulated position that militarisation is an appropriate, proportionate and necessary response to an urgent situation.", "As such, this Perspective Piece is a timely first step towards sparking a dialogue about militarisation of conservation, not least because more militarised approaches are gaining traction in other sectors such as forest protection (Asiyanbi, 2016).", "More thorough engagement and dialogue between different perspectives will help conservation practitioners to develop more robust and socially just policy approaches to the current poaching crisis.", "First, we examine why it is important to engage critically with the militarisation of conservation.", "Second, we set out the main themes which characterise the emerging literature on this topic: the importance of understanding how poaching is defined, the experiences of local communities, the experiences of rangers, the challenges of conservation in areas of armed conflict, and finally the political economy of the link between conservation and militarisation.", "To conclude we suggest that failure to engage more critically with militarisation risks making things worse for the people involved and leads to poor conservation outcomes in the long run.", "Militarised approaches to conservation appear to be expanding, becoming institutionalised and normalised in a growing number of places and amongst particular conservation non-governmental organisations (NGOs) and donors (Duffy, 2016; Marijnen, 2017; Büscher and Fletcher, 2018).", "Part of the reason for a shift towards militarised conservation is that some conservationists feel pressure to act urgently to prevent extinctions.", "This can be especially acute in conflict zones or if conservationists determine that poachers are adopting more aggressive tactics (Büscher and Ramutsindela, 2016; Marijnen and Verweijen, 2016; Kelly and Gupta, 2016; Lombard, 2016).", "Of course, there are many arguments offered in favour of militarised conservation: that it is the only effective solution in an urgent situation, that conservationists need to defend themselves and the last remaining populations of endangered species against armed threats, and that use of more forceful approaches can reduce poaching leading to growing wildlife numbers, amongst others (for example see Mogomotsi and Madigele, 2017; Henk, 2005, 2006; Hübschle and Jooste, 2017; Hillborn et al., 2006; Emslie and Brooks, 1999).", "However, our aim is not to recount the many high profile and well-covered arguments in favour of militarised conservation.", "Instead our aim is to intervene in the debate by changing the focus to address important counter-arguments and concerns in more detail.", "While recognizing that militarisation will be varied in its details from place to place, we argue that militarised conservation as a model, even when it might result in conserving some animals and enforcing some protected areas, is fundamentally unjust.", "Moreover, if it is not regarded as a just and legitimate policy it will be difficult to enforce in the longer term, even with enhanced levels of resources (Wright et al., 2016).", "This Perspective piece draws on research that critically engages with militarised conservation published in books, reports, and over 29 distinct journals that span a range of disciplines from geography, sociology, criminology, anthropology, political science, political ecology, conservation biology, amongst others.", "In addition, conservation social scientists, conservation and enforcement practitioners themselves have also pointed to the limitations of a top-down and violent approach to anti-poaching (Barichievy et al., 2017; Bennett, 2011), especially if not combined with efforts to address local socio-economic inequalities and injustices (Annecke and Masubele, 2016; Cooney et al., 2017; Haas and Ferreira, 2018).", "Indeed,even those who argue that militaries, such as the Botswana Defence Force (BDF), have been successful in leading anti-poaching in the country, caution against the use of military approaches in conservation (Henk, 2005, 2006).1", "A case in point concerns the development of technological solutions, like surveillance technologies aimed at detecting poachers, as part of militarised conservation.", "Conservationists may find these technologies compelling, but being overly enchanted with them risks obscuring how they can waste or divert scarce resources; indeed there is a lack of transparency about the effectiveness of such technologies, the costs of which run into millions of dollars2 (Lunstrum, 2014; Hahn et al., 2017; Gore (2017).", "Furthermore, many conservationists do caution against adopting technologies that have military origins, recognizing that they can be expensive and possibly divert important investment in other aspects of conservation, and that they do not address the underlying causes of dwindling wildlife numbers (Berger-Tal and Lahoz-Monfort, 2018, 5).", "Reflecting on the negative implications of militarised conservation is vitally important precisely because of its (generally) positive presentation by NGOs, international donors, and national governments.", "Proponents of militarised conservation often present forceful approaches as a noble or heroic quest to save species (Marijnen and Verweijen, 2016; McClanahan and Wall, 2016).", "From this perspective, criticism may appear as an unhelpful distraction from the urgent operational challenges faced by practitioners in the field.", "Critics have been portrayed as naïve, lacking in understanding, as pseudo-scientists or even as hostile towards conservation (Hübschle and Jooste, 2017; Mogomotsi and Madigele, 2017).3", "Such portrayals obscure the nature and value of (critical) social science work on conservation (see Bennett et al., 2016; Charnley et al., 2017).", "As Sandbrook et al. (2013) point out, there is sometimes a false distinction drawn between those who work on and in conservation.", "Indeed, researchers often work closely with practitioners, and have provided an important alternative avenue for them to draw attention to and express their concerns, criticisms and frustrations (Barbora, 2017; Bennett et al., 2016; Lunstrum, 2014; Massé et al., 2017a, 2017b.", "Critical engagement can facilitate a better understanding of militarised conservation, its short and long-term implications, why alternatives are needed and what these might look like.", "As such, this Perspective piece aims to provide a basis for further debate and dialogue in conservation to enable conservationists to improve practices, and help develop more effective and more socially just forms of conservation.", "A vital first step in this is to identify and discuss the range of negative impacts of militarisation as a strategy to address wildlife poaching that are found in the literature.", "One of the criticisms that has been raised about militarised conservation is that it does not address the underlying reasons for why people engage in poaching and trafficking; instead it focuses on tackling the symptoms (poaching and trafficking) of a much deeper and complex structural contexts that is at the root of these practices (Duffy et al., 2015; Hübschle, 2017; Witter and Satterfield, 2018).", "Reflecting on the body of more critical scholarship on militarised conservation can help conservationists to step back and consider the wider dynamics of what produces poaching in the first place.", "Scholarship also shows how the portrayal and treatment of poachers as criminals could be ineffective, and even counterproductive.", "The history of poaching is central to this debate: there is a well-established body of literature exploring how some forms of hunting, and not others, became defined as poaching and the importance of examining how poverty, inequality, historical grievances and the continuing effects of colonial and racial discourses shape understandings of poaching (see Challender and MacMillan, 2014; Duffy et al., 2015; Hübschle, 2017; Neumann, 2004; Peluso, 1993).", "Similar arguments about the need to understand and address these underlying historical and structural factors to tackle environmental and wildlife crime effectively are also made by some green criminologists, such as Gore (2017) Wyatt (2013) and Cao and Wyatt (2016).", "Recent scholarship on the militarisation of conservation also focuses on unpacking and analysing the portrayals of illegal wildlife trade issues by interested parties.", "Central to this task is the need to interrogate the discursive and visual representation of poaching.", "Militarised conservation is characterised by a process of ‘moral boundary drawing’ between rangers as heroes and poachers as villains (Neumann, 2004).", "Such boundaries are drawn through a variety of representational practices in policy literature, academic articles and fund-raising activities (Marijnen and Verweijen, 2016; Massé, 2018).", "These boundaries can be used to explain and justify the use of coercion and (deadly) violence against poachers (Neumann, 2004; McClanahan and Wall, 2016).", "There are direct parallels between the present day criminalisation of poachers and colonial era initiatives to control or outlaw hunting by African communities that produced deeply held grievances and animosity towards wildlife conservation (Duffy et al., 2015; Duffy et al., 2016; Ramutsindela, 2016).", "Many of these grievances still persist today and forms part of the reason for why young men might enter the poaching economy (Hübschle, 2017), as discussed in the next section.", "The example of Virunga National Park in eastern DRC shows why representations matter for the practice of conservation.", "The communications team of the NGO managing the park heavily deploys hero versus villain categorisations to attract donations, for instance for the ‘Fallen Rangers Fund’.", "In its messaging, it consistently repeats the figure of ‘175 ranger deaths in twenty years’.", "Yet these numbers are presented in a decontextualised way: for example, it is rarely mentioned that people other than rangers were also killed, or that rangers were engaged in destroying homes or fields as part of their operations, (Verweijen and Marijnen, 2018; Vikanza, 2011).", "Instead, park communications simply portray the rangers as heroes battling against undefined armed groups, without outlining which specific group is involved and why they attack the park guards.", "This deliberate erasure of abuses committed by rangers gives external audiences the impression that rangers are always the heroes and contributes to a more general lack of understanding for why local communities may resist conservation initiatives (also see Massé, 2018).", "Identifying and tackling abuses committed by rangers (environmental and human rights abuses) is vitally important – failure to do so undermines trust between conservation authorities and people, and does a disservice to those rangers who conform to very high standards of personal conduct.", "In sum, moral boundary drawing between rangers as conservation heroes and evil poachers is problematic for three reasons.", "First, it obscures how some rangers are involved in activities that have a negative impact upon people and biodiversity (Neumann, 2004; McClanahan and Wall, 2016)–failure to recognise this, means that economic and training support for militarisation can increase the capacity to engage in such abuses (Lombard, 2016).", "Second, it ‘traps’ rangers in a particular role, rendering it more difficult to bring to light the complex variety of actual ranger stories and experiences needed to improve their working conditions and effectiveness (as discussed below).", "Third, by decontextualising the death of rangers, the park is able to generate more financial support for a military-style response, even though this has fostered more direct attacks by rebel groups against the park (and park rangers), ultimately leading to a cycle of violence (Verweijen and Marijnen, 2018).", "Critical engagement with militarised conservation also means exploring and revealing the everyday challenges and problems encountered by people living in and around areas affected by militarised conservation, including the ways it can infringe upon their rights and daily lives.", "Militarised conservation can mirror and recreate past injustices, which risks alienating inhabitants of conservation spaces.", "For example, militarised conservation tactics in specific contexts in South Africa often resemble apartheid-era counterinsurgency practices, where efforts to win the support of local people also coincide with tactics of intimidation and use of violence.", "These tactics also currently extend into Mozambique, and include: the development of informant networks, co-option and development of cultures of mistrust within communities (Annecke and Masubele, 2016; Lunstrum, 2015; Massé et al., 2017a, 2017b); raiding and invading people's homes in operations to uncover evidence of wildlife crimes (Ramutsindela, 2016; Massé et al., 2017a; Büscher, 2018); and active displacement of communities for conservation (Massé and Lunstrum, 2016; Witter and Satterfield, 2018).", "More forceful approaches to conservation can also be accompanied by new incentive schemes, such as the provision of game meat to schools and water reticulation programmes.", "However, such interventions can simply serve as stop-gap measures, or as distractions which do not address the systemic problems which produce incentives to engage in illegal hunting in the first place.", "There is a debate in conservation about whether it can or should address poverty and inequality; this is important because efforts to address these problems underpin ‘hearts and minds’ approaches.", "However, these approaches are then systematically undermined by practices of intimidation, violence and surveillance which can be part and parcel of militarisation (Massé and Lunstrum, 2016; Massé et al., 2017a; Ramutsindela, 2016).", "As discussed in more depth below, addressing inequalities and ensuring that conservation does not exacerbate them is necessary to tackle the underlying causes of poaching in the longer term (see for example Cooney et al., 2017; Haas and Ferreira, 2018; Hübschle, 2017; Hübschle and Shearing, 2018; Duffy et al., 2016; Duffy et al., 2015).", "Local inhabitants' negative experiences of these more forceful forms of conservation are also inadequately incorporated into portrayals of rangers as heroes.", "When any type of conservation practice is presented as inherently good, it becomes difficult to investigate and address alleged abuses by conservation staff (Moreto et al., 2015).", "This can lead to a loss of accountability and legitimacy in the eyes of local people and the international community.", "Despite often being crucial to the success of conservation efforts, the experiences of the people living in the areas concerned are overlooked in debates about the militarisation of conservation.", "Understanding what militarisation means for those people can shed light on how the drive to save species by more forceful means has counterproductive effects in the longer term.", "For example, one of the most significant problems with militarisation is that it has the capacity to alienate local communities who object to the use of force to protect wildlife, the development of cultures of surveillance and their continued (often violent) exclusion from protected areas; such approaches will lose the support of the very people who are central to conservation efforts in the longer term (Duffy et al., 2015; Cooney et al., 2017; Hübschle, 2017; Massé et al., 2017a, 2017b).", "Further, a recent study by Holden et al. (2018) details emergent findings that demand reduction campaigns coupled with sustainable livelihood approaches are more effective at tackling poaching for ivory than enhanced policing and enforcement alone.", "This further calls into question the long-term sustainability of militarised approaches.", "There is also a need to gain a better understanding of the lived experiences of those involved in implementing militarised conservation, notably rangers.", "A growing number of researchers are engaged in this task (Massé et al., 2017a, 2017b; Moreto, 2015a, 2015b; Moreto et al., 2015; Moreto et al., 2017; Gore, 2017).", "Not claiming to speak for rangers, these researchers instead offer avenues to draw attention to the problems and challenges that rangers face, and their own concerns and criticisms of militarised conservation.", "In so doing, they assist with and support documenting ranger concerns, and act to amplify them.", "This is important work because rangers operate in complicated power hierarchies, and often fear that they will lose their jobs if they criticise conservation authorities, so they cannot necessarily speak out publicly (Annecke and Masubele, 2016; Massé et al., 2017a, 2017b; Moreto, 2015a, 2015b).", "Moreover, understanding rangers' experiences relates directly to an understanding of their practices in the field and their motivations, as the recent WWF Ranger Perception surveys show.4", "The idea that rangers do what they do simply because they love nature, and that they willingly engage in hard-line approaches to anti-poaching does not map well on to their actual experiences of carrying out the practices of militarised conservation (Moreto, 2015a, 2015b).5", "For example, there are rising rates of Post-Traumatic Stress Disorder (PTSD) amongst rangers in Kruger National Park, and there are reports of many more staff diagnosed with other stress-related conditions.6", "We also see a re-direction of ranger duties away from ‘their typical role as conservationists to become active players in guerrilla warfare, putting their lives in constant jeopardy’.7", "Indeed, rangers are increasingly targets of violence themselves (Lunstrum, 2014; Massé et al., 2017a, 2017b), which is especially harsh given that militarised anti-poaching may not be what they signed up for when they entered the profession (Annecke and Masubele, 2016).", "The entire spectrum of ranger experiences is important.", "Rank-and-file staff involved in militarised conservation often express concern about what they are expected to do, and there can be a (perceived or genuine) lack of transparency of senior staff and the wider institutions engaged in designing and implementing militarised conservation (Moreto et al., 2015).", "One reason for such opacity is that conservationists face immense pressures to demonstrate that they are making in-roads against poaching.", "In the Kruger National Park, key performance assessments of management staff are narrowed to reducing the rate of rhino killed per day, irrespective of the pressures faced by conservation managers.", "Such pressures have snowballing effects that can lead to the use of excessive force, torture, and even extra judicial killings of suspects, as is well documented in Tanzania (Carlson et al., 2015; Mabele, 2016).", "The negative effects of this pressure also extend to conservation staff themselves, as a culture of suspicion and mistrust can lead to toxic work environments, increasing workplace stress.", "There is often an unacknowledged racial politics running through conservation circles as well.", "Conscious and unconscious bias amongst white staff, which leads to unfair and incorrect assumptions about fellow black staff prevents the development of effective working relationships.", "This undermines and unravels any gains made in terms of increasing diversity and equality in the workplace in the longer term (Moreto, 2015a, 2015b; Mbaria and Ogada, 2016).", "Rather than reducing rangers to a singular category of ‘conservation heroes’ it is important to highlight and understand the complex realities faced by rangers in order to improve their well-being and working conditions (Massé et al., 2017a, 2017b; Moreto, 2015a, 2015b).8", "Yet, many aspects of ranger experiences remain understudied, including: How do rangers regard the use of tracking technology, which monitors their movements during the working day?", "What are the implications of such work place surveillance for labour relations?", "Are rangers paid adequately and on time?", "Do rangers feel they have the right equipment, and are there sufficient and appropriate pathways through the profession?", "What are their other options for employment?", "What kinds of pressures do their families face?", "Addressing these questions requires thorough and sustained research from the social sciences, and could benefit from developing an analysis which is more firmly anchored in debates about labour relations rather than conservation per se.", "One of the strongest arguments in favour of militarised conservation is that it is the best (or only) workable option in areas of intense armed conflict.", "However, the intersections between conservation and wider dynamics of armed conflict remain ill understood.", "Yet there is an emerging body of work, often drawing on insights from a range of academic disciplines such as development, and peace and conflict studies, that specifically examines how militarised conservation intersects with conflicts and violence.", "(Lombard, 2016; Verweijen and Marijnen, 2018).", "In this Perspectives piece we argue that militarisation of conservation is in itself fundamentally problematic precisely because it can serve to embed conflict dynamics further, rather than resolve them (Marijnen, 2018).", "A pertinent question which requires further debate is whether militarised conservation ultimately contributes to rising levels of violence in contexts of armed conflict.", "When conservationists operate in conflict zones, they often face intense pressures and can feel directly threatened by armed groups and by heavily armed poachers.", "When faced with such threats it can seem a ‘common sense’ response for rangers to resort to the use of force to protect wildlife and themselves.", "However, when readily using force, it may occur that groups (including rangers), which are armed for conservation purposes, are simply regarded as another armed group engaged in a conflict.", "This can lead to an escalation in arms and in levels of violence, and once such a dynamic is generated it is difficult to de-escalate (Duffy, 2016; Humphreys and Smith, 2014).", "Another key issue is the well publicised and high profile claims that poaching is a crucial funding strategy for militias, rebel groups and even terrorist networks.", "While conservation social scientists do not deny that poachers may be heavily armed in certain places, especially in conflict zones, they do examine and question the claims that there is a clear link between illegal wildlife trade and funding for armed conflict.", "For example, the Elephant Action League controversially claimed Al Shabaab use ivory to fund terrorist activity.", "This narrative was taken up by a range of NGOs, philanthropic foundations, governments and media outlets (Duffy, 2016).", "However, several studies show that these claims are poorly evidenced and are based on false assumptions (Duffy, 2016; Smith & Haenlein, 2016; Sommerville, 2016; White, 2014; Kelly et al., 2018).", "While there has been a recent move away from making such explicit claims about Al Shabaab because of the lack of evidence, it has not stopped the circulation of the problematic narrative that ivory (and illegal wildlife trade more generally) is used to fund conflict and specifically militant groups such as Janjaweed, the Lord's Resistance Army and Boko Haram (Kelly et al., 2018).", "For example, Achim Steiner when he was head of UNEP, Prince William and the #Whosesideareyouon campaign of United for Wildlife, Wildlife Conservation Society's 96 Elephants Campaign and Conservation International's Direct Connection campaign all drew links between (broadly defined) international terrorism and the illegal wildlife trade (Duffy, 2016; Sommerville, 2016).", "Even while lacking evidence, such claims increase the sense of urgency to save species in the face of intense or growing pressures of armed conflict, and are thus used to justify a shift towards more militarised forms of conservation.", "Increasing collaborations between conservationists, national armies and UN Peace Keeping Operations (UNPKO) could also benefit from greater critical reflection.", "This could draw on long standing debates in politics and international relations about the risks and challenges of working with these kinds of military institutions.", "For instance, much of the literature on UNPKOs highlights that peacekeeping and peace enforcement often means taking sides, and that it is not possible (or even necessarily desirable) to be ‘neutral partners’ in a conflict zone (Verweijen, 2017; Fassin, 2012).", "This is an important point for conservationists seeking to partner with UNPKOs: working with military actors means becoming more deeply embedded in the very conflict dynamics that undermine conservation efforts.", "Collaborations between military actors and conservationists might also spark tensions: military actors are trained in a particular type of approach and rules of engagement that are geared towards overlapping areas of defence, counter-insurgency and pursuing warfare.", "This approach differs from the role and purpose of conservationists.", "It cannot be assumed that government forces have a clean record on conservation issues either.", "In specific places, there have been accusations of direct involvement of military personnel in poaching or other forms of illegal and damaging natural resource extraction, even using poaching to fund their operations (Carlson et al., 2015; Ellis, 1994; Titeca, 2013).", "There are also risks in providing military training and equipment to rangers because there are instances where the new skills and weaponry are turned back on wildlife and local communities.", "This occurred for example in CAR where a park guard who received paramilitary training funded by the European Commission became a rebel leader with the Seleka movement, and many of the other externally trained park guards joined as well (Lombard, 2016).", "Conservationists also need to be aware of the human rights record of their collaborators.", "In some instances, soldiers from national armies engage in human rights abuses and are regarded as a repressive and hostile force.", "Working with military partners can therefore contribute to a more negative view of the work of park rangers amongst those who bear the brunt of these abuses (Verweijen and Marijnen, 2018).", "Collaborating with military partners with records of social and ecological abuses is potentially problematic to conservationists because it can inflict significant reputational damage on the international stage, thereby undermining international support for conservation.", "These considerations highlight the importance of studying military collaborations and the effects on conflict dynamics in zones of armed conflict, in particular when conflict dynamics are central to justifications for the need to militarise conservation in the first place.", "One central, but often overlooked, question in arguments about militarisation of conservation is: who wins and who loses from increasing militarisation?", "Who profits from the development of more militarised approaches, and how does that shape or produce specific conservation strategies?", "Answering these questions requires examining the political economy of militarisation of conservation.", "As a growing number of studies show, the militarisation of conservation can be driven by the demand for profits from private sector actors seeking to expand into new markets.", "For example, Devine (2014) examines the intersections of ecotourism, conservation and militarisation in Guatemala, drawing out the ways in which ecotourism development has become a means by which the Guatemalan state has revived and repurposed tactics of counterinsurgency warfare derived from the country's civil war.", "Massé and Lunstrum (2016) argue that anti-poaching strategies in and around Kruger National Park constitute ‘accumulation by securitisation’ whereby anti-poaching security and training offers lucrative avenues for the private sector (Lunstrum, 2018).", "Such accumulation is evident in the number of Private Security Companies (PSCs) offering their services for training or for direct engagement in anti-poaching operations.", "Critics of using PSCs in conservation point out that it is important to examine past examples of poor practice.", "For example, Neumann (2004) discusses the case of Liwonde National Park in Malawi, where rangers trained by a PSC were implicated in human rights abuses over a two-year period.", "In the past, a number of conservation NGOs have been engulfed in scandals involving intelligence gathering for anti-poaching via contracting private companies staffed by ex-special forces.", "One of the best known examples is Operation Lock during the mid 1980s, which was carried out by KAS Enterprises (a private military company).", "While intended to conduct wildlife crime sting operations, it was later revealed that they also gathered intelligence on anti-apartheid activists for the South African state.", "As WWF-International had provided funds to this operation, the NGO's reputation was damaged (Rademeyer, 2012).", "Despite these negative experiences, several conservation NGOs today continue to hire private intelligence companies staffed by former operatives from intelligence services, including the former Bureau of State Security (BOSS) and Mossad (Massé et al., 2017a).", "The concerns raised about the role of PSCs and partnering with intelligence specialists, especially external or ‘foreign’ operatives, are shared by professional bodies.", "In 2017, the Game Rangers Association of Africa issued a statement expressing concerns about the activities of security agencies from outside Africa.", "They noted these agencies' lack of coordination, lack of understanding of the operating environment, lack of ecological sensitivity, lack of knowledge of the legal frameworks that rangers operate in as well as the lack of proper vetting of foreign security agents and profiteering by military equipment manufacturers.9", "Conservation NGOs themselves are also becoming more involved in surveillance, intelligence gathering and developing informant networks (Massé et al., 2017a, 2017b; Sandbrook, 2015).", "The risks related to these practices are not sufficiently acknowledged by their proponents.", "For instance, NGO staff may not have the training to collect and store sensitive data securely in order to ensure that participants or informants are not put at increased risk (although there is now more attention on this theme).10", "Additionally, the process of gathering information may itself be problematic if informants feel physically threatened or fear losing their jobs.", "Furthermore, in certain areas where it is practiced, intelligence-led approaches to conservation have already fomented intra-community tensions resulting in violent attacks (Biggs et al., 2016).", "Practices of intelligence gathering by NGOs and private security sector partners are embedded in and shaped by the attitudes and approaches of the individuals and organisations involved in it, which may not align with local attitudes (Massé et al., 2017a, 2017b; Roe et al., 2015).", "Another dimension of the political economy of militarised conservation is that this practice can become commodified in itself.", "For instance, it may become a form of ‘spectacle’ that is central to fundraising efforts by enticing ‘consumers’ to directly fund armed conservation efforts.", "An example is the multi-media campaign launched by the Virunga Foundation after the release of ‘Virunga, the movie’ whereby the viewer is asked to become part of ‘Virunga's epic fight’ by donating money, for instance to fund patrols or dog-tracking teams, without understanding the on the ground effects of these strategies in this conflict zone (Marijnen and Verweijen, 2016).", "The redistribution of resources, attention, and focus resulting from green militarisation and the political economy of fundraising that has emerged around it also has broader ecological impacts.", "This is especially the case in areas where (para)military actors are at the helm.", "Massé (2018), for example, demonstrates how many conservationists in areas where former and current military personnel have increasing decision-making power are concerned about the impacts this has on broader conservation activities.", "Similar concerns are voiced with regards to shifts in fundraising practices that focus on simplistic understandings of poaching and the responses needed.", "The result is an increasing allocation of scarce resources to (para)militarised enforcement approaches, and away from other conservation priorities that may be less spectacular, but no less important.", "Indeed, officials from South African National Parks draw attention to how rangers in Kruger spend 90% of their time hunting potential rhino poachers at the expense of their more traditional conservation management and monitoring roles like basic ecological and landscape monitoring assessments (Annecke and Masubele, 2016; Hübschle and Jooste, 2017).", "This is coupled with a shift in ranger training away from holistic conservation and ecological management towards more narrow paramilitary and counter-insurgency tactics (Ibid.; Lunstrum, 2014).", "Our research indicates similar trends in Mozambique and elsewhere in Sub-Saharan Africa.", "This is a worrying development for the broader state of biodiversity conservation now and in the long term, as resources are diverted away from activities that are essential to the ecological integrity of protected areas.", "Therefore, more research into how the trend of conservation's militarisation risks jeopardizing the broader ecological health of protected areas is needed.", "It is vitally important to reflect on militarised actions and interventions in conservation.", "Failure to do so, especially in urgent situations, can lead to a greatly enhanced willingness to use violence, with counterproductive and unjust outcomes for people and for wildlife.", "The sense of urgency produced by concerns that wildlife poaching and trafficking will lead to extinctions has led to the argument that there is a need to act before it is too late.", "Moreover, this urgency feeds into appeals that the ends (saving species) justify the means (use of force, including deadly force).", "However, conservationists must not simply accept this as a stark ‘no choice’ pathway for tackling trafficking and poaching.", "Amidst the sense of urgency to save wildlife and prevent extinctions via any means necessary, those who favour and support militarisation have not paid enough attention to how it will fundamentally reshape conservation in the longer term; in this Perspective piece we have cast light on the wider dimensions of, and potential problems with, relying on a militarised approach.", "In order to conserve species and develop socially just and sustainable strategies we need critical, imaginative, and often uncomfortable thinking to get us out of the ‘urgency’ of the moment and put things, literally and figuratively, in perspective.", "This Perspectives piece is intended as a first step towards that: the negative effects of militarisation and the criticisms of it need to be made clear in order to build effective alternative approaches.", "Developing such alternatives also means that it is essential that we develop an understanding of all the steps that lead to the specific moment when rangers encounter poachers, think about how and why people engage in poaching and what the effects of using forceful strategies are on rangers, their families and wider social networks.", "In addition, we should further analyse the political economy of militarised conservation, learn lessons from the past (including the colonial legacies that produced poaching), think through the challenges of conservation in contexts of armed conflict and consider how rangers and communities in and around protected areas experience militarisation.", "Further research inspired by this kind of thinking can facilitate the design of conservation policies that aim to conserve species in ways that are sustainable, effective and locally acceptable.", "Failure to engage more critically with militarisation will make things worse for the people involved, and could lead to poor conservation outcomes as well.", "The use of forceful and violent strategies in conservation can be counterproductive and can lock conservationists into an escalation of violence, a dynamic that also risks undermining other conservation priorities.", "Yet, we only have a partial grasp on the full range of implications of the militarisation of conservation, and its intersections with broader social, political and economic contexts.", "Social sciences (as well as Arts and Humanities) researchers therefore have much to offer in developing future research on militarised conservation.", "Such research provides important opportunities for dialogue to develop better conservation practice, with more positive outcomes for wildlife and for people." ]	[ "Draws together key themes in the critical literature in militarisation", "Shows why the expansion of militarisation needs to be challenged", "Argues militarisation can have counter-productive and unjust outcomes", "Indicates how militarisation can negatively affect rangers", "Examines the political economy of militarisation" ]
Comparison of bubble detectors and size distribution estimators	Detection, counting and characterization of bubbles, that is, transparent objects in a liquid, is important in many industrial applications. These applications include monitoring of pulp delignification and multiphase dispersion processes common in the chemical, pharmaceutical, and food industries. Typically the aim is to measure the bubble size distribution. In this paper, we present a comprehensive comparison of bubble detection methods for challenging industrial image data. Moreover, we compare the detection-based methods to a direct bubble size distribution estimation method that does not require the detection of individual bubbles. The experiments showed that the approach based on a convolutional neural network (CNN) outperforms the other methods in detection accuracy. However, the boosting-based approaches were remarkably faster to compute. The power spectrum approach for direct bubble size distribution estimation produced accurate distributions and it is fast to compute, but it does not provide the spatial locations of the bubbles. Selecting the most suitable method depends on the specific application.	[ "COMP" ]	[ "Boosting-based detection", "Bubble detection", "Circular arrangements", "Convolutional neural networks", "Pulping", "Size distribution estimation" ]	[ "1702", "1707", "1711", "1712" ]	[ "This paper focuses on estimating the size distribution of bubbles, or more generally, transparent approximately spherical objects in a liquid.", "The research is driven originally by the pulpmaking industry, in particular the development of the pulp delignification process.", "Pulp delignification with oxygen is a very energy-intensive operation.", "To optimize and control the process, it is essential to be able to characterize the process, especially the sizes of the oxygen bubbles.", "The recent progress in camera and illumination technologies has made it possible to capture images inside the process machines.", "Mutikainen et al. [29] present an imaging setup applied to the pulp mill environment.", "From the produced images (see Fig. 1), the bubble size distribution could be determined by manually marking the individual bubbles.", "However, manual analysis of the images is very time-consuming, which motivates the development of automatic methods for estimating the bubble size distribution.", "The automatic determination of the bubble size distribution can be performed either by detecting and characterizing each bubble in an image or by estimating the size distribution directly using, for example, frequency information.", "Of these two approaches, the one based on detection is more common.", "The bubble detection problem is not easy to solve because the bubbles are transparent and the illumination conditions inside the process machines are challenging, which causes the bubble appearance to vary.", "In the images, the bubbles appear as roughly circular objects which motivates to solve the problem by detecting circles.", "Two common approaches are used to detect circular objects: geometry-based and appearance-based approaches.", "In the geometry-based approach, a circular model parameterized by its center and radius is fitted to the image edge map.", "These methods typically utilize a voting technique, such as the Hough Transform (HT) [10] or its modifications [23].", "To address the main weaknesses of the HT, such as computational complexity and storage requirements, several revisions of the basic HT have been proposed (see e.g. [27]).", "However, despite the progress the geometry-based approaches suffer from a large number of false positives and are sensitive to noise.", "Moreover, they often fail to detect small blob-like bubbles that do not have a ridge edge expected by the model [35].", "The appearance-based approaches use typically a sliding window in combination with a classifier/regressor in order to detect the objects of interest.", "This class of methods include boosting-based detectors [9,36], HOG-based detectors [8,12], or recently emerging convolutional networks [15].", "These approaches require a large amount of annotated training data covering all possible conditions and appearances of target objects.", "Insufficient data makes detector performance worse, causing lower detection rates and higher false positive rates.", "Several bubble detection methods that focus on resolving the overlaps between bubbles exist.", "For example, Karn et al. [21] proposed a method that utilizes morphological operations and the watershed transform to detect individual bubbles and Zhong et al. [39] makes use of concave points.", "However, these methods are developed for high quality images where the bubbles are clearly distinguishable from the background.", "Therefore, the methods are not applicable for noisy industrial images.", "The problem of cell segmentation shares common characteristics with bubble detection.", "Methods developed for cell segmentation include intensity thresholding, morphological operations, region growing, feature detection, and deformable model fitting [26].", "There are, however, some important differences between the bubble detection problems and the cell segmentation problems: the cells contain more complex shapes while typical bubbles are close to perfect circles, cells have smaller variation in size assuming only certain cell type is considered, a small bubble can appear fully inside larger bubble, and bubbles are more transparent.", "The cell detection is often solved as blob detection problem.", "Bernardis and Yu [4] proposed a method to detect blobs defined as small round regions in an image.", "The detection is carried out by solving a region segmentation problem where the blobs form one region and the areas between blobs form the other.", "Arteta et al. [1] further developed a similar blob detection approach by proposing a more complex statistical model that uses detected blobs as object candidates.", "The blob detection approach, however, cannot be applied to bubble detection as such since bubbles typically have distinguishable edges, but are otherwise transparent.", "Therefore, bubble detection should be based on edge point, instead of detection of uniform blobs.", "Direct methods for the bubble size distribution estimation also exist (e.g., [19]).", "They do not require individual bubbles to be detected.", "Such methods usually rely on the assumption that the image does not contain other objects besides bubbles and the size distribution is estimated by analyzing the frequency information.", "The direct methods are often fast to compute and are not sensitive to frequent overlaps between bubbles, but may become unreliable if the conditions (e.g., lighting or the amount of bubbles) change considerably compared to the method training phase.", "Optical granulometry is a process of computing a size distribution of grains from images.", "The classical approach obtains the size distribution by applying a series of morphological opening operations [24].", "It is, however, only applicable for binary images.", "More sophisticated modifications of the original method have been proposed since (see, e.g., [33]) and have been applied to, for example, biometric image analysis [16] and grain size analysis of sediments [14].", "However, the granulometry based methods are not likely suitable for estimating the size distribution of the bubbles since the bubbles appear as overlapping rings rather than regions with approximately constant color.", "In this work, different promising methods to detect bubbles and to estimate the bubble size distribution are compared.", "The set of methods is selected so that the different approaches to solve the problem are covered including geometry-based methods, appearance-based methods with both hand-crafted and learned features, as well as, a direct method for the size distribution estimation.", "Both specific methods developed particularly for bubbles or circular objects as well as general purpose object detectors are included.", "The experiments were carried out using a novel and extensive data set consisting of challenging images from real industrial environment.", "The CCA-based bubble detection [35] starts with the computation of the oriented edge map by filtering image with second derivative zero-mean oriented Gaussian filters in eight directions followed by non-maximum suppression [6].", "After that, the CCA hypotheses are generated by sampling from the edge map using procedure similar to RANSAC [13].", "A CCA hypothesis is generated from the arc if it satisfies the following requirements: (i) it receives sufficient support from the pixels located within the CCA area and (ii) the support comes from a sufficient number of directions.", "The center location and the radius of the hypothesis are optimized by maximizing the support with the Neadler–Mead Simplex method [30].", "Finally, the non-maximum suppression is performed on the CCA parameters to prevent multiple hypotheses for a single bubble.", "Object detection based on Boosting (AdaBoost algorithm or its modifications) is one of the most widely known and used technique today.", "It has been successfully applied to detection of faces [36], pedestrians [9], and other object categories including bubbles in a preliminary study [34].", "The main advantages of this method are the simplicity of implementation and the speed of detection of objects.", "Detectors based on AdaBoost were successfully implemented on GPUs and programmable hardware [38].", "During the last decade, much attention has been given to different aspects of the method in order to handle multiview detection, improve computational performance and detection accuracy, specifically as follows: Training algorithm – Discrete/Real AdaBoost [36], GentleBoost, VectorBoost [18], etc; detector structure – cascade [36], soft-cascade [5,37], constant soft-cascade [9] and various tree structures [18]; weak classifiers – Haar features, Local Binary Patters, Local Rank Differences, decision trees; and to underlying image features used for classification – grayscale image, HOG, decorelation filters, etc.", "The particular choice depends on the task at hand.", "The detector works on the sliding window principle.", "On pyramidal image representation, each level is scanned with the detector, and each image sub-window is classified by the model H. Classification speed can be improved by making the rejection decision after the evaluation of every ht based on the tentative sum of H [9].", "Background areas (which are easy to classify), are rejected by the first few weak classifiers, and more time is spent on the areas which are harder to classify.", "Finally, positive responses are clustered by a non-maxima suppression method in order to obtain the locations and sizes of detected objects.", "In this work, we consider Soft-cascade detectors operating on grayscale images or more advanced aggregate feature channels [9] (HOG+L channels specifically).", "As weak classifiers we use shallow decision trees, as they have proved to be efficient in many tasks [3,25], and Local Binary Patterns, as they outperform the commonly used Haar features and were efficiently implemented on hardware platforms [38].", "Fig. 3 shows the overview of our detector architecture.", "Convolutional Neural Networks (CNNs) have been used to achieve state-of-the-art performance in various image analysis applications, such as image recognition [17] and object detection [32].", "A typical CNN consists of convolutional layers that are formed by neurons which look at small overlapping regions of the input image/feature map, a pooling layer used for downsampling, and fully connected layers for the final decision making.", "Unlike the traditional methods where the decision is made using preselected features, CNNs learn the filters used for feature extraction.", "In order the solve the bubble detection problem, the CNNs can be applied in a sliding window manner.", "For each window, the CNN is applied to compute the probability whether the window contains a bubble or not.", "This creates a probability map for bubble detections (detection image).", "To detect bubbles with different sizes, the detection image is computed in multiple scales.", "A real bubble creates a circular patch of high values, usually in few neighboring scales.", "To detect these, the detection image is, first, thresholded with a threshold tc to create binary images.", "Connected components that do not meet the following conditions are rejected: 1) area is larger than ta, 2) the proportion of the pixels in the convex hull that are also in the region is larger than ts, and 3) the ratio of minor and major axis is larger than tm.", "This means that only roughly circular blobs with a size large enough are accepted.", "After this the detection images in different scales are scaled to a common scale, and a detection cube with detection images from all scales is created.", "The centroids of the blobs in the detection cube are then detected.", "The location of the centroid in the depth direction represents the scale (radius) of the detected bubble.", "Finally, bubbles which are found only in one scale are rejected.", "The final step is not applied for the smallest scales (highest resolution) due to the fact that a circular arrangement of small bubbles occasionally looks like a large bubble, but they are usually detected only in one scale.", "It should be noted that much faster CNN-based object detection methods exist, such as Faster R-CNN [32] that uses computationally efficient ways to create region proposals that are classified by using a CNN, and You Only Look Once (YOLO) [31] that uses a CNN to predict bounding boxes by only processing the image once.", "However, the sliding window approach, while slow to compute, can be seen as a method to obtain the upper limit for the performance of approaches based on region proposals.", "If the same CNN is used for classification, R-CNN can exceed the performance of the sliding window approach only if the method to generate the region proposals does not miss any bubble that the sliding window approach found.", "YOLO is not suitable for large images with hundreds of objects (e.g., bubbles) due to the limited number of predicted bounding boxes and known problems on detecting objects with a small size [31].", "Given the radii of the detected bubbles, computing the bubble size distribution is straightforward.", "However, it is possible to estimate the bubble size distribution directly from the image without detecting the bubbles.", "In [11,22], a method was presented for determining the bubble size distribution using a stationary planar boolean model and two-point set statistics.", "The method, however, requires a binary image where the bubbles and background are separated, and it is, therefore, unsuitable for noisy industrial image data.", "Ilonen et al. [19] proposed a method to estimate the bubble size distribution directly from the images.", "This approach makes the detection of bubbles unnecessary and, therefore, the problems related to overlapping bubbles and computation time caused by large amount of bubbles can be avoided.", "Fourier transform of two signals (here bubbles) is the same as taking the Fourier transform for them separately and adding them up.", "Therefore, the Fourier transform of an image consisting of bubbles is the same as Fourier transforms of images of separate bubbles.", "However, the power spectrum of two combined signals is not the same as their separate power spectra combined because the phase (location) difference can cause them to nullify each other.", "In the case of a large set of bubbles located randomly, it is reasonable to argue that their phases overlap predictably on average so that the distribution can still be determined with a good accuracy from the power spectrum.", "The method consists of the following steps:", "Calculate the power spectrum of an image using L frequency bands.", "Use principal component analysis (PCA) to reduce the data to M dimensions.", "Use multivariate linear regression to learn the dependency between the power spectrum and bubble count or volume distribution.", "The power spectrum is a vector of the portion of a signal power falling into specified frequency bins.", "It is acquired by applying the 2-D discrete Fourier transform to the signal (image) and computing the energy belonging to L linearly spaced frequency bins, producing a vector Pi for an image i.", "The frequency range is limited at the lower end because the maximum size of the bubbles is known, and therefore, the lowest possible frequencies caused by actual bubbles is defined.", "Images were gathered from the oxygen delignification process of a pulp fiber line that provides a difficult imaging environment resulting challenging data for bubble detectors and size distribution estimation methods.", "The imaging setup developed for the purpose has been described in [28] and [29].", "The experiments were performed with varied mixer rotor speeds from 900 rpm to 1380 rpm (see examples in Fig. 1).", "Increasing the rotor speed increases the amount of bubbles and decreases their size.", "Therefore, the images taken with a high rotor speed are more difficult to analyze.", "All other process variables were kept constant during each measurement session.", "20 images were captured with 6 different rotor speeds.", "The size of the original images was 2588 × 1940, but only the central 1482 × 1482 pixel areas of the images were used.", "The ground truth (bubble contours) was manually marked by an expert.", "Due to the large amount of bubbles in each image and low image quality, the ground truth is imperfect.", "However, it provides a representative set of bubbles to train the detection methods and to estimate the bubble size distribution.", "The images were divided into separate training and test sets so that the training set contained 72 images and the test set 48 images.", "As a baseline method we selected the phase coding based circular Hough transform (CHT) method by Atherton and Kerbyson [2].", "CHT parameters were selected by using a grid search and minimizing the volume estimation error in the training set.", "We trained three models that differ in the training algorithms used and features they extract from the input image.", "The ACF model was trained for reference.", "We used Piotr’s Image and Video Toolbox1 with default settings, except for the channels and smoothing.", "We used HOG+L channels as no color information is available in our data, and we oversmoothened images with a 5 pixel triangle kernel prior to shrinking and feature channel computation in order to handle the noise in images.", "The model size was 52 × 52 pixels with the shrink factor of 2.", "We applied 3 rounds of bootstrapping with detectors of length 32, 128 and 512, loading 10,000 hard negatives in each round.", "The final model contained 2048 decision trees of depth 2.", "The WaldBoost+Trees model is similar to ACF.", "We used the same settings for the channels and model size.", "The differences are twofold: 1) training algorithm – WaldBoost [37] – bootstraps hard negative samples in each training round, and 2) splits in the decision trees are pixel differences instead of simple pixel values.", "The WaldBoost+LBP model is the simplest one.", "Here we used just the grayscale image, Local Binary Pattern features, and no shrinking.", "The model size was 26 × 26 pixels.", "The LBP features were composed from 3 × 3 adjacent cells, each of size up to 2 × 2 pixels.", "The feature is parametrized by its position in a sliding window and the size of its blocks.", "During detection, a pyramid with 8 levels per octave is created, feature channels are calculated, and shrinking is applied.", "Each pyramid level is scanned with the trained model, and positive responses are clustered using a simple overlap-based non-maxima suppression algorithm, producing final locations of the detected bubbles.", "To train the CNN, the Caffe framework [20] was used.", "The first convolution layer had 40 outputs with the kernel size of 9 and the second convolution layer had 100 outputs with size 5.", "Max-pooling was used.", "Training was performed using image patches of size 28 × 28.", "If the bubble radius was small enough, the patch was taken directly from the image.", "For larger bubbles, a larger patch was cropped and resized.", "From each ground truth bubble in the training data, 10 positive examples were generated (two scales with factors 1.25 and 1.5, shifts in 4 directions and one without a shift).", "Negative examples (background) were created by selecting 20 random patches so that they were not too close to the actual bubbles.", "To avoid learning that a bubble border is a bubble, additional 10 randomly selected patches from the border of each large bubble (radius larger than 40 pixels) were included to the negative examples.", "This way the training data contained 148,860 positive and 317,732 negative examples.", "Examples of bubble detection results are shown in Fig. 4.", "It was noted that the methods found several apparent bubbles that were not by the expert.", "In order to reliably estimate the true positive and false positive detections, all the unmarked detections were given to the expert for validation who made a binary decision whether the detection was a correct detection or background.", "The new improved ground truth was used only for the evaluation and not for method training.", "To compare the methods, three performance measures, True Positive Rate (TPR) and Positive Predictive Value (PPV), and F-measure (the harmonic mean of TPR and PPV) were used.", "A detection was considered as a true positive (TP) if the overlap (intersect divided by union) between the detected bubble (circle) and the ground truth bubble was higher than 0.5.", "Multiple detections were not allowed, i.e. only the detection with the greatest overlap is considered as TP, others are treated as false positives.", "The results with the improved ground truth are shown in Table 1.", "As it can be seen, the CNN outperformed the other methods by detecting more correct bubbles and producing less false detections.", "It should be noted that the numbers of false negatives can be slightly underestimated assuming neither the expert nor any of the methods detected certain bubbles.", "Therefore, the true positive rates can be slightly overestimated.", "The results on bubble size distribution estimation are shown in Figs. 5–7.", "CHT produces a huge amount of false positives with small bubble sizes.", "Therefore, CHT is left out from the figures to keep the presentation clear.", "Chi-squared distances between the estimated distributions and the ground truth distributions are shown in Table 2.", "Both the distances between the combined distributions over all rotor speeds (Combined) and the mean distances over distributions for different rotor speeds (Mean) are listed.", "Among the bubble detection methods, the CNN produced the most accurate bubble size distribution.", "WaldBoost+LBP, ACF, and WaldBoost+Trees suffered from a large amount of false positives.", "This, together with the small numbers of bubbles in the larger size groups causes noise to the size distribution estimation witch appears as a zigzag pattern in the histograms.", "Moreover, a limited number of detection scales (window sizes) might cause the sliding window based methods to struggle with certain bubble sizes.", "ACF resulted a count distribution with a low chi-squared distance to the ground truth although it overestimates the number of bubbles.", "This is due to the fact that the chi-squared distances are computed for normalized histograms (the sum of bins is equal to 1), i.e., it measures the shape of the histograms instead of the count estimates.", "ACF fails to estimate the volume distribution.", "The power spectrum based bubble size distribution estimator provided an accurate estimate over all rotor speeds.", "However, as can be seen from Fig. 6 it slightly underestimates the number of the bubbles with high rotor speeds, and overestimates the number with low rotor speeds.", "Also, since the method estimates the distribution directly from the image, the user cannot be sure if the method is working correctly.", "The exact execution times of different methods are not comparable due to the different programming environments (MATLAB, C, etc.) and degrees of performance optimization.", "However, indicative conclusions can be made.", "ACF and WaldBoost+LBP can be efficiently implemented on PC and programmable hardware allowing real-time performance.", "The current implementation of the CNN takes approximately one hour per image to compute.", "Although the method performance could be optimized considerably, it is still not possible to reach real-time performance with large images with several hundreds small bubbles, and the CNN is, therefore, only valuable in off-line use.", "The execution times for CCA and CHT are few seconds per image.", "The power spectrum method is fast to compute and it is also fast to train making online training possible.", "We compared various bubble detection and size distribution estimation methods with industrial image data from a pulpmaking process.", "The CNN-based approach outperformed the other methods in the bubble detection task and provided an accurate estimate for the bubble size distribution.", "However, the method is slow making it unsuitable for in-line use.", "The WaldBoost+LBP and WaldBoost+Trees methods are fast to compute and provided reasonably accurate bubble detection results.", "Out of these two, WaldBoost+Trees was able to detect more bubbles, but it suffered from a slightly higher number of false positives.", "However, the amount of false positives was similar in different bubble size categories making WaldBoost+Trees a better method for the size distribution estimation.", "The power spectrum approach for direct bubble size distribution estimation produced accurate distributions and it is fast to compute.", "However, it does not provide the spatial locations of the bubbles.", "Moreover, from the user perspective, the method is essentially a black box, and therefore, the user cannot be sure if the method is working correctly.", "This is not the case with the bubble detection methods where the user can easily see if the method is detecting bubbles correctly.", "Selecting the most suitable method depends on the application.", "If real-time performance is not needed, a CNN is the best choice.", "For in-line measurements WaldBoost+Trees and the power spectrum based size distribution estimator provide the most promising solutions." ]	[ "We review the different approaches to detect bubbles from noisy images.", "We review methods to estimate the bubble size distribution.", "We compare five methods for bubble detection with challenging image data.", "We compare the detection methods with one direct size distribution estimation method.", "The best methods achieve a good accuracy on both detection and estimation tasks." ]
Local-scale forcing effects on wind flows in an urban environment: Impact of geometrical simplifications	Wind flow in urban areas is strongly affected by the urban geometry. In the last decades most of the geometries used to reproduce urban areas, both in wind-tunnel (WT) tests and Computational Fluid Dynamics (CFD) simulations, were simplified compared to reality in order to limit experimental effort and computational costs. However, it is unclear to which extent these geometrical simplifications can affect the reliability of the numerical and experimental results. The goal of this paper is to quantify the deviations caused by geometrical simplifications. The case under study is the district of Livorno city (Italy), called “Quartiere La Venezia”. The 3D steady Reynolds-averaged Navier-Stokes (RANS) simulations are solved, first for a single block of the district, then for the whole district. The CFD simulations are validated with WT tests at scale 1:300. Comparisons are made of mean wind velocity profiles between WT tests and CFD simulations, and the agreement is quantified using four validation metrics (FB, NMSE, R and FAC1.3). The results show that the most detailed geometry provides improved performance, especially for wind direction α = 240° (22% difference in terms of FAC1.3).	[ "ENER", "ENGI" ]	[ "CFD simulations", "Geometric uncertainties", "Model detailing", "Statistical performance", "Urban wind flow" ]	[ "2105", "2205", "2210" ]	[ "The complex morphology of cities renders the analytical description of wind flow in urban areas very difficult.", "Analytical wind flow models are generally well established over flat open terrain, where the wind profiles mainly depend on the aerodynamic roughness of the surface and on thermal stratification.", "In contrast, wind flow in urban environments is governed by a variety of complex factors, such as the heterogeneous geometry of buildings, flow impingement, separation and recirculation and local thermal effects.", "The region above the buildings, usually defined as the urban boundary layer (UBL), is influenced by continuously changing surface roughness, so that the wind flow never reaches a homogeneous equilibrium condition.", "The situation is even more complex within the urban canopy layer (UCL), where streets give rise to complex canyoning effects that are strongly dependent on the canyon orientation with respect to the incoming wind.", "Many researchers have investigated different aspects of urban flows (see e.g. reviews by Britter and Hanna, 2003; Fernando, 2010; Fernando et al., 2010), but at present the UBL is not yet completely understood although WT tests and CFD simulations are frequently used in urban physics and wind engineering to gain increased understanding.", "In the last decades, several studies have focused on numerical modeling of wind flow over random urban-like obstacles (e.g. Xie et al., 2008), uniform and staggered building arrays (e.g. Coceal and Belcher, 2004; Xie and Castro, 2006; An et al., 2013; Razak et al., 2013), idealized urban surfaces (e.g. Cheng and Porté-Agel, 2015), semi-idealized urban canopies (e.g. Hertwig et al., 2012) and actual urban environments (Blocken et al., 2012; Janssen et al., 2013; Montazeri et al., 2013; García Sánchez et al., 2014).", "Errors and uncertainties can be related to the geometrical model precision, the approximate form of the governing equations (RANS, LES), the turbulence models, the discretization schemes and the boundary conditions such as the inflow conditions and the surface roughness (Franke et al., 2007; Blocken et al., 2007a; Hargreaves and Wright, 2007; Tominaga et al., 2008a, b; Emory et al., 2013; Gorlé et al., 2015).", "Carpentieri and Robins (2015) analyzed the impact of morphological parameters on wind flow in the UCL.", "Their results show how the building height variability, the angles between street canyon orientations and incoming wind and other local geometrical features can strongly influence the characteristics of the urban flow.", "Several authors (Chang and Meroney, 2003; Fernando, 2010; Barlow, 2013) stressed that the complexity of the UBL requires studies with multiple approaches with complementary strengths and weaknesses.", "In particular, the joint use of WT testing and CFD simulations has enabled a better understanding of urban aerodynamics and has enhanced the performance of both (Murakami, 1990; Stathopoulos, 1997; Baker, 2007; Tominaga and Stathopoulos, 2013, 2016; Blocken, 2014, 2015; Meroney, 2016).", "However, results of both techniques are strongly dependent on geometrical model details (AIAA, 1998; Casey and Wintergerste, 2000; AIJ, 2004; Franke, 2006; Blocken, 2014).", "Nevertheless, geometrical simplifications are frequently adopted and often even required in both techniques, and the question therefore arises to what extent different levels of geometrical simplification can affect the results.", "The aim of this study is to quantify the so-called uncertainty (AIAA, 1998; Oberkampf et al., 2004; Versteeg and Malalasekera, 2007) caused by geometrical simplifications of the urban model, both intentionally imposed (e.g. to limit experimental manufacturing cost or computational cost) and arising from a limited knowledge of the system to be analyzed (e.g. the epistemic uncertainty).", "For this purpose, a historical district called “Quartiere La Venezia” in Livorno city (Italy) is selected as case study.", "This area is chosen because the nearby port has been monitored by different measurement instruments in several locations, which will provide input data for the present study.", "They consist of anemometric stations and a LiDAR wind profiler installed in the framework of the European projects “Wind and Ports” (Solari et al., 2012) and “Wind, Ports, and Sea” (Burlando et al., 2015a; Repetto et al., 2017).", "The location is particularly interesting since the highest wind velocity observations generally occur for Western winds, in which case the incoming velocity profile in the district can be measured by the aforementioned LiDAR station (Fig. 1).", "Moreover, the transition from sea to land is expected to strongly influence the vertical wind profile in this area.", "WT tests on a model of the district at scale 1:300 were performed and reported in an earlier study (Ricci et al., 2017).", "In the present paper, 3D steady-state RANS simulations using the realizable k-ε turbulence model are performed at the same scale as the WT tests in two different steps.", "First, CFD simulations are performed on a single block of Quartiere La Venezia with three different levels of geometrical simplification, i.e. the so-called simplified, approximated and detailed geometrical models, where the latter has an identical geometry as the WT model.", "Second, simulations are performed for the whole urban model of Quartiere La Venezia for two of the three levels of geometrical simplification, i.e. the simplified and the approximated geometrical models.", "The paper is organized as follows.", "Section 2 contains a short description of the WT tests used to validate the CFD simulations.", "Section 3 and Section 4 describe two steps of CFD analysis.", "In particular, Section 3 introduces the CFD models (simplified, approximated and detailed), the boundary conditions and the computational setup for the simulations for the single block of Quartiere La Venezia.", "On the basis of the obtained results, Section 4 describes the CFD simulations for the whole urban district adopting the simplified and approximated models, and shows the comparison of the WT and CFD results in terms of mean velocity profiles.", "In Section 5, the level of agreement between WT and CFD results is quantified using validation metrics.", "Finally, Section 6 (discussion and limitations) and Section 7 (summary and conclusions) conclude the paper.", "Only some main aspects of the WT tests are mentioned in this section; the reader is referred to Ricci et al. (2017) for more details.", "WT tests on a geometrical model of the whole urban district were performed in the atmospheric boundary layer (ABL) wind tunnel of the Department of Civil, Chemical and Environmental Engineering (DICCA) of the Polytechnic School of the University of Genoa, Italy.", "The wind tunnel at DICCA is a closed-loop subsonic circuit with a test section of 8.8 m long and a cross-section of 1.70 m (width) x 1.35 m (height) (Fig. 2b).", "A WT model of the case study - Quartiere la Venezia in Livorno city - was created at a scale of 1:300 using medium density fiberboard (MDF) of different thicknesses for the ground plates and buildings, and 3 mm closed cell PVC foamboard panels for roofs and bridges (Fig. 2a,c).", "The blockage ratio in the cross-section was kept below 3.5% for all wind directions.", "Tests were performed for three western wind directions (α = 240°, 270° and 300°), corresponding to the prevalent wind directions for the strongest winds in Livorno (Fig. 2a).", "The mean wind velocity scenarios obtained during the European project “Wind and Ports” (Solari et al., 2012) by means of the WINDS model (Wind Interpolation by Non-Divergent Schemes), anemometric data, and the digital land cover maps of the CORINE project (Bossard et al., 2000) were used as reference for the choice of the incoming flow profiles in the WT tests.", "Based on this, an ABL profile with aerodynamic roughness length z0 = 0.1 m (full scale) and friction velocity u* = 0.89 m/s was used for the WT tests.", "In the WT tests, the mean wind velocity was measured at two sets of positions (Fig. 3).", "In the first set, 10 positions inside “Canale Rosciano” (A11 - A52) were monitored at 15 heights in the range from 0.02 to 0.6 m above the wind-tunnel floor (corresponding to 6 m and 180 m full scale above mean sea level).", "Since these positions are fixed to the model geometry, their position in the WT is dependent on model orientation and hence on wind direction.", "The second set was distributed along a Cartesian grid laid out according to the WT local reference system, consisting of 15 measurement locations at 15 heights (from 0.02 to 0.6 m above the wind tunnel floor) aligned along three lines of five measurement positions each (L11-5, L21-5, L31-5).", "Both sets of measurements will be used to validate the CFD simulations performed on the urban district, Quartiere La Venezia, described in Section 4.", "A third set of measuring positions (L16-14, L26-14, L36-14), indicated in Fig. 3 by the green points, was used to investigate the wind velocity profile development in the downstream part of the computational domain.", "The results of this investigation are described in Section 4.", "In the first step, CFD simulations were performed on a single block of Quartiere La Venezia (indicated by the red outline in Fig. 2a) at the same scale of the WT tests (1:300) and for wind direction α = 240°, in order to preliminarily investigate the deviations caused by three different levels of geometrical simplification (i.e. called hereafter simplified, approximated and detailed geometrical models).", "Based on the CFD results obtained in this step, the criteria to realize the geometries of the whole urban district were chosen for the second step.", "Fig. 4 shows the relationship between the WT test-section and the CFD computational domain.", "The size of the computational domain was L × W × H = 5.5 × 1.70 × 1.35 m3, where the width (W) and height (H) are coincident with the WT cross-section.", "The three CFD geometrical models and the associated grids were realized using the software Gambit 2.4.6.", "The first one, termed simplified geometrical model, was obtained representing the single group of buildings as a single bluff body with a height equal to the arithmetic average height of that building group (Fig. 5a).", "The second one, termed approximated geometrical model, was obtained including buildings with their real ground plan and heights, but replacing pitched roofs with flat ones (Fig. 5b).", "To this purpose, the height of every building was calculated as the average between the heights of the peak and the eaves of the pitched roofs.", "Note that the pitch of the roofs of Quartiere La Venezia are probably less relevant from the aerodynamic point of view, since these are low-sloped roofs with slopes in the range of only about 5°–10°.", "The third one, the detailed geometrical model, was perfectly coincident with the WT geometrical model, including the real ground plan and heights of the buildings including pitched roofs (Fig. 5c).", "Grid generation for the simplified and approximated geometrical was performed using the surface grid generation technique presented by van Hooff and Blocken (2010) in order to achieve a high-level control over the grid layout.", "Grid generation for the detailed geometrical model was performed only in part using this technique due to the sloped roof geometry.", "All three grids were constructing adhering to the best guidelines (Franke et al., 2007; Tominaga et al., 2008a; Blocken, 2015).", "The local grid resolution was taken equal or higher than that in previous studies that used the same technique and employed certain grid resolutions based on detailed grid-sensitivity tests (van Hooff and Blocken, 2010; Blocken et al., 2012).", "The expansion ratio of the grid was kept below 1.2 everywhere in the computational domain and at least thirty cells were used along every building edge, which is far beyond the minimum number of ten mentioned in the best practice guidelines (Franke et al., 2007; Tominaga et al., 2008a).", "In order to avoid convergence problems and maximize numerical accuracy, only hexahedral and prismatic cells were used for the simplified and approximated geometrical models in line with van Hooff and Blocken (2010); conversely, hexahedral, prismatic and tetrahedral cells were employed in the detailed model because of the pitched roof geometries.", "The construction of the computational grid of the detailed geometrical model was found to be about five times more computationally demanding (user time and processing time combined) than the other two geometrical models.", "The resulting grids counted 5.9 million cells for the simplified geometrical model, 8.5 million cells for the approximated geometrical model, and 11.8 million cells for the detailed geometrical model.", "At the bottom, sides and top of the domain as well as on the building and bridge surfaces, the standard wall functions by Launder and Spalding (1974) with roughness modification by Cecebi and Bradshaw (1977) were employed.", "At the bottom of the computational domain, an equivalent sand-grain roughness height ks equal to 0.0013 m (0.39 m full scale) was imposed.", "This value was calculated in accordance with Blocken et al. (2007a) as ks = 9.793 z0/Cs, where Cs (the roughness constant) was taken equal to 2.5 in order to comply with the necessary condition yp > ks, where yp is the distance of the centroid of the first cell from the wall.", "The size of the first near-wall cell was chosen in order to obtain dimensionless wall unit values y+ in the logarithmic layer range, i.e. 30–300 (Blocken et al., 2007a,b).", "An overview of these boundary conditions is given in Fig. 7.", "At the sides and top of the domain as well as on the building and bridge surfaces, ks was equal to zero.", "At the outlet of the domain, zero static gauge pressure was imposed.", "The CFD simulations were performed using the open-source CFD code OpenFOAM 2.3.0 with the 3D steady-state Reynolds-Averaged Navier-Stokes (RANS) approach.", "The realizable k-ε turbulence model was adopted for closure (Shih et al., 1995).", "Second-order discretization schemes were used for the convective and viscous terms of the governing equations.", "The SIMPLE algorithm was adopted to couple pressure and velocity fields (Patankar, 1980; Ferziger and Perić, 2002).", "Numerical convergence was achieved when the residuals showed no discernible fluctuation and further decrease during the iterative process.", "All CFD simulations were performed on a High Performance Computing (HPC) system at DICCA, using a computer node with 32 cores running in parallel at 1.4 GHz.", "The amplification factor U/Uin,0.02m is defined as the local wind speed divided by the inlet wind speed at height z = 0.02 m (Uin,0.02m).", "The amplification factor U/Uin,0.60m is defined in the same way but with inlet wind speed at height z = 0.60 m. Fig. 8 shows contours of the amplification factor in a horizontal plane at 0.02 m and in the vertical centerplane through L2 (see Fig. 3a).", "The CFD results for the three geometrical models exhibit almost the same wind-flow patterns upstream of the building block, both in terms of horizontal and vertical contours (Fig. 8).", "In contrast, significant differences are observed for the approximated and detailed geometrical models, with respect to the simplified one, at the courtyard and in the wake (Fig. 8c and d).", "The differences can be investigated more in detail by the mean wind velocity profiles at the positions C1 - C8 (Fig. 9).", "In Fig. 9, the axes show the ratio U/Uin,0.6m (abscissa) vs. normalized reference height z/zref (ordinate), with reference height zref = 0.60 m.", "The deviations of the simplified and approximated geometrical models with respect to the detailed geometrical model are calculated in terms of differences between mean velocity ratios, U/Uin,0.6, i.e. Δsim = Usim/Uin,0.6 – Udet/Uin,0.6 and Δapp = Uapp/Uin,0.6 – Udet/Uin,0.6, at the positions C1 - C8 and for four different levels (0.05, 0.1, 0.15 and 0.2 of z/zref) above the bottom of the domain (Table 1).", "In general, the CFD results from the three geometrical models show the same wind velocity values above z/zref ≈ 0.2.", "In contrast, the approximated and detailed geometrical models exhibit significant differences with respect to the simplified geometrical model (below z/zref ≈ 0.2) at all positions except for C1 and C2 (upstream of the obstacle).", "In the central part of the computational domain, at positions C3, C4 and C5, larger differences in terms of mean velocity ratio are found between the three CFD models mostly inside the courtyards of the buildings (for the approximated and detailed geometrical models).", "The approximated model shows small overestimations at positions C3 and C4 below z/zref ≈ 0.1 with respect to the detailed model (Table 1).", "Below z/zref ≈ 0.1, the simplified model evidently does not provide any velocity values due to the absence of the courtyards (Fig. 9 and Table 1).", "At z/zref = 0.15 and at z/zref = 0.2, the Δapp values are smaller than Δsim (Table 1).", "Approximately the same trend of mean velocity ratio observed at positions C3 and C4 is also found at position C5 at levels 0.15 and 0.2 of z/zref (Table 1).", "In the downstream part of the computational domain, at positions C6, C7 and C8, very substantial discrepancies in mean velocity ratio are found between the simplified and the other geometrical models (Fig. 9 and Table 1).", "A flattening of the three curves is observed at the positions C6 and C7 between 0.05 and 0.15 of z/zref, which increases with decreasing height of the buildings (Fig. 9).", "As a matter of fact, in the simplified model the height of the single block (constant and equal to 0.07 m) is lower compared to the tallest building of the approximated and detailed models, respectively equal to 0.09 and 0.098 m.", "The results are clearly confirmed by the corresponding values of Δsim and Δapp reported in Table 1.", "Overall, a satisfactory agreement is found, in terms of velocity values, between the approximated and detailed models.", "Conversely, the simplified model shows large discrepancies with respect to both finer models at about all central and downstream positions.", "Based on the satisfactory agreement between the approximated and detailed geometrical models, and considering that the construction of the detailed model is found to be about 5 times more computationally demanding (user time and processing time combined) than the approximated model, only the simplified and approximated models are retained for further study and are applied to the whole urban district Quartiere La Venezia.", "In this second step, CFD simulations are performed on two geometries with different level of precision of the urban district Quartiere La Venezia, i.e. the simplified and approximated geometrical models.", "A computational domain with the same size (L x W x H = 5.5 × 1.70 × 1.35 m3) as the one used in the first step (Section 3) is employed also in the current step.", "As already discussed in Section 3, since the grid generation is computationally demanding, a detailed grid-sensitivity analysis for each computational model and domain is not performed.", "However, in order to provide grid independent results, a very high-resolution of the grids is adopted based on the previous similar works published by van Hooff and Blocken (2010), Blocken et al. (2012) and Janssen et al. (2013) that are based on a detailed grid-sensitivity analysis.", "The computational grids are constructed using the surface-grid extrusion technique presented by van Hooff and Blocken (2010) in order to achieve a high-level of control over the grid and to adhere to the best guidelines (Franke et al., 2007; Tominaga et al., 2008a; Blocken, 2015).", "The expansion ratio of the grid was kept between 1.05 and 1.1 in the whole domain and at least twenty cells along every edge of buildings were used (Fig. 10).", "Three computational grids are generated for each of the geometries, one for every wind direction.", "In order to avoid convergence problems and maximize numerical accuracy, only hexahedral and prismatic cells were used.", "Key statistics for each grid are shown in Tables 2 and 3.", "It can be seen that the approximated models are more than 50% more computational expensive than the simplified ones, in terms of the number of cells, grid generation and required runtime.", "Fig. 11 compares the geometry of the actual urban area, the WT model and both CFD models.", "Concerning the other computational settings, the same boundary conditions, inlet conditions, turbulence model, discretization scheme for the equations, algorithm solver and cluster machine are used as in the first step (Section 3).", "Contours of the amplification factor are analyzed in various horizontal and vertical sections at different heights and widths of the computational domain.", "Fig. 12 shows the amplification factors Uin,0.02m, for both CFD models and for three wind directions.", "In Fig. 12a–b (α = 240°), Canale Rosciano is almost aligned with the approach-flow wind direction and, as a result, the wind flow is funneled through the canal.", "Separation and reversal zones are not observed along the canal for this wind direction.", "Inside the canal, the wind velocity in the simplified model is substantially higher than in the approximated one.", "In Fig. 12c–d (α = 270°), the Canale Rosciano is more sheltered from the approaching wind compared to α = 240°.", "The simplified model shows higher wind velocities in the central part of the domain (to the North of the Canale Rosciano) than the approximated one.", "In Fig. 12e–f (α = 300°), the Canale Rosciano is more perpendicular to the approach-flow, and it is therefore to a large degree sheltered by the upstream buildings.", "The approximated model shows higher wind velocities and more extensive leeward zones at the beginning and the end of the canal, respectively.", "Especially for this wind direction, the CFD simulations are found to be quite sensitive to the geometric detailing.", "Important differences between the two levels of detail employed in this study are found inside the narrow street and canal, as shown in Fig. 13 for the amplification factor Uin,0.6m.", "Fig. 14 shows contours of the amplification factor Uin,0.6m for both CFD models at the centerline of the computational domain for α = 240°.", "Despite the fact that model detail can heavily modify the wind-flow pattern inside the urban district, overall, the thickness of the UBL seems similar in both CFD models.", "Indeed, Fig. 14 shows almost the same horizontal development of stratification for the simplified and approximated models, although the maximum height of the buildings is different for both models, i.e. about 0.10 m and 0.15 m (32 and 45 m at full scale) respectively.", "This is useful information in our understanding of the physical interpretation of displacement height.", "To analyze the streamwise (horizontal) homogeneity of the approach-flow profiles, an empty domain with the same size as that for the urban model (see Section 4.1) is employed.", "The profiles at the inlet face and at the position of the first building of the urban model are compared along the lines L2 (centerline of computational domain) and at 0.02 m above the bottom, yielding a relative difference of only 0.91% (with an increase of the velocity), indicating sufficient horizontal inhomogeneity (Blocken et al., 2007a).", "Fig. 15 shows dimensionless mean wind velocity profiles obtained from the WT and CFD models for the three wind directions at four locations.", "The set of measuring positions placed at the central line of the Canale Rosciano (A22-A52) were chosen in order to understand the canyoning effects inside the urban district (see Fig. 3b).", "Fig. 15a (α = 240°): at position A22, where the incoming flow is aligned with the canal, the agreement between CFD and WT results is quite satisfactory.", "At position A32 the CFD model predictions are less accurate.", "Intentionally chosen, this position is located directly leeward of the important bridge near the city center and in the middle of a crossroads.", "At position A42 the level of detail of the CFD model plays a key role.", "In this part of the canal the incoming flow is no longer perfectly aligned with it, and the wake effects due to the presence of the buildings can be extremely dependent on their shape.", "Whereas the mean velocity profile of the approximated model shows a very similar trend compared with the WT profile, the simplified model displays a larger gap to the WT results between 0.11 and 0.28 of z/zref.", "In the last position A52, which is located in the middle of a canal curve, the approximated model again leads to a better prediction than the simplified one.", "Overall, for this wind direction, the approximated model shows better agreement with WT results than the simplified one.", "This also holds true for the measuring positions that are not reported here.", "Fig. 15b (α = 270°): at position A22 the agreement between the CFD models and the WT results is very close in the higher part of the domain down to z/zref = 0.1 (18 m full scale).", "However, the discrepancy between WT and CFD results is substantial close to the ground.", "At positions A32 and A42 important differences are found between 0 and 0.2 z/zref.", "As previously stated, these positions are located directly leeward of some buildings (see also Fig. 12c–d), where the results are affected by the underestimation of the turbulent kinetic energy k due to the steady-state RANS approach here adopted.", "As an example, Fig. 16 shows the underestimation of the turbulent kinetic energy for all wind directions at position A32.", "The quantitative agreement between WT and CFD results is unsatisfactory for this wind direction, although the qualitative wind profile development of the models inside the Canale Rosciano (from position A22 to A52) is somewhat similar.", "This was probably due to the canyoning effects along the water canal.", "The worst agreement between the WT and CFD results occurs at position A22 below z/zref = 0.15, and at positions A32, A42 and A52 below z/zref = 0.30.", "Fig. 15c (α = 300°): the worst performance of the CFD models is obtained for this wind direction.", "The buildings located at the edge of the Canale Rosciano represent a barrier for the wind flow.", "Only for position A22, located windward of the canal, the comparison is satisfactory.", "For the rest of the positions (A32, A42 and A52) the CFD profiles are quite different from the WT ones.", "The wind flow is not channeled along the Canale Rosciano and canyoning effects occur in the simulations behind the buildings placed at the edge of the channel (see Fig. 12e–f).", "This aspect explains the trend of the profiles at different positions.", "The worst agreement between the WT and CFD results is observed at position A22 below z/zref = 0.1, and at positions A32, A42 and A52 below z/zref = 0.40.", "Finally, in order to understand the different wind flow patterns produced by two CFD models (i.e. simplified and approximated models) in the downstream part of the computational domain, mean velocity profiles of two CFD models are compared at the 27 positions (L16-14, L26-14, L36-14) already shown in Fig. 3a.", "The mean velocity profiles monitored along the centerline of the computational domain (from L26 to L214) and for the three wind directions considered, are graphically displayed in Fig. 17.", "The axes show the ratio U/Uin,0,6m (abscissa) vs. normalized reference height z/zref (ordinate), with the incoming wind velocity Uin,0,6m measured at a reference height of zref = 0.6 m as normalization factor.", "Overall, the two CFD models show, for each wind direction, a different wind velocity profile development in the downstream part of the computational domain, from the last building of the urban district (at about the position L25 and L26) to the outlet face (Fig. 17).", "It is worth to note that, for the simplified model, where the tallest building (about 0.10 m) is lower than the equivalent of the approximated model (about 0.15 m), the wind velocity profile reaches almost a complete equilibrium condition, i.e. it does not change anymore, in the downwind distance (from the position L26 to the outlet face of the domain).", "In contrast, for the approximated model, for which the mismatch of roughness heights is larger than the simplified model, the mean velocity profile needs a larger downwind distance to reach an equilibrium condition.", "A comparison between the CFD and WT test results is made along vertical lines at the positions A21 and A41 (see also Fig. 3b) for 15 heights and three wind directions.", "Normalized mean wind velocities in the CFD results and WT data are plotted against each other in Fig. 18.", "The abscissa reports the ratio between the mean wind velocity measured at height z and the reference mean wind velocity measured at z = 0.6 m (Uref) in the WT tests; the ordinate axis shows the same ratio for the CFD results.", "Fig. 18a shows better agreement between the approximated model and the WT model than between the simplified and the WT model.", "In the latter case the velocity ratio is substantially overestimated compared to the WT data.", "In contrast, the approximated model shows some underestimation of the velocity values close to the ground.", "For the inlet wind direction α = 270°, shown in Fig. 18b, discrepancies are observed between the CFD and WT velocity ratios, especially at position A41 near the ground.", "This is found for both CFD models, probably due to the flow reversal zones close to the walls (see Fig. 12c–d).", "The results shown in Fig. 18c are consistent with those for the central line of the Canale Rosciano (Fig. 15c).", "The large deviations between CFD and WT results highlighted at position A21 for this inflow direction (α = 300°) can be due to the zone of flow separation present at the entrance of the canal (see Fig. 12e–f).", "Underestimation and overestimation of the velocity ratios occurs at position A41, in the lower and higher part of the profiles, respectively.", "Fig. 18 clearly shows one of the limitations of this study.", "As a matter of fact, the accuracy that can be achieved is most likely limited by the deficiencies of the 3D steady state RANS approach.", "The use of a more accurate geometry may therefore not lead to improved predictions within the areas where non-stationary wind-flow patterns occur.", "This limitation was already pointed out, for instance, by Murakami (1993), Tominaga et al. (2008b) and Blocken et al. (2016), who discussed this issue for building aerodynamics and by Burlando et al. (2015b), who discussed this issue using an analogous CFD model to simulate the flow around a vertical axis wind turbine.", "The validation metrics were calculated in horizontal planes at 15 heights (z) from 0.02 m to 0.6 m above the bottom (corresponding to 6 and 180 m above sea level at full scale) for all measuring positions A and L (overall 25 points) and for all inlet wind directions (α = 240°, α = 270° and α = 300°).", "Results for z = 0.02 m above the bottom are given in Table 4 for each considered wind direction.", "The results of Table 4 are based on data which are graphically displayed in Fig. 19.", "On the abscissa, the mean wind velocity value UWT measured at all available positions is displayed, normalized with respect to the mean inlet wind velocity magnitude taken at reference height Uin,0.02 (6 m above sea level at full scale).", "The equivalent ratio UCFD/Uin,0.02 of the WT data is reported on the ordinate.", "For a wind direction of α = 240° the FB values of the approximated model clearly shows a tighter distribution around the diagonal compared to the simplified model, indicating better agreement.", "For α = 270° the same FB value is obtained for both CFD models while for α = 300° the simplified model performed better than the approximated one.", "A similar trend is observed for the NMSE which shows better performance of the approximated model for α = 240°.", "Its performance becomes comparable to that of the simplified model for α = 270° while it is outperformed by the simplified model for α = 300°.", "The R of the approximated models is in general quite satisfactory.", "For α = 240° and α = 300° the approximated model clearly performed better than the simplified model while they showed similar performance for α = 270°.", "Finally, the metric FAC1.3 is used to understand how many data positions fall within 30% of WT data.", "No difference in the performance of both CFD models is observed in this metric for α = 270° and α = 300°, while the approximated model performed much better for α = 240°.", "In order to assess the effect of the geometrical simplifications on the total drag (D), this parameter was calculated for both the simplified and approximated models (Dsim and Dapp) and for the three wind directions considered.", "As expected, for all the CFD cases the most significant contribution to the total drag is given by the pressure drag associated to the bluff bodies (i.e. buildings and bridges), whereas the viscous drag contribution is confined between 2% and 6%, depending on the CFD case considered.", "In Table 5 the ratio between D and Dsim, 270° (chosen as reference value) is shown for all the CFD models.", "For all the wind directions considered, the drag for the approximated model is higher than the simplified one, i.e. +20.2% for α = 240°, +19.0% for α = 270° and +8.1% for α = 300°, as reported in the last row of Table 5, where the relative difference between two CFD models is calculated as ((Dapp/Dsim,270°)-(Dsim/Dsim,270°)/(Dsim/Dsim,270°)).", "This is possibly attributable to the higher mean height of the buildings, which yields a higher contribution of the pressure drag as well.", "In addition, the total drag also changes according to the wind direction considered.", "In this case study, the 3D steady-state RANS approach with the realizable k-ε turbulence model was applied to simulate mean wind-velocity patterns in a single block of Quartiere La Venezia and in the whole urban district reproduced at reduced scale (1:300).", "A portion of the WT section was reproduced by the computational domain in order to facilitate the comparison between WT and CFD results.", "This study is based on several assumptions:", "The study was performed for only a single city district.", "Nevertheless, the selected area may be considered representative of many historic towns including different types of buildings and narrow and curved streets.", "WT tests and CFD simulations were performed only for a neutrally stratified ABL flow, which typically occurs at the highest wind velocities.", "Three geometries with different degrees of precision were analyzed only for a single block of Quartiere La Venezia, since the detailed model of the whole urban district was found to be extremely computational demanding.", "The method used to estimate the height of the buildings (the average between the heights of the peak and eaves of roofs) of the approximated model may be not completely representative of buildings with roofs particularly slanted.", "The CFD simulations were performed using the 3D steady-state RANS approach, which is known to be deficient especially in separation zones.", "Large-Eddy Simulation (LES) are currently planned in order to investigate the impact of this limitation.", "Only a limited number of measuring positions (25 positions in the horizontal plane) was taken into account during the WT tests and subsequently used to validate the CFD results.", "For each point, however, 15 different heights were monitored, so that the overall number of points is actually equal to 375.", "These points, in turn, were measured for three wind directions, which means 1125 independent measurements.", "In spite of these limitations, the CFD simulations of the urban district showed a satisfactory agreement with the WT tests for wind direction α = 240° when the finer geometry (approximated model) was used.", "In this paper the local-scale forcing effects on wind flows in an urban environment were evaluated.", "In the first step, 3D steady-state RANS simulations were performed on a single block of a selected case study - Quartiere la Venezia in Livorno city (Italy) - for one wind direction (α = 240°) and three geometries with different degrees of precision, i.e. simplified, approximated and detailed models.", "Based on the numerical results found in the first step, a simplified and approximated model of the urban district Quartiere La Venezia were generated and simulated in the second step, using the same computational settings employed in the first step (dimension of domain, boundary conditions, turbulence model, discretization schemes for the equations, algorithm solver).", "In order to investigate to which extent geometrical model details can affect the CFD results, the mean wind velocity profiles of both CFD models at 25 positions for 15 heights each and three different wind directions were compared with the WT results.", "In order to quantify the agreement between the WT and the CFD results, the statistical performance was evaluated using four different validation metrics: FB, NMSE, R and FAC1.3.", "From the first step of this study, the following observations can be made:", "The simplified model showed large difference in terms of mean velocity with respect to the approximated and detailed models, mostly in the lower part of the wind velocity profiles (Section 3.4).", "The approximated and detailed models showed a satisfactory agreement both upstream and downstream of the single block (Section 3.4).", "The small differences in terms of mean velocity found mostly in the courtyard of the buildings, between two CFD models, do not justify probably a very high computational effort required to realize and simulate an eventual detailed model of the entire urban district (Section 3.4).", "From the second step of this study, the following observations can be made:", "The mean velocity contours showed the sensitivity of the simulations to the different degrees of model precision.", "The largest differences between the flow fields in the approximated and simplified models were found in the narrow streets and canal, especially for the wind direction α = 240° (Section 4.2.1).", "Although the geometric detail can affect the wind-flow pattern inside the urban district, the thickness of the UBL was similar for both CFD models (Section 4.2.1).", "For the wind directions α = 270° and α = 300°, corresponding to a decrease of the flow alignment with respect to the entrance of Canale Rosciano, the agreement between CFD and WT results decreased (Section 4.2.2).", "The CFD results showed an unsatisfactory correspondence between the CFD and WT results in locations where non-stationary phenomena occurred.", "It is likely that here the 3D steady-state RANS approach becomes the main source of error, in which case a more detailed geometry will not improve the flow prediction.", "The validation metrics (FB, NMSE, R and FAC1.3) confirmed that the finer geometry (approximated model) on average assures notably better performance than the coarse one (simplified model) (Section 5.2).", "The geometrical simplifications applied to the urban model of Quartiere La Venezia affected the results also in terms of total drag.", "The approximated model resulted in a higher total drag with respect to the simplified model for all wind directions considered (Section 5.3).", "Overall, wind flow modeling in urban areas is affected by many errors and uncertainties related to the inlet conditions, boundary conditions, numerical approach (RANS, LES, DNS), turbulence models, etc.", "A discussion of all these errors and uncertainties is beyond the scope of the present paper, but the future intention is to quantify their relative importance for the urban area investigated in this paper.", "In particular, numerical investigations on the same urban model (at scale 1:300) are in progress to quantify the uncertainties concerning inlet conditions as well as the numerical approach (RANS, LES, …)." ]	[ "Wind-tunnel tests have been performed on a reduced-scale urban district.", "3D steady-state RANS simulations have been performed for a single block of the reduced-scale urban district (as wind tunnel).", "3D steady-state RANS simulations have been performed for the whole urban district (as wind tunnel).", "The modeling errors caused by geometrical simplifications have been quantified.", "The agreement between wind-tunnel and numerical results has been quantified using four validation metrics." ]
Microarray data and pathway analyses for human microvascular endothelial cells supplemented with low dose vitamin D or niacin during lipotoxicity	Low dose niacin and vitamin D can directly improve human microvascular endothelial cell angiogenic function under lipotoxic conditions Peters et al.,2019. Despite exerting similar benefits on in vitro angiogenic function, these vitamins are known to signal through independent receptors, raising the possibility that differential changes in gene expression may underlie these effects. Here we provide data collected using Affymetrix GeneChip microarrays to compare gene expression in human microvascular endothelial cells treated for 16 h with growth medium containing BSA alone, or BSA complexed with the saturated fatty acid palmitate, and supplemented with 10 μM niacin or 10 nM vitamin D (1,25-dihydroxyvitamin D3). Data sets of differential gene expression included many genes involved in cellular stress responses. Pathway analyses of genes specific to vitamin D treatment identified a robust overrepresentation of pathways related to the cell cycle and DNA replication and repair.	[ "MULT" ]	[]	[ "1000" ]	[ "Microarray analyses comparing mRNA isolated from human microvascular endothelial cells (HMVEC) following 16 h treatment in growth medium containing bovine serum albumin (BSA) or 0.5 mM palmitate supplemented with 10 μM niacin or 10 nM vitamin D revealed significantly (p < 0.05) altered expression of many cellular stress response genes.", "These included genes involved in ER stress, oxidative stress, and inflammation, at a fold change of greater than 2 [1] (Supplemental material).", "These data were consistent with subsequent Reactome pathway analyses (Figs. 1–3, and Supplemental material).", "Pathway analyses of differentially expressed genes specific to vitamin D treatment identified robust overrepresentation of genes involved in cell cycle regulation, DNA replication, DNA repair, and signal transduction (Fig. 2, and Supplemental material).", "Primary HMVEC (Lonza, Walkerville, MD USA) were maintained in Medium 199 (Life Technologies, Carlsbad, CA, USA) supplemented with EGM-2MV SingleQuots (Lonza) and subcultured according to manufacturer's protocol.", "For experiments, cells from a single donor were used at subculture 5.", "For lipotoxic treatments, cells were exposed to media supplemented with 0.5 mM palmitic acid complexed to bovine serum albumin (BSA) at a molar ratio of 2:1.", "Cell monolayers at 80% confluence were treated for 16 h with media containing either BSA, palmitate, palmitate with 10 nM vitamin D (1,25-dihydroxyvitamin D3; Sigma Aldrich), solubilized in dimethyl sulfoxide (DMSO), or palmitate with 10 μM niacin, solubilized in cell culture grade water.", "DMSO (2 μl/ml of media) was used as the vehicle control.", "A total of twelve samples (three BSA, three palmitate, three palmitate plus vitamin D, and three palmitate plus niacin) were generated for subsequent gene expression analyses.", "Following treatment, cells were lysed and RNA was isolated following a standard QIAGEN protocol as previously described.", "RNA concentrations and purity were measured using a NanoDrop spectrophotometer (Thermo Scientific).", "Samples were diluted to a concentration of 200 ng/μL in nuclease-free water and RNA was stored at −80 °C prior to microarray analyses.", "All sample labeling and GeneChip processing were performed at the London Regional Genomics Centre (Robarts Research Institute).", "RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and the RNA 6000 Nano kit (Caliper Life Sciences).", "End labeled single stranded complementary DNA was prepared from 200 ng of total RNA and hybridized, for 16 hours at 45 °C, to Human Gene 2.1 ST arrays, as described in the Affymetrix Technical Analysis Manual.", "All liquid handling steps were performed using the GeneChip Fluidics Station 450 and GeneChips were scanned with the GeneChip Scanner 3000 7G (Affymetrix) controlled by Command Console v3.2.4.", "Microarray probe data were imported into Partek Genomics Suite v6.6 using the robust multi-array average (RMA) algorithm [2].", "Partek was used to determine gene level ANOVA p-values and fold changes.", "Filtered gene lists were generated for expression changes of greater than 2.0-fold and having a p-value of less than 0.05 (Supplementary material).", "Lists of genes exhibiting at least a 2.0-fold change in expression with a p-value less than 0.05 were used for gene ontology (GO) and KEGG pathway enrichment analyses (Supplementary material).", "Over-represented transcripts were also imported into the open-source Reactome curated pathway data base (v58) (http://www.reactome.org/) for further analyses.", "Pathway overview schematics, indicating significantly represented (p < 0.05) nodes (pathways) and relationships between nodes were generated for each list of differentially expressed genes." ]	[]
A critical review on analysis in pretreatment of lignocelluloses: Degree of polymerization, adsorption/desorption, and accessibility	The pretreatment of lignocelluloses results in changes in the different properties of these materials. In a recent review (Karimi and Taherzadeh, 2016), the details of compositional, imaging, and crystallinity analyses of lignocelluloses were reviewed and critically discussed. Changes in the cellulose degree of polymerization, accessibility, and enzyme adsorption/desorption by pretreatments are also among the effective parameters. This paper deals with the measurement techniques, modifications, and relation to bioconversions, as well as the challenges of these three properties. These analyses are very helpful to investigate the pretreatment processes; however, the pretreatments are very complicated and challenging processes. It is not easily possible to study the effects of only one of these parameters and even to find which one is the dominant one. Moreover, it is not possible to accurately predict the changes in the bioconversion yield using these methods.	[ "CENG", "ENER", "ENVI" ]	[ "Accessibility", "Degree of polymerization", "Enzyme adsorption/desorption", "Pretreatment" ]	[ "1502", "2105", "2305", "2311" ]	[ "Lignocelluloses are a very complicated matrix of different polymers and chemicals, affecting the yield of their bioconversions (e.g., Karimi and Pandey, 2014).", "When the hydrolytic enzymes or microorganisms attack the lignocellulose’s carbohydrates, various factors affect their effectiveness.", "In a recent review (Karimi and Taherzadeh, 2016) some of the factors, including those that affect the analytical results of the composition of lignocelluloses, imaging, and crystallinity, were reviewed and critically discussed.", "This article is dedicated to the remaining factors.", "The length of the carbohydrate chains is among the factors that highly affect the hydrolysis.", "Among the carbohydrates, the major constituent, cellulose chains are typically larger and more difficult to hydrolyze.", "The larger the cellulose chains, the lower the hydrolysis yields; thus, the degree of polymerization of the cellulose is known to be a critical factor in the hydrolysis (Karimi et al., 2013).", "On the other hand, the carbohydrate polymers need to be accessible for the hydrolysis.", "Besides accessibility, the enzyme or microorganisms should be adsorbed on the polymer chains, and after the hydrolysis they should be desorbed to start the hydrolysis of the other chains.", "These factors are known as determining factors in the bioconversion of the lignocelluloses (Kumar and Wyman, 2009, 2013).", "These analyses help to give different interpretations in the pretreatment processes; however, there are some challenges with the analysis and in correlating these factors to the lignocelluloses bioconversions.", "This review paper introduces the different analytical methods, their strengths and weaknesses, modifications, and its possibility to be used for correlating with the pretreatment efficiency.", "The DP of cellulose, the number of glucose in the molecule, is known as a key parameter contributing to the enzymatic hydrolysis of the lignocelluloses (Hallac and Ragauskas, 2011) (Fig. 1).", "Long cellulose chains contain more hydrogen bonds and are difficult to hydrolyze (Zhang and Lynd, 2005).", "Furthermore, the DP reduction by a pretreatment process is accompanied by the formation of more cellulose ends available to the exoglucanase (Yang et al., 2011).", "In addition, the shorter cellulose chains are more reactive to the enzymes, as the shorter chains containing lower hydrogen bonding have weaker networks and are easier for the enzymes to access (Hallac and Ragauskas, 2011).", "Thus, it can be expected to increase the susceptibility of the cellulose to hydrolysis by decreasing the cellulose DP (Zhang and Lynd, 2005).", "Nonetheless, the information that is available on the effects of the DP on hydrolysis is still limited.", "Moreover, no clear relation has been detected between the DP and hydrolysis (Yang et al., 2011).", "For instance, Sinitsyn et al. (1991) decreased the DP of pure cellulose and bagasse by more than fivefold using the irradiation pretreatment, in which the other properties including the crystallinity were not altered.", "They found higher synergy between some types of endoglucanase and cellobiohydrolases at a lower cellulose DP.", "However, no significant change in the enzymatic hydrolysis yield was observed by changing the DP.", "In the pretreatments that reduce the cellulose DP, e.g., dilute acid hydrolysis, chains with different sizes are formed, including soluble and insoluble cellulosic polymers and oligomers.", "Insoluble cellulose molecules have a DP from 100 to 20,000, while the soluble cellulose has a DP in the range of 2–12.", "Cellulose chains with a DP of less than 6 are soluble and those in the range of 6–12 are slightly soluble (Zhang and Lynd, 2004).", "The activity of β-glucosidase is reduced by low DP cellulose chains (Lee and Fan, 1980).", "This can be one of the negative effects of the pretreatment and the DP reduction on the enzymatic hydrolysis.", "Generally, limited information is available regarding the effects of the different cellulose with different degrees of polymerization on the enzymatic hydrolysis.", "Moreover, the effects of the DP on a number of other parameters and properties, e.g., the cellulase adsorption and desorption (Yang et al., 2011).", "Viscometric technique is the most widely used method for the cellulose DP measurement (Hubbell and Ragauskas, 2010).", "It is probably the easiest and cheapest way.", "It needs dissolution in a cellulose solvent, such as cuproethylenediamine, measuring the intrinsic viscosity, e.g., by using a capillary viscosimeter and calculating the DP by applying a correlation such as the Immergut formula (Pala et al., 2007).", "A modified and more accurate viscometric method for the DP measurement was recently presented by Kumar et al. (2009).", "In this method, the lignocellulose powder is dissolved in the copper diethylene amine under continuous nitrogen flushing.", "The cellulose DP is then determined using the intrinsic viscosity measurement, according to the ASTM standard D1795.", "The effect of the hemicellulosic polymers on the viscosity is also excluded, resulting in a better approximation of the cellulose DP.", "This method is suitable for comparing the effects of the pretreatment of lignocelluloses and resulted in more reasonable values, although the absolute values are not as accurate as the more advanced methods.", "In spite of its simplicity, this method is suggested for the comparison of the lignocellulosic materials.", "Even the comparisons of the hydrolysis and the DP of the different lignocelluloses measured by viscometry make sense.", "For example, the DP of wheat straw, rice straw, poplar, and aspen measured by viscometry was 2660, 1820, 3500, and 4581, respectively (Hallac and Ragauskas, 2011).", "It corresponds to the general agreement that agricultural residues are more amenable to hydrolysis than wood residues.", "The DP can also be calculated by the molecular weight distributions of the derivatized cellulose (e.g., tricarbanilate cellulose).", "The number- and weight-average degree of polymerization (DPn and DPw, respectively) can be obtained by gel permeation chromatography (GPC) and be simply related to the DP (Hubbell and Ragauskas, 2010).", "The lack of availability of the cellulose tricarbanilate, as a standard, is one of the drawbacks; however, the GPC results offer detailed information, suitable for following the cellulase mode of action on the cellulose (Pala et al., 2007).", "Most of the methods used for the DP measurement of cellulose are based on the cellulose dissolution.", "The dissolution can be obtained using either metal complex solutions (e.g., cuam, cuen, and cadoxen) or preparing easily soluble derivatives of the cellulose (e.g., by nitration and tricarbanilation).", "After dissolution, besides the viscometry and GPC, the DP can also be measured by membrane osmometry, vapor pressure oscomometry, cryoscopy, ebullioscopy, and chromatography, although they are time consuming and need a number of preparations (Zhang and Lynd, 2004).", "An alternative approach to measuring the number-average DP is to divide the number of glycosyl residues by the number of chain ends, by measuring the available reducing end sugar assays, e.g., 3,5-dinitrosalicylic acid (DNS), Nelson–Somogyi, or 2,2′-bicinchoninate (BCA) methods.", "A rapid procedure presented by Zhang and Lynd (2005) can be used to determine the DP of the pure cellulose using the ratio of the glucosyl monomer concentration measured by the phenol-sulfuric acid method) to the reducing-end concentration measured by a modified BCA method.", "BCA was modified in order to avoid breaking the glucosidic bond cleavage.", "However, this method has limitation for real biomass, as compounds such as protein react with the BCA reagent and may overthrow the numbers.", "Light scattering techniques can also be used for determination of the cellulose molecular weight and DP without the cellulose derivatization.", "The typical system uses the size exclusion chromatography coupled with a multi-angle laser light scattering.", "However, the method is suitable for pure cellulose and less for typical celluloses containing an appreciated level of lignin, as a result of the difficulties with dissolution (Yanagisawa and Isogai, 2007).", "For the DP measurement of the lignocelluloses, the cellulose in the biomass must be isolated.", "Nevertheless, it is difficult or let us say impossible to separate the cellulose without changing its structure (Hallac and Ragauskas, 2011).", "Because of the restrictions in the measurement and interference with other effective factors, it has been stated in most studies that “more investigations are necessary” (Kumar and Wyman, 2013).", "Browning (1967) presented a chemical method for the isolation of the cellulose from the lignocellulose, during which the length of the cellulose chains is not significantly changed.", "However, the DP change in this purification method depends on the species and the composition of the lignocelluloses (Hallac and Ragauskas, 2011).", "The main problem in investigating the effects of the cellulose DP change following pretreatment is simultaneous changes in a number of properties, including changes in the lignin and hemicellulose content, lignin–carbohydrate complexes, accessibility, and crystallinity (Shafiei et al., 2015).", "The structure of the cellulases is adapted to enter the insoluble substrates.", "The hydrolysis of cellulose starts with the adsorption of the enzymes onto the cellulose molecules.", "Thus, the enzymes adsorption/desorption is one of the main properties of a lignocellulose, affecting the rate of hydrolysis.", "After hydrolysis of a cellulose segment, the cellulases should be desorbed to start the hydrolysis of a new segment or move to the next parts.", "Thus, desorption is also an important factor affecting the hydrolysis.", "Nonspecific adsorption of the cellulase, e.g., on lignin, can play a major limiting role (Kumar and Wyman, 2009, 2008).", "Instead of cellulose, cellulases are adsorbed on lignin, making fewer enzymes available for the hydrolysis.", "Adsorption/desorption of the cellulases is suggested, as properties of lignocelluloses matched well with the hydrolysis (Fig. 2).", "A number of properties, e.g., crystallinity, accessible surface area, particle size, and lignin and hemicellulose content, are involved in the cellulase adsorption/desorption.", "In other words, cellulase adsorption/desorption is a suitable representative for a number of affecting parameters.", "Physical adsorption of the cellulases on the cellulose reaches the equilibrium state within a very short time (e.g., 30–90 min), as compared to the time required for the hydrolysis (e.g., 72 h).", "Several equilibrium models and dynamic models have described the adsorption experimental data (Zhang and Lynd, 2004).", "Among the applied models, the Langmuir isotherm model fits well with the equilibrium data in most of the cases, even in the presence of lignin (Suvajittanont et al., 2000).", "Several modifications to the Langmuir isotherm model have also been presented (Zhang and Lynd, 2004).", "Despite the well-fitting data, there are several assumptions regarding the Langmuir equation that may not comply with the cellulase–cellulose system, including the partial irreversible adsorption (particularly on the high lignin content binding sites), cellulase component interactions, multiple types of adsorption sites on the cellulose, and enzyme entrapment in the cellulose pores.", "Furthermore, cellulase is a mixture of different enzymes, and each enzyme has different Langmuir constants (Zhang and Lynd, 2004).", "In spite of these drawbacks, the adsorption equilibrium constants have a strong correlation with the yield and rate of the hydrolysis (Lynd et al., 2002), which supports the hypothesis that the hydrolysis is primarily controlled by the cellulase adsorption onto the cellulose surface (Kumar and Wyman, 2009).", "The adsorption/desorption experiments are easy to perform.", "However, to avoid the hydrolysis of cellulose, they should be carried out at 4 °C (Lee et al., 1982).", "The cellulase adsorption/desorption analysis can be performed in a 1–10 ml media similar to the hydrolysis experiment (i.e., 50 mM sodium citrate buffer, pH 4.8) for 10 min to 2 h with vigorous stirring or shaking, gentle shaking for 6 h, or for 12 h without shaking (Gao et al., 2014; Kumar and Wyman, 2008; Lee et al., 1982; Zhu et al., 2009).", "For direct measurement of the absorbed cellulases, the biomass may be washed with water to remove the non-adsorbed enzymes (Zhu et al., 2009) or without washing (Kumar and Wyman, 2008).", "Furthermore, a very small amount of pretreated or untreated sample, e.g., 0.05 g, is necessary (Noori and Karimi, 2016).", "Free sugars may be removed before the adsorption analysis.", "Besides inhibiting the enzymatic hydrolysis, the free sugars, e.g., glucose and particularly cellobiose, can severely and negatively affect the cellulase adsorption (Kumar and Wyman, 2008).", "This should be considered for the lignocelluloses containing high free sugars, e.g., sweet sorghum.", "Desorption can be conducted easily by dilution with water, citrate buffer, or caustic water and equilibrating for 10 min to 2 h (Hu, 2009; Kumar and Wyman, 2009).", "A typical way is dilution with an equal amount of citrate buffer (50 mM, pH 4.8) and equilibrating at 4 °C for 2 h.", "A number of other solutions, e.g., citrate buffer at a higher pH, detergents, salts, and polyhydric alcohols were also used to improve the cellulase desorption.", "Among them, high concentrated ethylene glycol (EG) (e.g., 72–75%) is among the most suitable solution for the effective desorption (Hong et al., 2007; Zhu et al., 2009).", "In order to compare the desorption results between the untreated and the pretreated substrates or different lignocelluloses, desorption in a citrate buffer similar to the one used in the hydrolysis gives reasonable results (Kumar and Wyman, 2008).", "The cellulases adsorbed on the cellulose can be determined based on the difference of the initial cellulase and the free cellulase.", "The concentration of the free enzymes can be measured by traditional methods, e.g., Lowry, Biuret, bicinchoninic acid (BCA), and Bradford.", "When some free sugars are present in the biomass, the proteins can be precipitated by the acetone and re-dissolved in the citrate buffer before the analysis (Lee et al., 1982).", "However, the enzymes adsorbed on/in the cellulose cannot be directly measured by these methods.", "The Kjeldahl method, a favorable method for determining the nitrogen and protein content in many industrial products particularly in food and fertilizer industries, was used for the direct measuring of the protein adsorbed on the biomass and shown to be not suitable for this purpose (Kumar and Wyman, 2008).", "Different direct or indirect measurement methods are available to measure the adsorbed proteins, as summarized here:", "The adsorbed cellulases can be washed with a large volume of a buffer, and the protein mass concentration is assayed by the traditional protein assayed methods.", "The major challenge is that of very low concentrations of free enzyme, resulting in inaccuracy in the protein assay.", "Moreover, the method is not applicable to lignocelluloses as a result of interference with lignin and hemicelluloses (Zhu et al., 2009).", "Another method is measurement of the released cellulases using activity measurement.", "This method is very time consuming, sensitive, and accompanied by changes in the composition of the cellulases (Zhang et al., 2006).", "One of the major disadvantages of the indirect methods is that the accuracy of the methods cannot be checked by the material balances.", "Sensitivity to the free sugars, lignin, chemicals, salts, color, and other components are also among the problems (Kumar and Wyman, 2008).", "Different direct measurement methods are presented.", "Using radio- or fluorescent-labeled cellulases, high-throughput fast protein liquid chromatography (HT-FPLC), nitrogen element analysis, and hydrolysis of the absorbed enzymes to the amino acids and their analysis, are among the methods.", "Kumar and Wyman (2008) presented a direct method for measuring the cellulase absorbed on the cellulose.", "This method is based on the nitrogen element analysis, using a CHNS/O analyzer, which is accurate, but costly.", "Since the lignocelluloses contain nitrogen components, the nitrogen in the biomass should also be measured and subtracted.", "The nitrogen content measured is then converted into the corresponding protein content using a nitrogen factor (NF), which requires some more measurements to give an accurate value.", "The NF for cellulases and xylanase is similar or greater than that of plants’ protein (NF = 6.25).", "A simple method is also presented based on the hydrolysis of the absorbed enzymes to the amino acids and their analysis.", "The hydrolysis of the cellulases can be performed by autoclaving with 10 M of NaOH.", "Without interfering with the free sugars, cellulose, hemicellulose, and lignin, the ninhydrin assay can be used to quantify the total amount of amino acids released from the hydrolyzed cellulase.", "The proteins in the untreated and the pretreated samples can be measured by the ninhydrin assay and considered in the calculations (Zhu et al., 2009).", "Radio-labeled cellulases have also been successfully used to study the cellulase adsorption/desorption as well as investigate the reversibility of the adsorption/desorption (Palonen et al., 2004).", "However, this method is very expensive and needs enzyme purification and labeling.", "The Langmuir model has two parameters, maximum adsorption capacity (σ, or specified adsorption capacity) and single adsorption equilibrium constant (Kd).", "They can be used to compare the kinetic properties of the various cellulase–cellulose systems, e.g., pretreated and untreated lignocelluloses.", "Maximum adsorption capacity can be related the binding capacity of biomass.", "The adsorption equilibrium constant is related to the inverse of the binding affinity (liters per gram of cellulase).", "A strong direct relation was reported between the maximum adsorption capacity and the enzymatic hydrolysis (Gao et al., 2014).", "One of the main fundamental problems with the adsorption/desorption analysis is performing the adsorption analysis at 4 °C, avoiding the hydrolysis of the cellulose.", "Conversely, the temperature at which the hydrolysis is performed is 37–50 °C.", "Both adsorption and desorption are highly affected by temperature.", "At 4 °C, the rate of adsorption on the cellulose and lignin is more or less the same, whereas the cellulase adsorption capacity of the lignin is less than one-tenth of that of cellulose.", "On the other hand, at 50 °C, the adsorption of the cellulase on the cellulose is much faster (reaching an equilibrium in 1 h with a peak at 0.25 h), while the cellulase adsorption on the lignin is much slower and took more than 12 h to reach equilibrium (Zheng et al., 2013).", "Therefore, the adsorption/desorption data obtained at 4 °C should not always be expected to match well with the enzymatic hydrolysis data at the high temperatures (e.g., 37–50 °C).", "One of the main objectives of almost all pretreatments is to increase the cellulose accessibility for the hydrolytic microorganisms or enzymes (Fig. 3).", "Even the main goal of the lignin/hemicellulose removal is to increase this accessibility (Shafiei et al., 2015; Taherzadeh and Karimi, 2008).", "Cellulose accessibility was reported to be the most important factor affecting the digestibility of the lignocelluloses, among the delignification, destruction of the hydrogen bonds, breakdown of intramolecular hydrogen bonds, cellulose crystallinity, and lignin and hemicellulose content (Huang et al., 2010).", "In anaerobic digestion for the biomethane or biohydrogen production, four sequential phases are conducted with different microorganisms.", "The first stage, which is the limiting stage in the case of recalcitrant lignocelluloses, is hydrolysis by hydrolytic bacteria (Kabir et al., 2015).", "The sizes of these bacteria are in the range of between 0.2 and 20 μm, despite their different shapes.", "Therefore, the biomass substrate should have enough pores, with at least 0.2–20 μm to be digested by these bacteria.", "The cellulolytic bacteria are truly efficient hydrolyzers of the cellulose/hemicellulose, more efficient than the free enzyme systems (Shoham et al., 1999).", "As they reach the cellulose surfaces, the bacteria produce extracellular multi-enzyme complexes, so called cellulosomes, localized at the interface between the cell and the cellulose/hemicellulose, and they degrade the cellulose and the hemicellulose to soluble products that are then directly absorbed (Shoham et al., 1999).", "These intricate enzymes are designed for efficient hydrolysis of the carbohydrates.", "Cellulosome is a very complex system and composed of different functional domains and subunits, interacting with each other as well as the cellulose/hemicellulose (Shoham et al., 1999; Dykstra et al., 2014; Salehi Jouzani and Taherzadeh, 2015).", "One of the cellulosome subunits is a large glycoprotein, which is non-catalytic, called scaffolding, containing different types of cellulases and hemicellulases, with the optimized ratio and order of the component (Schwarz, 2001).", "It adheres to cellulose/hemicellulose by cellulose/carbohydrate-binding domains.", "Cellulosome is typically attached to the cell wall of the bacteria; however, it can also be present freely in the solution (Bayer et al., 2004).", "The initial stage in the degradation of the lignocellulose is the physical binding of the entire microorganism to the cellulose substrate, as the cellulosome formation is induced and occurs at the cell-substrate interface (Shoham et al., 1999); thus, the direct physical contact between the microorganisms and lignocellulosic surfaces is a requirement to start the hydrolysis.", "Therefore, lignocelluloses with only exterior surfaces are not suitable for the microbial digestion, due to the low accessible surface area for the microbial digestion.", "This can be simply observed by a 60-day anaerobic digestion of a piece of wood, resulting in no considerable weight loss or change in its appearance.", "This discussion is also true for the improvement of the digestibility of the lignocelluloses, e.g., rice straw, for the animal feed production as well as for the composting of the wood wastes.", "Cellulase and hemicellulases cocktails can be produced by different microorganisms, mainly fungi and bacteria.", "Different types of these enzymes are purified and commercially available with reasonable prices.", "Hydrolysis with cellulase involves three stages (Taherzadeh and Karimi, 2007): (a) enzyme adsorption to the cellulose surface, (b) hydrolysis of the cellulose, and (c) desorption of the adsorbed enzymes into the liquid.", "Thus, similar to the microbial hydrolysis, direct physical contact of the enzymes and cellulose is necessary; and, accessible surface area for the enzymes is among the most effective factors affecting the rate and yield of the cellulose/hemicellulose enzymatic hydrolysis.", "Cellulases are composed of a core and a long tail, like a tadpole, including a catalytic domain, linker, and a carbohydrate-binding module (CBM), which is significantly varied by the strains produced.", "As a simple approximate, it can be considered as an ellipsoid core with around 6 nm diameter and approximate length of 20 nm (Srisodsuk et al., 1993).", "For diffusion of a molecule to a pore, the size of diffusing component cannot be equal to the pore size, as the wall confinement plays an important role.", "For instance, the maximum accessible particle size is 1.31 nm for a 3 nm cylindrical pore.", "Thus, considering the wall confinements, pores with 40–60 nm width limit the cellulases diffusion to a high degree (called critical width).", "Practically, even 200 nm may also limit the enzymes diffusion to a certain extent, because of the confinement as well as the pore clogging effects by previously bound enzymes, irreversible enzymes adsorption, jamming, and protein aggregates (Bubner et al., 2012).", "On the other hand, the most severe inhibitor of the cellobiohydrolase is the cellobiose, resulting in the feedback inhibition of the cellulases.", "When the pores are very narrow, the β-glucosidase, the cellulase enzyme that is mainly responsible for the cellobiose hydrolysis, cannot accompany the cellobiohydrolases in the pores.", "Thus, synergistic action of the different types of cellulases, which is the requirement for efficient and fast hydrolysis of cellulose, needs some more spaces (Bubner et al., 2012).", "Lignocellulosic materials have external and internal surfaces, where the total accessible surface area is the sum of these areas.", "External surface depends on the size and shape, and its area can be increased by size reduction, that is, a typical physical pretreatment of the lignocelluloses.", "On the other hand, the internal surfaces of a lignocellulose depend on the pore sizes and distributions.", "Lignocelluloses have a very small internal surface, especially after drying.", "A suitable pretreatment should significantly increase the internal surfaces (Shafiei et al., 2015).", "Accessible surface area is considered as an important factor for the digestibility of a lignocellulose.", "The accessible surface area is gradually increased by the enzymatic hydrolysis, as a result of the partial cellulose and hemicellulose removal.", "Thus, a more porous biomass is formed at the latter stages of the hydrolysis.", "However, hydrolysis is usually much faster at the beginning of the enzymatic hydrolysis and considerably slower at the latter stages, despite the availability of higher surfaces.", "This indicates that the surface area is not the main controlling factor for the hydrolysis.", "Easier hydrolysis of available amorphous cellulose is related to the faster hydrolysis of the biomass at the first stages and the slower rate related to the hydrolysis of the higher crystallinity regions, deactivation of enzymes, and increasing the concentration of the lignin.", "Consequently, the accessible surface area is not the only factor and should be considered in addition to the other effective factors affecting the hydrolysis (Khodaverdi et al., 2012).", "Nitrogen adsorption, mercury porosimetry, and solute exclusion are among the methods used for accessible surface area measurement.", "Pores are generally classified as micropores (>2 nm diameter), mesopores (2–50 nm diameter), and macropores (>50 nm diameter) according to their sizes (Sing et al., 2008).", "Main limiting features of lignocelluloses to enzymatic hydrolysis is the lack of accessibility, which can be related to the pore sizes.", "It should be noted that different methods give considerably different results, as the principles of the measurement are different.", "As an example, solute exclusion analysis showed a mean pore diameter of 3 nm for pine Kraft fibers, whereas this diameter was reported as 13 nm as detected by NMR (Suurnakki et al., 1997).", "However, for comparison purposes, all of them may make sense.", "Long ago, Grace and Maass (1931) presented a method for the sorption of the different vapors on wood and cellulose, which was then further used for the measurement of the total surface area and the pore size distribution of the wood.", "Volume, specific surface area, and the size distribution of the lignocelluloses pores can be determined by this method (Thode et al., 1958).", "The Brunauer–Emmett–Teller (BET) equation can be used for the calculation of the specific surface area, while the pore size distribution is determined using the isothermal data of either the adsorption or desorption.", "Details of the calculations are presented by Barrett et al. (1951).", "One of the advantages of using the nitrogen adsorption method is that it measures only the open pores, but not the closed interior pores, as the cylindrical pore model is assumed.", "However, the pore network can affect the desorption isotherm, used in the determination of the pore size distribution.", "For obtaining the desorption data, the pressure is reduced for the evaporation of the liquid; however, the liquid is not uniformly evaporated, as the liquid in the narrower channels remain filled.", "The evaporation can also result in changes in the pore distribution.", "Moreover, the distribution of the pores and their sizes may be significantly changed during the treatment with the liquid nitrogen at a reduced temperature (e.g., at −196 °C) (Westermarck, 2000).", "Washburn (1921) introduced MP as a suitable method for determining the pore size distribution of porous materials, which has subsequently been used to analyze the pores in cement, catalysts, and adsorbents.", "This method is also used for the analysis of the pores in the lignocelluloses.", "Similar to nitrogen adsorption, surface tension, capillary forces, and pressure are the basis of the method.", "The method was first used for the determination of the macropore-size distribution in the range of 85,000–200 Å pore diameter (Drake, 1949).", "MP was further improved to perform at low and high pressures.", "Depending on the equipment used, low-pressure can be suitable for the determination of the macropores (14–200 μm) and high-pressure for the mesopores and macropores (3 nm to −14 μm) (Tantasucharit, 1995).", "Commercial types of MP are now available for the determination of different powder properties, including total pore volume, incremental volume, differential volume, log-differential volume, total pore surface area, mean pore diameter, incremental area, pore size distribution, and percentage of porosity.", "The MP method can analyze a wider range of pore sizes in a shorter time than nitrogen adsorption; however, nitrogen adsorption is more widely used than MP (Allen, 1997).", "Pores with 0.3–300 nm diameter (mesopores and macropores) can be analyzed by nitrogen adsorption, while mercury porosimetry may be suitable for pores with 14–200 μm (macropores).", "However, the results from both of these methods are more or less the same (Conner et al., 1986).", "MP measurement is rather fast and easy.", "First, all the gas in the sample is evacuated, and then mercury is forced to enter the samples under a vacuum and pressure process to obtain intrusion-extrusion curves.", "The curves are then used for the calculation of the pore structure.", "The total introduced volume of mercury at the highest pressure is considered as the total pore volume.", "The area above the intrusion curve is used as the total pore surface area.", "Based on the assumption of the cylindrical shape, the mean pore diameter and volume pore size distribution can be calculated (Ritter and Erich, 1948).", "One of the disadvantages of MP is the compression and damage to the sample during the high pressures forcing the mercury to enter the pores.", "Another problem is the overestimation of the volume for the smallest pores (Auvinet and Bouvard, 1989).", "The major drawback of the nitrogen adsorption and mercury porosimetry methods is measuring the available surface area using the molecules that are very much smaller than the hydrolytic enzymes and the microorganisms.", "Another disadvantage is the need to use a dried sample, while the internal structure is changed during the drying process (Tantasucharit, 1995).", "Stone and Scallan (1967) developed the SE method, which was further established for the estimation of the accessible surface area for an enzyme as well as the pore volume distribution of the lignocelluloses (Tarkow et al., 1966).", "An experimental procedure complete with caveats was then presented by Van Dyke (1972).", "This method is quite simple and does not need special equipment; only a refractometer or polarimeter is necessary.", "In this method, the lignocellulose is added to a known weight of indicators, called “probes”, dissolved in water and mixed.", "Three scenarios are possible: (1) the concentration of the probes will not change, when all the pores are accessible to the probes; (2) the probes will be slightly less diluted than in the first scenario, when larger probe molecules are used; (3) the inaccessible water equals the total water of swelling or fiber saturation point, when the probe molecules are very large and cannot enter the pores.", "A series of probes with different sizes can be used.", "The probes should not be uncharged and physically or chemically adsorbed onto the pores, should have a narrow molecular weight distribution, and be available over a wide range of molecular weights.", "Probes should be spherical and have a known size in the solution.", "Different probes are suggested for this method.", "Among them, polyethylene glycol as well as dextrans and related sugars are the most applied ones (Tantasucharit, 1995).", "In this method, it is assumed that the concentration of the probes in the accessible pores is equal to that in the solution out of the pores.", "Moreover, when the probe diameter is less than the pore diameter, complete penetration of the probe is assumed (Lin et al., 1985).", "Furthermore, the pores are assumed to be parallel slits with a similar width between the multiple lamellae (Stone and Scallan, 1968; Tantasucharit, 1995), which is questionable.", "The main advantage of this method is the possibility of performing the analysis in the same solution media as used in the enzymatic hydrolysis.", "In spite of the assumptions, promising results and a reasonable relation between the rate of porosity by the SE and the enzymatic hydrolysis have been observed.", "For instance, the accessible surface area of the cotton linters to molecules with 40 Å sizes was compared with the initial rate of the enzymatic hydrolysis, and a linear relationship was observed (Stone et al., 1969).", "Moreover, a linear correlation was found between the accessible area of the hard- and soft wood to the molecules with 51 Å diameters, and the initial enzymatic hydrolysis was observed (Grethelin, 1985).", "However, this method is very time consuming, involving several solution preparation, weighting, agitation for several hours, settling without agitation, centrifugation, optical measurement, and need for special care and attention to obtain accurate results.", "Simon (1950) developed a method for the study of the interior structure of the fibers fibrillation and mechanical damage using two sensitive color probes.", "He used a mixed solution containing the two probes, a blue dye with a small molecular size and low affinity for cellulose, and an orange dye with a large molecular size and high affinity for cellulose.", "According to this method, the damaged fibers were stained orange, observable under an optical microscope.", "The original method is very time consuming and has some limitations.", "As the method has the possibility to indicate the large and small pores of the fibers, it was further improved in order to be used for the measurement of the interior and exterior surface area of the lignocelluloses, with a relatively high sensitivity to changes in the cellulose accessibility (Chandra et al., 2008; Goshadrou et al., 2013).", "The blue probe used has a 1 nm molecular diameter and a well-defined chemical formula, entering all the pores larger than 1 nm.", "However, the orange probe, obtained by condensation of 5-nitro-o-toluenesulfonic acid in an alkali solution, has a molecular diameter range of 5–36 nm.", "The orange dye has a higher affinity to the hydroxyl groups on the lignocellulosic surface.", "The ratio of these adsorbed probes (orange to blue dyes; O/B ratio) is used to analyze the amount of large and small pores and subsequently as an indication of the cellulose accessibility to the hydrolytic enzymes.", "Furthermore, it was shown that lower enzyme loading is required for efficient hydrolysis when the substrate has a higher O/B ratio (Arantes and Saddler, 2011; Chandra et al., 2008).", "One of the modifications to this method was conducted when it was found that the high molecular weight fraction of the orange dye used by Simon had a high affinity to cellulose and that the low molecular weight part had a similar affinity to the cellulose as the blue dye.", "Then, the low molecular weight part of the orange dye was removed by ultrafiltration.", "The accessible area was then well correlated to the enzymatic hydrolysis of the lignocelluloses (Esteghlalian et al., 2001).", "The method is rather simple and inexpensive.", "It needs some chemicals, a simple filtration set up, a spectrophotometer, and an optical microscope; all are available in almost all laboratories.", "However, the method is rather time consuming, even after the modification in which the incubation time was reduced from around 50 h to around 6 h and needs different sample and dye preparations.", "Moreover, the method is suitable for comparing the pore sizes of the untreated and pretreated lignocelluloses.", "However, the accuracy of the method is not as high as the NMR methods.", "The Simons’ staining analysis is also very sensitive to the pore inlet size.", "As indicated by the NMR analyses, there are some large pores in the lignocelluloses with small entrances (ink-bottle shaped pores), in which the dyes cannot enter and detect them.", "In addition, some of the pretreatment methods result in increased pore tortuosity within the biomass that negatively affects the Simon’s staining evaluations (Meng et al., 2013).", "NMR-based methods are powerful methods for the analysis of accessible surface areas of the lignocelluloses.", "NMR cryoporometry (NMRC) is among the best methods for detailed study of the pore size distribution in the lignocelluloses.", "The method is a perturbative method that is independent of the spin interactions and offers direct measurements of the pore sizes and volume.", "The pores on the scale of 2 nm to 2 μm can be analyzed.", "The ability to analyze the wet samples are among the main advantages of this method, as drying which is necessary for most methods, e.g., nitrogen adsorption, can significantly change the pore sizes and the volumes (Östlund et al., 2010).", "Cryoporometry is based on the fact that small crystals of a liquid molecule in the pores melt at a lower temperature than molecules in the bulk of the liquid.", "A liquid is charged to the pores, cooled to frozen, and warmed to melted.", "NMR is used to quantify the liquid inside the pores that has melted as a function of the temperature.", "The pore sizes are inversely proportional to the liquid melting point depression.", "The method can be used to compare the pore sizes obtained by pretreatments and compare it with the untreated samples.", "A clear image of the pore size changes can be observed (Meng et al., 2013).", "Besides NMRC, different thermoporometry and cryoporometry methods are also presented for the analysis of accessible surface area and pore size distributions of the lignocellulose.", "All of them have the same basis of melting a small region of a solid at a lower temperature than the bulk biomass.", "The detection of the melting can be done by differential scanning calorimetry (DSC), neutron scattering (ND), and NMR.", "Other NMR based methods, e.g., NMR relaxometry and NMR diffusometry, are also suggested to investigate the details of the changes in accessibility upon pretreatment of the lignocelluloses.", "Here, the probe molecule is water.", "The nature and strength of the association-adsorbed water localized within the pores is directly related to the ultrastructural and chemical state of the lignocelluloses (Felby et al., 2008; Menon et al., 1987).", "The main disadvantages of the NMR based methods are long experiment time and requirement for expensive instrument and complicated setup (Meng et al., 2013).", "If water cannot enter the pores, then it is for certain that no enzyme can enter.", "Thus, WRV, also known as water swelling capacity, is used as an indication of the accessible interior surface area and the suitability of the lignocelluloses to enzymatic hydrolysis (Noori and Karimi, 2016).", "This property of a biomass, which is the ability to swell in the presence of water, can be measured rapidly and easily, in most of the laboratories (Jeihanipour et al., 2010; Shafiei et al., 2014).", "Ogiwara and Arai (1968) tried to find relationships between the enzymatic hydrolysis and the WRV of different cellulose fibers.", "They reported a linear relationship between the WRV and the hydrolysis rate of untreated and pretreated substrates with different chemical compositions and crystallinity.", "However, they observed that the slope of the regression line was changed with the pretreatment method.", "It is interesting that the results of this simple and inexpensive method correlated well with the improvement observed in the enzymatic hydrolysis by pretreatment.", "Its results are comparable to the results of more advanced methods, e.g., NMR and Simons’ staining (Chandra et al., 2009; Östlund et al., 2010).", "Recently, Ju et al. (2013) reported that higher a xylan content increases the swelling, while lignin decreased the WRV.", "An increase in WRV corresponded to the increase in the efficiency of the enzymatic hydrolysis.", "Being able to use the same media as used for the enzymatic hydrolysis is also another advantage of the method.", "However, in some cases, the WRV showed limited sensitivity to be correlated to the enzymatic hydrolysis results changed by pretreatment (Chandra et al., 2009).", "Degree of polymerization is a parameter that can be related to the bioconversion of the lignocelluloses.", "However, there are some challenges in the DP measurement methods.", "Prior to the DP measurements, the cellulose should be isolated from the lignocelluloses structure.", "The isolation results in some changes in the DP.", "Moreover, it is difficult or may be impossible with the current knowledge to investigate the effects of the DP individually.", "It is also very difficult to investigate the relative importance of the different changes that have occurred due to the pretreatment.", "Cellulase adsorption/desorption is a property that is related to the bioconversion of the lignocelluloses.", "Reliable methods are available for its measurement; however, special attention should be paid to the measurement of the enzyme absorbed on the biomass.", "One of the major drawbacks of the cellulase adsorption/desorption analysis is when conducting the measurement at 4 °C, to avoid the hydrolysis of the cellulose.", "This temperature is far from the hydrolysis temperature (>37 °C).", "Nevertheless, the enzyme adsorption is highly dependent on the temperature.", "The kinetics of the cellulase adsorption and desorption on the cellulose and lignin are very different at 4 °C compared to that at high temperatures (e.g., 37–50 °C).", "Substrate accessibility is another important property of the lignocelluloses, which is a desirable parameter in all pretreatments.", "External surface can be increased by the size reduction and changing of the particle shapes.", "Increase in the internal surface of the lignocelluloses is followed by typical chemical and biological pretreatments.", "It can be measured by different methods, including nitrogen adsorption, mercury porosimetry, solute exclusion, Simon’s staining, NMR, and water swelling capacity; however, all methods have their specific drawbacks.", "Water swelling capacity is among the simplest methods, with its changes typically corresponding to the changes in the hydrolysis.", "Overall, it is not possible to exactly predict the effectiveness of a pretreatment with any of the mentioned analysis.", "However, it is possible to find the reason and the mechanisms for the changes in the bioconversions by the above-mentioned methods, analyze the changes, and try to improve the effectiveness of the pretreatments." ]	[ "Change in the cellulose degree of polymerization is an important factor in pretreatment.", "Adsorption/desorption and accessibility of the enzymes are also important features.", "Methods, strengths and weakness, and some details of the analysis are reviewed.", "The methods are helpful to understand and improve the pretreatment techniques.", "It is impossible to investigate the effects of just one factor in the pretreatments." ]
Preventing damage of germanium optical material in attenuated total reflection-Fourier transform infrared (ATR-FTIR) studies of living cells	Germanium is an important element for ATR-FTIR spectroscopy. It has often been selected as a substrate for live cells FTIR spectroscopy because of its low toxicity for most human cells. However, there is a lack of study on the physical interactions between germanium and the living cells. In this study, we have shown that germanium surfaces are easily damaged when living cells are grown directly in contact for more than 20 h on the element surface. We then proposed a methodology to reduce the damage by coating the surface of the element with a thin layer of biological materials and tested for their capacities to protect polished germanium surfaces from living cell cultures. Fibronectin, collagen and gelatine coated and non-coated, newly polished Ge plates were subjected to 48 h culture of HeLa cells for six times and the degree of erosion by the cell was monitored using optical and atomic force microscopy (AFM). The cells showed normal attachment and morphology to either the treated or untreated surfaces and the viability of the cells were confirmed by trypan blue assay. Furthermore we have tested with an ATR FTIR measurement with a multi-bounce Ge element and found that the coatings were thin enough not to interfere with the measurements. However the degree of erosion (measured by the roughness of the Ge plate after cell culturing) was found to depend on the types of coating used where gelatine coating showed the best protection.	[ "CHEM" ]	[ "Attenuated total reflection", "Extracellular matrix", "FTIR", "Live cell", "Microscopy" ]	[ "1607" ]	[ "Fourier transform infrared (FTIR) spectroscopy is a powerful label-free analytical technique.", "It is an ideal method for analysing biological materials [1].", "Infrared radiation is a low energy wave so that the measurement can be non-destructive and without any influence on the sample.", "Several studies of FTIR imaging of fixed biological material have shown significant differences between different cell types [2], cancer cells and normal cells [2,3], as well as cancer cells response to chemotherapy drugs [4–6].", "However, using living cells not only avoids artefacts due to the fixation process, but also enables to study the living material through time.", "To measure living cells, the issue about the strong absorbance of water in the mid-IR region has to be overcome.", "For this reason, transmission cells with very small path lengths have been designed [7–9] and synchrotron radiation is often employed for the measurement of living cells [10–15].", "Attenuated total reflection (ATR) was also found to be a suitable measurement mode for this purpose [16,17].", "In ATR mode measurements, samples are probed by the evanescence wave which is generated on the measuring surface when the infrared light undergoes an internal reflection (or multiple internal reflections for a multi-bounce ATR) through the high refractive index ATR element [18].", "The ATR FTIR measurement of live cells was achieved by seeding and culturing cells directly on the measuring surface of the ATR element such that the cells are measured in a similar geometrical environment as in a typical cell culturing flask [19].", "The path length are small and easily controlled and the sampling depth, which is ∼2–3 time the depth of penetration, is approximately 0.5–1.5 μm which probes deep into the attached living cell but without significant interference from the bulk medium above the cells.", "The development in the ATR FTIR measurement of live cells allows their study under treatment of drugs in situ for time-resolved FTIR spectra of the living cells to be recorded [20].", "One of the promising applications of ATR FTIR study is the imaging of living cells in micro ATR mode [21].", "Imaging in micro ATR mode has the added advantage of the increased numerical aperture where the light is further focused when approaches the sample through the high refractive index ATR element.", "A four times improvement in spatial resolution has been demonstrated when using Ge, which has a refractive index of 4, as the ATR element of the objective [22].", "Using this approach, it has been shown that major cell organelles such as nucleus and endoplasmic reticulum can be resolved [22].", "Ge is an important ATR element and it is universally used for micro ATR imaging.", "The surface properties of Ge are different to the standard cell culturing flasks which living cells may interact differently.", "A previous study has shown that cells can be cultured on Ge surface with no observable difference in morphology when compared to cell grown in tissue culture flask [23] and the attachment of cells was improved when the ATR surface was coated with a thin layer of collagen [21].", "However, the coating of Ge appears to be more important than simply improving the attachment of cells.", "In this work, we report on the possible damage to the bare Ge ATR element by the attached living cells.", "We have assessed the degree of damage by visible microscopy as well as atomic force microscopy after a single and repeated cell culturing on the optical element.", "This is important because the degree of damage is thought to be directly related to the scattering of light and loss of throughput.", "We have also measured the protective effect of various extracellular matrix components routinely used in tissue culture such as gelatin, collagen and fibronectin [24,25] as coating on Ge in order to propose a solution to reduce the degree of damage to the Ge surface.", "Newly polished germanium windows of 1 mm thick and 25 mm of diameter (surface area ∼491 mm2) were used as obtained from Crystan Ltd (UK).", "The coatings applied to the Ge windows were gelatine type 2 tissue culture grade (2% solution from Sigma, UK), collagen from calf skin type I for tissue culture (Sigma) and fibronectin from bovine plasma (Calbiochem, UK).", "Gelatine coating was applied at 2 mg/cm2, or 490 μL of 2% solution per window.", "Fibronectin was applied at a 5 μg/cm2, by applying 245.4 μL of a 0.1 mg/mL solution per window.", "Collagen was dissolved as recommended by the supplier to a working solution of 0.01%, which 490 μL were applied to obtain a coating of 10 μg/cm2.", "After application of the coating solutions the windows were left to dry for 2 h at 35 °C.", "For FTIR measurements of live cells, a temperature controlled multi-(i.e. 20) bounce ATR accessory trough plate (HATR, Pike technologies) and germanium ATR element (80 mm × 10 mm × 2 mm, Pike technologies) with an angle of incident of 45° were used.", "The path length in the living cells produced from this accessory in the 2000–1000 cm−1 region is approximately 3–6 μm with a depth of penetration of ∼0.32–0.64 μm.", "The trough plate has a measurement area of ∼500 mm2 where the live cells can attach and be measured.", "The coatings of the ATR plate were applied similarly to the optical windows but volumes adjusted to the growth area of 500 mm2.", "HeLa (ATCC#CCL-2) and PC3 (ATCC#CRL-1435) were maintained in T25 cell culture flasks using RPMI medium supplemented with 10% FBS and 2 mM L-glutamine, in a 5% CO2 environment at 37 °C.", "The cells were trypsinised and harvested when they reached ∼80% confluence.", "The cells were then centrifuged into a pellet and re-suspended in L15 medium (Sigma Aldrich) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin.", "L15 was used during the ATR measurement to allow closed chamber culturing with air as the gaseous phase, and therefore it was also used when cells were seeded on germanium windows.", "The cell suspension was diluted in medium to reach a cell density of ∼2.4 × 106 cell per mL.", "Germanium windows were placed in 6 well plates and 0.5 mL of the resulting cell suspension were seeded on the windows.", "The cells were allowed to settle for one hour, after which 2 mL of medium were added to each well and the cells were left to grow at 35 °C.", "The confluence was verified using reflective optical microscope with a 10× objective (NA 0.25) coupled with a camera.", "The cell culture was maintained till 48 h after seeding; the cells were then stained with trypan blue to test the viability, and then removed by washing the windows with distilled water using a micropipette.", "The surface of the washed windows was imaged by optical microscopy using a reflective microscope and by atomic force microscopy (AFM).", "Detailed procedures can be found in the previous work [19].", "In brief, two millilitres of culture medium were applied directly onto the multi-bounce, temperature controlled ATR (at 35 °C) and a spectrum was measured.", "Five hundred microlitre of cell suspension (i.e. ∼1.2 million of cells) were seeded directly onto the ATR trough which was then sealed and maintained at 35 °C, covered with a lid (also temperature controlled at 35 °C) to prevent evaporation.", "After incubating overnight, the cell seeding density ensured continual growth without reaching confluence (<90%) of cells adhered to the measuring surface.", "The attachment of cells and confluence were verified using reflective optical microscope.", "For assays on the response of cells to doxorubicin, 1 μM doxorubicin was applied to the HeLa cells cultures after the overnight growth on the ATR surface, similarly to the previous reported26 but instead of using a ZnS ATR element as in previous work, a Ge ATR was used.", "Continuous scan FTIR spectrometer (Frontier, Perkin Elmer Ltd, UK) fitted with a room temperature deuterated triglycine sulfate (DTGS) detector was used to acquire the IR spectra with a scanning time of 11 min, a spectrum was measured every 20 min.", "All measurements were acquired with a spectral resolution of 8 cm−1 and a spectral range of 4000–900 cm−1 and a mirror speed of 0.2 cm/s.", "The strong Norton-Beer apodization function and self-phase correction was chosen for the process of the interferogram.", "Spectra were analysed without further data retreatment except those for principal component analysis where spectra were baseline corrected based on wavenumber 1990, 1797 and 940 cm−1 using the Spectrum 10 software (Perkin Elmer).", "Full spectra of the cells were obtained, using as background the spectrum of culture medium, on the coated or uncoated Ge as stated.", "Water vapour peaks were subtracted from each spectrum using previously obtained spectra of water vapour, by capturing a sample-free spectrum in ambient conditions against a background with the environment dried with desiccant.", "AFM was carried out in tapping-mode using Bruker (ex-Veeco) Dimension ICON high-end SPM, Nanoscope V controller (Veeco, Cambridge, UK) with a Silicon AFM tip of resonant frequency of 160 kHz and a force constant of 5.5 N/m, to image an area of 93 μm × 93 μm with a resolution of 512 × 512 pixels and a scan rate of 0.7 Hz.", "All AFM images were performed in air.", "Surface roughness parameterroot mean square (Rq) was calculated, as well as the 95% maximum depth.", "(Bruker’s Nanoscope V and Microsoft® Excel 2010) The 95% maximum depth was obtained by excluding the 2.5% lowest and highest measurements as possible outliers.", "All measurements were taken in triplicate and results are presented as average ± standard deviation.", "Microsoft® Excel 2010 was used and the results were expressed as means ± standard deviation.", "Additional analysis of variance (ANOVA) was performed with P = 0.05.", "PCA was carried out using in-house software developed for analysis of spectra for a previous study [26], using the python programming language with numpy and scipy for calculations, and matplotlib for visualization.", "Spectra were truncated to 2000 cm−1 and 900 cm−1 wavenumber followed by baseline correction before the PCA.", "FTIR measurement results of the cells attaching and growing on the multi-bounce Ge ATR element are shown in Fig. 1 where culture medium was used as the background spectrum to highlight the attachment and growth of the cell on the Ge surface.", "During the first few hours after seeding the cells on the bare Ge ATR element, the absorbance associated with cellular materials was increasing (Fig. 1A) demonstrating that the cells were attaching and spreading on the ATR surface, forming and achieving a good contact.", "The spectral data obtained showed similarities to a previous study where breast cancer cells were grown on an ATR surface [27].", "A separate study has shown that due to the small probing depth of ATR measurements, there will be no significant absorbance from the cells if they are not attaching to the ATR surface even if they sediment to the surface of the ATR element [26].", "The absorbance of the cells reached a plateau after 60 mins (Fig. 1B) marking the end of the cell attachment process.", "After the attachment of the cells, there is a continual but small increase in the absorbance of the cell spectrum from cell growth (Fig. 1B), as observed in a previous study [19].", "All these observations are similar to the cells grown in tissue culture flasks and previous results from multi-bounce ATR study of HeLa cells using a ZnS ATR element [26].", "Fig. 2A shows the FTIR spectra of the cells with a clean Ge element as the background.", "The spectrum of the seeded cell immediately after seeding shows mostly the spectrum of water in the medium because cells were not yet attached.", "The spectrum of living cells in medium can be clearly seen 5 h after seeding which has a baseline that is similar to the spectrum measured immediately after seeding.", "However, approximately 20 h after seeding the living cells, a significant increase of a non-specific absorbance, in the form of a baseline shift, can be observed which continued throughout the experiment.", "After 3 days of cell culturing, the non-specific absorbance or baseline lift reached over 0.2 at 4000 cm−1 (Fig. 2).", "The baseline shift, highlighted by the difference spectra between the cells seeded for 5 h and 72 h is shown in Fig. 2B and is compared to a control measurement when medium without cells was incubated.", "The difference spectrum shows amide I and amide II bands on a strong baseline which resembles a typical feature of a non-specific scattering where a greater loss of IR intensity is found on the shorter wavelength than the longer wavelength.", "The difference spectrum showed a small decrease in the OH stretching mode absorbance because as the cells continue to grow, as shown by the increase in amide II band, the amount of water within the sampling volume decreases.", "The image of the surface of the Ge ATR element captured after removal of the cells by trypsin (Fig. 3) shows large degree of damage by the growing cells.", "The mechanism of the attack of Ge by living cells could be an interesting topic of biochemical study but it is beyond the scope of this work.", "The damage by the cells created a roughened surface on the Ge ATR element which increased the scattering loss of IR radiation.", "A reduction of the overall throughput of IR light through the Ge ATR element by ∼40% can be observed even when the cells were removed and the Ge surface has been cleaned, showing that the permanent loss of throughput is related to the damage of the ATR surface by the cells.", "This is also supported by the fact that on other materials that are not damaged by cell culturing the decrease of throughput is not observed, as can be seen on the spectral data of previous studies from our group using HeLa cells on a ZnS ATR element [26].", "The lack of scattering loss in the first 20 h of incubation indicates that the damage of Ge by living cells happens after the cells have been attached for over ∼20 h which explained why this damage was not observed in previous studies [21] where a much shorter duration of cell incubation was used.", "However, many drug-cell interaction studies expose cells to drug in a period of 48 h or longer.", "A method to reduce cellular damage to the optical material is needed for the future application of micro ATR imaging study of drug-cell interactions.", "The method proposed here to protect the Ge surface from damage is through coating the germanium with materials that would also enable cell attachment and cell measurement by ATR-FTIR.", "Three materials used commonly in tissue culture were chosen and HeLa cells used as an example which is shown to be able to attach and grow on Ge coated with gelatine, collagen or fibronectin, as shown in Fig. 4 where the images were captured 48 h after seeding.", "ATR-FTIR spectra of the coatings were collected after the drying step, before and after adding culture medium.", "As it can be seen in Fig. 5, the spectrum of each of the three coatings was higher when the coatings were dry than when the medium was added, which is due to the dissolution of the coating material (water-soluble protein) in the medium.", "However, the absorbance remained at 0.05 after 24 min which is higher than what is expected should the protein dissolve completely (if the protein dissolved completely the concentrations should be 490 μg/mL for gelatine, 12.4 μg/mL for fibronectin and 2.45 μg/mL for collagen, all of which resulted in an absorbance of lower than 0.005 using the same accessory), which means that a thin layer of adsorbed protein remained on the Ge element.", "The spectra of the three coatings 24 min after the medium was added reached very similar amide II absorbance values, suggesting that the coatings have similar final thickness on the Ge element in the medium (Fig. 5B), in spite of having been applied at different concentrations.", "From the morphology of the cell it is clear that HeLa cells proliferated on the Ge surface to high confluence showing no signs of toxicity by the bare germanium or by germanium with any of the coatings.", "Trypan blue was added to the cell culture and as can be seen in Fig. 4 where the cells attached to the surfaces, most cells are not stained indicating that they are not undergoing apoptosis.", "Note that the main difference observed in the images between Fig. 4A and B–E are due to the different background.", "Fig. 4A was measured against the transparent background of the tissue culture flask whereas Fig. 4B–E were measured against the reflective background of Ge which resulted in the higher contrast.", "The stained cells over the Ge discs show very dark round structures with a small light spot in the centre while on the tissue culture flask, they were shown with a much lighter spot in the centre.", "Other darker cells on top may be due to the ageing of the culture, as it was confluent for approximately 24 h.", "Cells were cultured 6 times in newly polished uncoated and coated Ge windows (as in Fig. 4), and after each time the cells were removed with trypsin and the surfaces were recoated for re-seeding.", "This is to show if the damage on Ge can be prevented for multiple usage.", "Exposing the newly polished Ge window in trypsin for 5 min did not show any observable effect on the surface.", "After the 6th culture, the surfaces were washed and observed in the optical microscope.", "As shown in Fig. 6, marks were visible where the cells were anchored, especially in the bare window (Fig. 6A).", "These marks have been noticed before (Ge crystal used for several ATR cell experiments shown in Fig. 3) and increase light scattering, and consequently the noise in the spectrum.", "Similar marks can be observed on the Ge coated with fibronectin and collagen but appeared to be less extensive in the latter.", "Ge coated with gelatine (Fig. 6C) showed the smoothest surface which has a similar appearance to the control Fig. 6B. To quantify the roughness of the germanium plates, the plates were analysed by AFM after the 6th culture (Fig. 7).", "It can be seen from the image that the bare surface showed the largest variations in height across the imaged area while the coated surface showed smaller variation in height after cell culturing.", "The size of these defects from peak to trough as shown in the AFM images is on the order of 15 μm.", "The roughness was quantitatively compared by the parameters Rq (root mean square of values) and the 95% highest depth (Fig. 8).", "The 95% highest depth consists in the difference between highest and lowest values, discounting the 2.5% highest and 2.5% lowest values to account for possible outliers due to noises.", "The values observed approach a normal distribution.", "All calculations were based on non-smoothed AFM data because the roughness was found to be dominated by the micron-sized features.", "Smoothing of the AFM maps, which removes the nano-sized roughness, did not result in significant difference in the calculated values.", "After being subjected to cell culture for 6 times, both Rq and 95% largest depth are lower for the coated Ge plates compared to the control plate.", "Fig. 8 showed that the Rq and 95% largest depth values for the gelatine coated Ge are similar to the plate treated with no cells.", "The result has shown that, while fibronectin and collagen may protect to a certain extent against the living cells, gelatine offers the best protection with the roughness measurement values similar to the plate without cells grown on top.", "This is in agreement with the visible images shown in Fig. 7.", "This suggests that the gelatine coating could protect the surface of the germanium crystal for multiple exposures to the culture conditions.", "In fact, scratches in the uncoated germanium surface, not submitted to cell culturing, were observed in the optical microscope and can be seen in Fig. 6B, while in the gelatine coated plate these scratches seem less evident (Fig. 6C).", "The depth of the marks on the surface measured by AFM also confirms that the depth of the deepest scratches in the uncoated window not subjected to cells was 17 nm higher than the deepest marks on the gelatine coated surface (Fig. 8).", "As the purpose of coating the germanium is to protect ATR crystals during FT-IR measurements, the coatings were tested to confirm that not only the cells could attach to the coated Ge surface, but also the spectra of the live cells could be obtained without strong interferences.", "As shown in Fig. 9A and B, the spectra of the cells are similar in the presence of any of the coatings under evaluation and with the uncoated germanium ATR crystal (control) showed <10% stronger absorbance of the cell and an elevated baseline.", "The results show that the coating is thin enough to allow majority of the signal from the living cells to be measured and offers protection to the Ge surface.", "HeLa cells grown on a gelatine-coated ATR surface were treated with 1 μM doxorubicin and subsequently analysed for 21 h, acquiring 1 spectrum/h.", "The concentration of 1 μM was chosen as for its clinical significance, as it is the therapeutic concentration of doxorubicin in the bloodstream [28].", "As can be observed in Fig. 9C, the difference spectra from the moment the drug was added resemble the spectrum of the whole cells, suggesting that the cells are detaching from the ATR crystal as all the absorbance due to their components are decreasing in the same proportion.", "These differences were confirmed by analysing the spectral data by PCA (Fig. 9D and E).", "PC1 shows that the overall absorbance of the cell decreased rapidly in the first 7 measurements (7 h) followed by smaller changes, represented by PC2, in the next 17 h.", "The results are in agreement with the previously reported for doxorubicin on uncoated ZnS surfaces [26], demonstrating that the coating did not affect the reaction of the cells to doxorubicin and the ability of ATR FTIR to observe the cellular changes in situ.", "The coating of germanium ATR elements would therefore allow microspectroscopy studies of live cells with improved spatial resolution.", "In recent years FTIR imaging studies have shown the potential of this technique in obtaining biochemical profiles of cells for biomedical sciences.", "Significant changes were found in cells in fixed tissue related with the response of melanoma cells to a chemotherapy drug [29], distinguishing cancer cells in lymph node metastases [3], distinguishing different types of healthy cells from breast tissue as well as differentiating cancer and healthy cells [2].", "The possibility of imaging living cells cultured directly on a Ge ATR element will allow in situ high-resolution measurements through a time course, resolving where and when the changes occur, and avoid possible artefacts due to the fixation and drying process.", "The present study suggests that the use of gelatine coatings can decrease the damage of Ge surfaces by cell cultures, enabling the analysis of cells growing on Ge surfaces for several days, and the repeated use of the Ge crystal for that purpose, as the cost of the optical materials, especially for micro-ATR measurements, is generally an important factor in spectroscopic studies.", "Germanium surfaces coated with tissue culture coating material prevents damage by culturing live cells in live cells ATR-FTIR studies.", "Gelatine, fibronectin or collagen coated Ge surfaces of optical elements enable cells to anchor and grow, and to take FT-IR measurements of the cells without noticeable interference.", "Gelatine coating showed the most efficient protection after repeated use, preventing not only the damage by the live cells but also the appearance of scratches in Ge surfaces." ]	[]
Cell structure, stiffness and permeability of freeze-dried collagen scaffolds in dry and hydrated states	Scaffolds for tissue engineering applications should be highly permeable to support mass transfer requirements while providing a 3-D template for the encapsulated biological cells. High porosity and cell interconnectivity result in highly compliant scaffolds. Overstraining occurs easily with such compliant materials and can produce misleading results. In this paper, the cell structure of freeze-dried collagen scaffolds, in both dry and hydrated states, was characterised using X-ray tomography and 2-photon confocal microscopy respectively. Measurements have been made of the scaffold's Young's modulus using conventional mechanical testing and a customised see-saw testing configuration. Specific permeability was measured under constant pressure gradient and compared with predictions. The collagen scaffolds investigated here have a coarse cell size (∼100-150 μm) and extensive connectivity between adjacent cells (∼10-30 μm) in both dry and hydrated states. The Young's modulus is very low, of the order of 10 kPa when dry and 1 kPa when hydrated. There is only a single previous study concerning the specific permeability of (hydrated) collagen scaffolds, despite its importance in nutrient diffusion, waste removal and cell migration. The experimentally measured value reported here (5 × 10-10 m2) is in good agreement with predictions based on Computational Fluid Dynamics simulation and broadly consistent with the Carman-Kozeny empirical estimate. It is however about three orders of magnitude higher than the single previously-reported value and this discrepancy is attributed at least partly to the high pressure gradient imposed in the previous study. Statement of Significance The high porosity and interconnectivity of tissue engineering scaffolds result in highly compliant structures (ie large deflections under low applied loads). Characterisation is essential if these scaffolds are to be systematically optimised. Scaffold overstraining during characterisation can lead to misleading results. In this study, the stiffness (in dry and hydrated states) and specific permeability of freeze-dried collagen scaffolds have been measured using techniques customised for low stiffness structures. The scaffold cell structure is investigated using X-ray computed tomography, which has been applied previously to visualise such materials, without extracting any structural parameters or simulating fluid flow. These are carried out in this work. 2-photon confocal microscopy is used for the first time to study the structure in hydrated state.	[ "BIOC", "ENGI", "MATE" ]	[ "Cell interconnectivity", "Cell structure", "Collagen scaffolds", "Specific permeability", "Specific surface area", "Young's modulus" ]	[ "1303", "1305", "1312", "2204", "2502" ]	[ "In tissue engineering, a highly porous permeable scaffold is required to provide appropriate void space for mass transport, for neovascularisation and to act as a template for de novo tissue formation.", "Many (natural and artificial) scaffolds are based on a collagenous matrix – collagen is the most abundant protein in the extracellular matrix – and they have a number of attractive features [1–5].", "However, collagen is a particularly challenging material to fabricate into fine structures, due to its hydrophilicity.", "Freeze-drying is a well-established method for producing collagen-based scaffolds with architectures complementary to tissue engineering applications [6].", "These collagen scaffolds have often been combined with glycosaminoglycans (GAGs), which are also constituents of the extracellular matrix.", "They have been found to promote angiogenesis and reduce foreign body reactions [7].", "Collagen scaffolds have been characterised in various ways in both dry and wet (hydrated) states, although of course they are always hydrated in vivo.", "They can be termed “cellular”, meaning that their structures are divided up into cells of some sort.", "A summary of the techniques employed for characterising the cell architecture, stiffness (Young’s modulus) and permeability of collagen-GAG freeze-dried scaffolds is shown in Table I.", "The scaffold structure, particularly the cell architecture, is very important, since it can influence both the biological response [8–11] and the mechanical and transport properties [12–17].", "The cell structure can be manipulated by varying the freeze-drying conditions.", "For example, by varying the freezing temperature, the cell size can be tailored [18].", "Introducing thermal gradients during the freezing process can lead to anisotropic cell structures [8,19,20], and hence affect biological cell organisation and scaffold properties.", "The cross-linking method [14,21] has also been found to affect significantly the Young’s modulus of the scaffold and biological cell response.", "Cross-linking typically involves either a physical dehydrothermal-based (DHT) or a chemical, carbodiimide-based (EDAC) process.", "Measured [12,13,16,21–24] Young’s moduli of freeze-dried (wet and dry) scaffolds tends to be very low – typically ∼1–40 kPa, with values usually towards the lower end of the range when wet (hydrated) and larger when dry.", "The main reason for (all) values being so low (i.e. orders of magnitude below those typical of rubbers), apart from the very high porosity level (often ∼95–99%), is that elastic deformation occurs primarily via bending of slender structural elements, such as cell walls.", "This allows large deflections under low applied loads – i.e. generates a low stiffness – and this is the basis of many types of (highly compliant) fibre network material [25–33].", "(There is, however, always the possibility of some kind of inelastic deformation occurring, as a result of plasticity or damage in the cell walls, giving an anomalously low stiffness: it is essential when measuring such stiffness to check that the deformation is genuinely reversible.)", "Rather surprisingly in view of its significance, there are relatively few experimentally-measured values reported in the literature for the specific permeability of scaffolds of this type.", "Furthermore, the values that have been reported cover a wide range, and some are very small indeed.", "The only data relating to collagen scaffolds of the type being investigated here appears to be that of O’Brien and co-workers [10], who reported values in the vicinity of 10−13 m2.", "This is a very low permeability, particularly in view of the fact that it relates to a cell structure that is relatively coarse and appears to be fairly open and inter-connected.", "In the present paper, the cell structure of freeze-dried collagen scaffolds is investigated, in both dry and hydrated states, and measurements are made of their Young’s modulus (E) and specific permeability (κ).", "Efforts are made to minimise the sample strain during these measurements, since excessive straining occurs easily with such highly compliant material and can produce misleading results.", "The inertia of the testing equipment is determined by the mechanical linkage system that is to be moved.", "In conventional testing machines, such effects can be significant.", "A customised see-saw set-up allowing such effects to be minimised has been designed here.", "The cell size and connectivity, and specific surface area of the scaffolds are explored using X-ray computed tomography.", "This technique has only been applied previously to such materials to visualise their architecture, without extracting any structural parameters or simulating deformation or fluid flow: all of these operations are carried out in the present work.", "To study the structure in hydrated state and approximate the in vivo situation, 2-photon confocal microscopy, involving laser-excited fluorescence and second harmonic signals, is used.", "The collagen-glycosaminoglycan (GAG) suspension was fabricated using a previously-described protocol [11].", "All chemicals were obtained from Sigma Aldrich.", "Insoluble type I collagen (0.5 wt%) and chondroitin sulphate salts (0.05 wt%) were suspended in 0.05 M acetic acid and homogenised at 15,000 rpm using a T81 UltraTurrax homogeniser (IKA, Germany).", "The suspension was poured into an aluminium mould and sealed in place.", "The mould suspension was then freeze-dried (Virtis advantage, SP Industries, USA), by cooling to −40 °C at a rate of −0.9 °C min−1, held for 60 min and then sublimated under a vacuum of 50 mTorr at 0 °C for 17 h. Subsequently, the scaffold was cross-linked in a 48-well tissue culture plate for 4 h, using the chemical 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC), alongside the catalyst N-hydroxysuccinimide (NHS) at a molar ratio of 2.5 M EDAC/M NHS.", "The cross-linking solution had a concentration of 6 mM EDAC per gram of collagen.", "The relative density of the collagen-GAG (termed CG) scaffold, ρ∗/ρs, was calculated from the dry density of the CG scaffold after freeze drying (ρ∗) and the known [11] dry density of solid collagen (ρs = 1.3 Mg m−3).", "The GAG was assumed to have a minimal contribution.", "The scaffold diameter and height were measured using a Keyence® dual-axis laser micrometer (model: TM-040, resolution of ±3 μm).", "Scanning electron microscopy (SEM), X-ray computed tomography (CT) and 2nd harmonic 2-photon imaging were used to investigate the scaffold cell structure.", "For SEM, the scaffolds were sliced with a scalpel, before being hydrated and freeze dried again, to ensure that the cells were not deformed.", "The scaffolds were then mounted, gold sputter-coated and imaged, using a Jeol-5800 SEM in secondary mode.", "These SEM images were thresholded, using a customised Matlab code, and the centre of each cell was computed.", "An ellipse-fit method was used and an equivalent cell diameter obtained, using an equivalent iso-volume sphere.", "About 250 cells were analysed.", "Fig. 1 shows typical SEM images at different magnifications, illustrating the pronounced “cellular” structure and a wide range of apertures (gaps) in the cell walls.", "The cell wall thickness appears to be of the order of 1 μm.", "For CT, two samples (10 mm diameter, 10 mm height) were punched from larger scaffold sheets, designated as CG-1 and CG-2.", "Tomography scans, with 3.4 μm linear resolution (voxel resolution 3.4 × 3.4 × 3.4 μm3), were acquired using a General Electric Phoenix X-ray Nanotom m system (GE Sensing & Inspection Technologies GmbH, Munich, Germany) equipped with a sub-micron focal spot X-ray source.", "The source voltage and current were set at 80 kV and 150 μA.", "A Mo target was used.", "The scans were recorded at projection angles between 0° and 360°, in steps of 0.2°.", "To increase the signal to noise ratio, 4 frames were captured and averaged for each projection on a 7.2 megapixel 14-bit detector array.", "To avoid edge effects, a sub-volume of 4 × 4 × 4 mm3 was analysed and, within this volume, 15 sub-sections (683.4 × 683.4 × 683.4 μm3) were analysed.", "These data were imported into myVGL2.2 (Volume Graphics GmbH), to divide the CT data into an image stack for further analysis.", "For the 2-photon work, four samples (5 mm diameter, 4 mm height) were punched from larger scaffold sheets.", "From each sample, one sub-volume of 1200 × 1200 × 250 μm3 (length, width and thickness respectively) was analysed.", "The samples were suspended in distilled water and imaged using a LaVision BioTec TriM Scope II system with an Insight DeepSee dual-line tunable laser equipped with a 25× water-immersion lens with a numerical aperture of 1.05.", "The laser was tuned to 880 nm and a power of 50 mW, to cause excitation of the 2nd harmonic of the collagen.", "The reading was obtained using a red-enhanced GaAsP detector, after the response was low pass filtered to allow collection of wavelengths below 510 nm.", "To increase the signal to noise ratio, the images were line-averaged twice.", "Voxel resolution was 3.13 × 3.13 × 3.13 μm3.", "CT and 2-photon datasets were analysed as follows.", "First, median filtering was applied to smooth the 3-D images, using a 3 × 3 × 3 kernel.", "The image stack was thresholded, using a customized Matlab program.", "To threshold the CT data, a threshold level was defined to give a 99.5% porous structure as calculated from experimental measurements.", "Any voxels of intensity below the threshold were assumed to represent void space whereas those above were scaled so the varying intensity of pixel represented the varying density of collagen.", "Fully saturated voxels represented 100% dense collagen whereas those just above the threshold level represented only 1% dense collagen.", "3-D cells were objectified using a voxel-growth constrained algorithm (Avizo Fire 3-D software).", "Each individual cell was then imported into Matlab, to calculate the cell volume, which was converted to an effective diameter using an equivalent iso-volume sphere.", "Fig. 2(a)–(c) show respectively a typical 2D tomographic reconstruction before and after thresholding, and after cell fitting.", "Comparing the cell-fitted structure to the original one, estimates of the cell interconnectivity (aperture size) were made.", "This involved working out the length (in μm) of each cell boundary and approximating it to a circular aperture – see Fig. 2(c).", "(Because of the image processing that has been carried out to clarify the cell structure, the cell walls appear thicker than they actually are.)", "The scale of the structure was characterised via the specific surface area, S, which was measured using a Brunauer–Emmett–Teller (BET) gas adsorption isotherm method on an ASAP 2020 Porosimeter (Micromeritics Instrument Co, Norcross, USA).", "Four scaffolds were sliced from a cast sheet, to give an approximate weight of around 0.2 g.", "The samples (scaffolds) were then re-suspended and washed in distilled water before re-freeze drying under the previously described protocol to give an undeformed clean sample.", "The sample was then placed into a long neck, round bottom sample tube and loaded into sample ports and evacuated to a high vacuum (2 μmHg).", "Nitrogen adsorption measurements were taken at 77 K, up to a pressure of 230 mmHg (pressure of 0.3 bar).", "Estimates of S were also obtained from 3-D tomographic and 2-photon confocal reconstructions.", "The CT and 2-photon images obtained were thresholded, as described in Section 2.2.5, and converted to a 3-D surface reconstruction using Avizo Fire (version 7.0).", "The total surface area of the scaffold was then measured and converted to a specific surface area (in m−1) by dividing by the total scaffold volume (scaffold plus void space).", "Cylindrical samples freeze-dried to dimensions of 7 mm (height) × 9 mm (diameter) were used for compressive testing.", "Compressive stress–strain curves were obtained using a screw-driven desktop Instron machine, fitted with a 5 N load cell.", "Three samples were tested in both dry and hydrated conditions.", "Tests were carried out under displacement-control, at a strain rate of ∼10−4 s−1.", "Displacement was measured using a Keyence® bi-axial laser micrometer.", "In the hydrated condition, scaffolds were immersed in Phosphate Buffer Solution (PBS) for 24 h, to ensure full hydration, and then remained immersed through testing.", "The through-thickness Young’s modulus was measured from the tangent slope of the unloading stress–strain curve (within the elastic regime).", "Four samples were tested in both dry and hydrated conditions, from which the average value was taken and standard deviation calculated.", "A customised “see-saw” set-up, with a lower inertia compared to a standard Instron machine, has been designed to measure the through-thickness Young’s modulus of the scaffolds – see Fig. 3.", "The set-up has a central pivot resting on a frictional support base that perfectly balances the free extending arms on either side.", "The end of one arm is fixed, with a small container to hold the liquid and transfer the load into the collagen sample aligned beneath a flat loading platen.", "Counter-weights are added in the hanging basket fixed to the other arm, in order to balance the arms such that the loading platen touches the sample surface.", "The load was ramped up at a constant rate of ∼10−4 N s−1, by pumping liquid (via a peristaltic pump) into the container.", "The resulting displacement was monitored using a Keyence® bi-axial laser micrometer.", "Four samples from each condition (dry and hydrated) were tested, from which the average value was taken and standard deviation calculated.", "Collagen scaffolds were scanned using a Skyscan 1172, high resolution desktop μCT scanner with a resolution of 810 nm.", "3-D cell and scaffold structures were rendered from (serial section) X-ray radiographs, using the Simpleware ScanIP software suite and built-in segmentation tools.", "A typical visualisation is shown in Fig. 5(a).", "The +FE module of the ScanIP software was used to mesh the cell volume, using about 32 million first order tetrahedral elements.", "The +FLOW module was then used to calculate the permeability of the scaffold, using a built-in Stokes solver (based on laminar flow).", "Boundary conditions included a pressure difference across the scaffold and a no-slip wall condition.", "A typical predicted flow pattern (for the structure of Fig. 5(a)), is shown in Fig. 5(b).", "As expected, higher flow rates are observed in regions where the structure is more permeable – see Fig. 5(a).", "The scaffolds used had a relative density ρ∗/ρs of 0.5% (porosity content ∼99.5%), assuming a value of 1.3 Mg m−3 for ρs [11].", "This value is probably an overestimate; such high porosity levels are rather unusual in a material as opposed to a latticework structure.", "SEM micrographs, such as those illustrated in Fig. 1, suggest that a porosity of around 95% is probably more realistic.", "It can be seen that the cell walls of the scaffolds are thin (∼a few microns), as well as being full of apertures (holes and defects).", "The equivalent cell diameter and cell wall connectivity for the two samples are shown in Fig. 6, as obtained from CT reconstruction analysis.", "The cell diameter is about 100–150 μm (SEM analysis gave a very similar value for the cell diameter – 119 ± 73 μm).", "The distribution of cell connectivity (aperture size) exhibits a peak at around 20 μm.", "There is no systematic difference between CG-1 and CG-2 – the variations just give an indication of sample-to-sample variations.", "In general, the structure is similar throughout.", "The scaffold structure when hydrated – i.e. under conditions close to those in vivo – was investigated using 2-photon confocal microscopy.", "Corresponding data to those in Fig. 6 are shown in Fig. 7.", "Specific surface area values have been obtained using three different techniques.", "Both the CT tomographic reconstruction analysis and the BET gave a value of about 4 × 104 m−1 for dry scaffolds, while the 2-photon optical reconstruction gave a value of about 3 × 104 m−1 for hydrated scaffolds – see Table II.", "It is of note that there is very good agreement between the BET and CT values (both for dry scaffolds), which gives confidence that the order of magnitude is correct.", "Typical compressive stress–strain curves, for dry and hydrated CG scaffolds, are shown in Fig. 8.", "These were obtained using a standard Instron machine.", "The curves suggest that, in the hydrated scaffolds, the cell walls tend to buckle and bend at a lower (plateau) stress, compared to the dry scaffolds.", "Also, the densification strain, above which deformed cell walls start to impinge on each other, is higher for the hydrated scaffolds.", "Young’s modulus values were obtained from the tangent of unloading curves giving 7.6 ± 0.6 kPa when dry and 0.57 ± 0.15 kPa when hydrated.", "A typical stress–strain curve showing load–unloading cycles for a dry scaffold, and corresponding inferred Young’s modulus values is shown in Fig. 9(a).", "Using the see-saw testing configuration, the measured Young’s modulus values were 9.6 ± 0.5 kPa when dry and 0.81 ± 0.08 kPa when hydrated.", "A typical stress-strain curve for a dry scaffold obtained using the see-saw set-up is shown in Fig. 9(b).", "The average measured value of the specific permeability κ was found to be 4.8 × 10−10 ± 2.2 × 10−10 m2.", "This is in good agreement with predictions based on Computational Fluid Dynamics (CFD) simulation of the flow of water through tomographically-captured structures which gave a value of about 2 × 10−10 m2.", "The effect of prolonged flow on the permeability was also assessed.", "Representative specific permeability data are presented in Fig. 10(a), which shows values obtained for three different (but nominally the same) scaffolds over an extended period of measurement (about 4 h).", "It can be seen that there is a tendency for the value to drop after passage of substantial quantities of water through the sample.", "However, in general the variations are relatively small.", "This is attributed to scaffold deformation (creep) even under the small pressure gradient applied in this study.", "In the present paper, the cell structure and specific surface area of freeze-dried collagen scaffolds is investigated, in both dry and hydrated states.", "Measurements are made of their Young’s modulus and specific permeability.", "Young’s modulus and permeability are a strong function of porosity level and cell structure.", "Permeability is also very sensitive to specific surface area.", "The cell diameter of dry scaffolds is about 100–150 μm and the cell connectivity is about 10–30 μm.", "These values are consistent with the general impression from Figs. 1 and 2 and show that the cellular structure is a quite heavily disrupted one, with a lot of defects and gaps (apertures) in the walls.", "By comparing the scaffolds in dry and hydrated states, it can be seen that hydration causes an increase in cell diameter (∼20%), while the connectivity has reduced by about 40%.", "These changes are presumably due to some swelling of the cell walls.", "In general, however, hydration does not appear to cause any dramatic changes in structure.", "CT tomographic reconstruction analysis and BET measurements gave a specific surface area of about 4 × 104 m−1 for dry scaffolds, while 2nd harmonic 2-photon confocal microscopy gave a value of about 3 × 104 m−1 for hydrated scaffolds.", "These values can’t be regarded as highly accurate, but the level of agreement gives confidence that the order of magnitude is correct (and that any change on hydration is relatively small).", "The Young’s moduli of the scaffolds, in both dry and hydrated states, were measured using conventional mechanical testing and a customised see-saw set-up.", "As expected, a drop in stiffness was observed on hydration, probably caused by weakening of hydrogen bonds within the molecular structure of the collagen.", "Fig. 9(a) shows that the Young’s modulus value obtained using a conventional Instron machine can vary, depending on the strain at which it is measured.", "There is a tendency for it to rise with imposed strain, probably due to changes in structure (elastic and/or plastic) that arise as the material is compressed.", "It can be seen in Fig. 9(b) that this effect was somewhat less pronounced with the see-saw testing configuration, even though the stress and strain ranges concerned were similar.", "This may be due to the lower inertia of the see-saw set-up, making it easier to interrupt the testing with minimal hysteresis.", "Furthermore, the see-saw setup is load-controlled (as opposed to conventional loading, which is usually displacement controlled).", "This means that as the loading increases and the scaffold compresses, the loading platen follows the compression so as to maintain the rate of loading.", "Load controlled tests are preferred when conducting tests at a very low rate, because an applied load can be kept constant for any given period of time.", "On the other hand, it can be seen that the values obtained for the Young’s modulus (∼5–10 kPa for dry and ∼0.5–1 kPa for hydrated) are similar with the two types of set-up, and values of at least this approximate magnitude have been found in most previous studies [12,13,16,17,21,22,24].", "The only previous study relating to collagen scaffolds of the type being investigated here [10], reports permeability values of 10−13 m2 – i.e. about six orders of magnitude lower than the Carman–Kozeny estimate.", "This is a very low permeability value, particularly in view of the fact that these scaffolds have a relatively coarse structure and large interconnectivity.", "Other workers have also obtained very low values, but these relate to scaffolds with lower levels of porosity.", "For example, Vikingsson et al. [41] obtained values of ∼10−17 m2 for polymeric scaffolds containing about 10–30% porosity, while Reinwald et al. [42] obtained 10−14 m2 for supercritical fluid-foamed scaffolds with porosity around 50–60%.", "It should perhaps be noted in this context that, with very compliant (low stiffness) materials, there must be a concern that the pressure gradients created during permeability measurements may have deformed the structure, perhaps compressing it to such an extent that the permeability dropped dramatically.", "This could occur even if the deformation were entirely elastic, although obviously plastic deformation might also have occurred.", "It is noteworthy that the permeability measurements of Lipowiecki et al. [43], carried out on much stiffer and stronger artificial bone scaffolds (although with porosity in the range 30–70% and a broadly similar structural scale and architecture to the materials of Vikingsson et al. [41] and Reinwald et al. [42]), gave values much closer to Carman–Kozeny predictions.", "A slightly more systematic comparison with other outcomes is shown in Fig. 10(b), in which specific permeability values are plotted against the solid fraction, (1 − p).", "Data from three other experimental investigations are included, of which only that by O’Brien et al. [10] was carried out on similar material to that of the current work – the other two studies relating to lower porosity structures that can be considered to contain sets of interconnected pores, rather than being “cellular”.", "For all four materials, however, the structural scale is similar, since the pores or cells were all about 50 μm in radius.", "Also shown are predictions from CFD simulation and the Carman–Kozeny equation (Eq. (1)), obtained using S ∼ 3/R.", "Of course, this Carman-Kozeny plot does nothing more than provide a very crude guide, and it should be recognised that they are expected to constitute an over-estimate for cellular structures (depending on the cell connectivity).", "Furthermore, care must be taken when using the Carman–Kozeny equation to predict permeability, since such predictions are highly sensitive to the porosity level, which therefore needs to be known quite accurately.", "Nevertheless, the three sets of values from previous work do appear to be unexpectedly low, particularly the one for the cellular structure.", "The most likely explanation for this is that the imposed pressure gradient (∼3 kPa mm−1) in the previous study [10] compressed the structures significantly, reducing the permeability, and there is no doubt that these cellular structures, which are highly compliant (E ∼ 10 kPa), will tend to be susceptible to this effect.", "The set-up used in the present work, involving a very low pressure gradient (∼10 Pa mm−1), was specifically designed to reduce this danger.", "In addition, a press-fit mount was used in contrast to the clamp mount employed in the previous study [10].", "The latter caused significant scaffold deformation, reducing the permeability.", "Collagen scaffolds are used as 3-D platforms for both in vitro and in vivo studies of cellular interactions and tissue biosynthesis, as well as in clinical repair of a number of tissues.", "Structure and property characterisation is essential if these scaffolds are to be systematically optimised.", "This paper outlines a procedure for quantifying the cell structure of freeze-dried collagen scaffolds in both dry and hydrated states using X-ray computed tomography and 2nd harmonic 2-photon confocal microscopy respectively.", "Measurements have been made of the scaffold’s Young’s modulus and specific permeability.", "The freeze-dried collagen scaffolds used in this study have an approximately isotropic cell structure and extensive connectivity between adjacent cells in both dry and hydrated states.", "This clearly has implications for both the stiffness and the permeability.", "The scaffold stiffness is very low, about 10 kPa when dry and 1 kPa when hydrated, and these values are consistent with most previous testing of similar scaffolds.", "There are far fewer data in the literature concerning the specific permeability of collagen scaffolds, but the value reported here is consistent with the CFD simulations, although it is about three orders of magnitude higher than the only previously-reported figure.", "A likely explanation for this discrepancy is that in the previous work the applied pressure gradient (which was substantially greater than the one employed here) and the scaffold mounting arrangement caused significant compression of the structure, reducing its permeability." ]	[]
Can high resolution 3D topographic surveys provide reliable grain size estimates in gravel bed rivers?	High resolution topographic surveys such as those provided by Structure-from-Motion (SfM) contain a wealth of information that is not always exploited in the generation of Digital Elevation Models (DEMs). In particular, several authors have related sub-metre scale topographic variability (or 'surface roughness’) to sediment grain size by deriving empirical relationships between the two. In fluvial applications, such relationships permit rapid analysis of the spatial distribution of grain size over entire river reaches, providing improved data to drive three-dimensional hydraulic models, allowing rapid geomorphic monitoring of sub-reach river restoration projects, and enabling more robust characterisation of riverbed habitats. However, comparison of previously published roughness-grain-size relationships shows substantial variability between field sites. Using a combination of over 300 laboratory and field-based SfM surveys, we demonstrate the influence of inherent survey error, irregularity of natural gravels, particle shape, grain packing structure, sorting, and form roughness on roughness-grain-size relationships. Roughness analysis from SfM datasets can accurately predict the diameter of smooth hemispheres, though natural, irregular gravels result in a higher roughness value for a given diameter and different grain shapes yield different relationships. A suite of empirical relationships is presented as a decision tree which improves predictions of grain size. By accounting for differences in patch facies, large improvements in D50 prediction are possible. SfM is capable of providing accurate grain size estimates, although further refinement is needed for poorly sorted gravel patches, for which c-axis percentiles are better predicted than b-axis percentiles.	[ "EART" ]	[ "Grain size", "Gravelometry", "Roughness", "Structure-from-motion" ]	[ "1904" ]	[ "The interaction of river channel morphology and hydraulics at the sediment-water interface influences both water and sediment fluxes through fluvial systems as well as in-stream ecological processes by providing habitat (Rice et al., 2010).", "As such, understanding and manipulating these interactions are often the focus of river restoration activities (Palmer et al., 2014).", "Bed material size represents one of the smallest scales of topographic variability within river channels.", "Analyses of grain size variability can yield information on flow history, sediment origins and patterns of previous transport and deposition (e.g., Folk and Ward, 1957; Friedman, 1979; Bui et al., 1989).", "Information on bed material size is essential for hydraulic modelling (Smart et al., 2004) and is required to predict boundary shear stress and sediment transport (e.g., Wilcock and Crowe, 2003) in morphodynamic models (e.g., Langendoen et al., 2016).", "Bed material size is also an influential determinant of the availability of suitable spawning habitat for fish and habitat for macroinvertebrates (Armstrong et al., 2003; Fryirs, 2015).", "Gravel bed rivers exhibit marked reach-scale variability in grain size distributions (e.g., Church and Kellerhals, 1978; Nelson et al., 2014), which necessitates sampling over large areas to represent accurately the heterogeneity of grain sizes (Church et al., 1998).", "Conventional and well-established methods of grain size analysis (e.g., Wolman, 1954), although relatively reliable (despite known biases; Hey and Thorne, 1983; Bunte and Abt, 2001), are limited in their ability to provide high resolution information of grain size variability owing to the time-consuming nature of physical sampling.", "Both Wolman pebble counts and even more laborious areal sampling methods are destructive techniques which limits the potential for repeated surveys and disrupts the habitat they are intended to quantify.", "To address the clear need for a fast, accurate and reproducible grain size measurement technique (Woodget and Austrums, 2017), recent studies have developed new methods of obtaining grain size information from remotely sensed data.", "Summary values of local grain size (e.g., D84 or D50, the size at which 84% or 50% of measured b-axes are finer) have been estimated from photographs (so-called ‘photosieving’ methods) using semivariograms of image texture (e.g., Carbonneau et al., 2004; Verdú et al., 2005), the autocorrelation of image intensity (e.g., Rubin, 2004; Barnard et al., 2007; Warrick et al., 2009) or other statistical properties of images (see Buscombe and Masselink, 2009; Buscombe and Rubin, 2012).", "Others have sought to identify and measure individual grains within an image (e.g., Graham et al., 2005; Detert and Weitbrecht, 2012), allowing the full grain size distribution to be estimated.", "While photosieving methods focus on the planimetric dimensions of grains, parallel developments have utilized the increased availability of high resolution Digital Elevation Models (DEMs) and other topographic data products to estimate the vertical scale of topographic variability attributable to grain size.", "Survey methods such as Airborne Laser Scanning (ALS) and Terrestrial Laser Scanning (TLS) can provide decimetre and millimetre scale 3D survey point accuracies, respectively (Gallay, 2013; Smith, 2015).", "In particular, TLS has been used at scales from individual grains and small patches of 100–101 m2 (e.g., Hodge et al., 2009a), to fluvial landforms of 102–103 m2 (e.g., Leyland et al., 2015), and even to reaches of 106–107 m2 (e.g., Williams et al., 2014).", "More recently, Structure-from-Motion with Multi-View-Stereo (SfM-MVS) methods have arisen as a flexible, low-cost alternative to TLS (Smith et al., 2015; Eltner et al., 2016; Carrivick et al., 2016) and have gained considerable traction in fluvial applications (e.g., Javernick et al., 2014; Smith et al., 2015; Woodget et al., 2015; Marteau et al., 2016).", "The focus of many fluvial geomorphic surveys is the broad-scale topography and quantification of surface changes (e.g., Milan et al., 2011).", "However, the high spatial resolution of TLS and SfM-MVS data has enabled sub-grid scale surface roughness to be investigated as a proxy for grain size (i.e., topographic scales smaller than the resolution of Digital Elevation Models which are typically decimetre-metre scale in fluvial studies) (Smith, 2014).", "The development of an empirical relationship between surface roughness and grain size essentially reverses the established practice of using measured grain size percentiles to estimate roughness height for use in flow resistance equations (e.g., Strickler, 1923; Clifford et al., 1992; Wilcock, 1996; Powell, 2014).", "These relationships, however, have been confounded by the range of particle sizes, shapes and packing-structures (e.g., imbrication) observed in natural alluvial channels (Furbish, 1987; Powell, 2014; Vázquez-Tarrío et al., 2017).", "Moreover, the presence of bedforms and multiple scales of topographic variability limits the usefulness of particle axes measurements to estimate flow resistance (Robert, 1990; Gomez, 1993; Smart et al., 2004).", "To some extent, the same confounding factors will hinder attempts to estimate grain size from surface roughness.", "The complex geometry of fluvially-formed surfaces cannot be described fully by a finite number of parameters (Smith et al., 2011) and as such no single property or parameter can be defined uniquely as surface roughness (Smith, 2014).", "Nevertheless, a single roughness metric has dominated the geomorphological literature for several years: the standard deviation of elevations (σz) (e.g., Grohmann et al., 2011; Brasington et al., 2012).", "Studies that relate sub-grid surface roughness to grain size (typically D50) also preferentially use σz, often detrended at the grid-scale (Smart et al., 2004; Aberle and Nikora, 2006; Hodge et al., 2009b; Brasington et al., 2012; Schneider et al., 2015; Westoby et al., 2015; Bertin and Friedrich, 2016) resulting in a single roughness value.", "However, estimates of grain size from surface roughness have not been subject to the same rigorous evaluation as photosieving approaches (e.g., Graham et al., 2010).", "Fig. 1A summarises the scatter observed across all these previously published studies; with all data combined an R2 of 0.49 is observed (n = 97; p < 0.001) though the 95% confidence intervals of the gradient (from 1.19 to 1.81) are sufficiently wide to preclude accurate prediction across a range of facies types.", "Table 1 provides details of each of these studies; while fluvial gravels dominate the literature, glacial moraines have also been sampled (e.g., Westoby et al., 2015) reflecting the fact that grain size classification is of benefit in a wide range of process environments beyond fluvial gravels.", "TLS data have dominated such studies, though the most recent studies have used SfM.", "Direct comparison of these results is complicated further by the different grain size measurement methods employed (which may require correction factors; Bunte and Abt, 2001; Graham et al., 2012), the different patch sizes (and hence different detrending scales) and the differences in methods used to extract roughness data.", "At individual study sites, linear relationships between D50 and σz are observed (typically R2 ~ 0.9; Table 1).", "Brasington et al. (2012) observed a strong linear relationship between σz and D50 for cobble-sized material at the River Feshie in Scotland (R2 = 0.92) that improved (R2 = 0.95) when additional data from Wales (beach sediment) and New Zealand (braided river) were added, though the parameters of the regression equation were different (Table 1).", "Conversely, on poorly sorted glacial sediment, Westoby et al. (2015) observed no significant relationship between σz and D50.", "Table 1 also describes studies that used two alternative roughness metrics for grain size prediction.", "Heritage and Milan (2009) avoided detrending patches over large scales by summarising the localised standard deviation of elevations within a moving window, equivalent in size to the largest clast (0.15 m in this case) and multiplied the result by two to generate the effective roughness equivalent (a convention maintained herein).", "Comparison of percentiles of the resulting roughness metric (given the notation 2σloc herein) with grain size percentiles yielded strong relationships in eight individual patches.", "To facilitate comparison with other studies, the median value of 2σloc for each patch has been plotted in Fig. 1B and a relationship between that and D50 summarised in Table 1.", "Baewert et al. (2014) used a similar approach (on c-axis data) with a smaller window size (~ 50 mm) while Vázquez-Tarrío et al. (2017) used a grid size of 1 m. Comparison of these D50-2σloc relations shows that they plot as expected with larger window sizes yielding higher roughness values; indeed Baewert et al. (2014) demonstrated this directly.", "Finally, Woodget et al. (2016) and Vázquez-Tarrío et al. (2017) outlined a third roughness metric (rh) which summarises the deviation from a fitted plane of all points within a given radius (the former study used the mean value within 0.2 m, the latter study used the median value within 0.5 m).", "In a comparison of all three roughness metrics, Vázquez-Tarrío et al. (2017) found rh to provide the best prediction of D50.", "While Woodget et al. (2015) plotted rh against D84 (for which there was the strongest linear relationship) rather than D50, this relationship remains very different from the rh-D84 relationship reported in Vázquez-Tarrío et al. (2017) (though note the calculation differences reported above).", "Although there are some differences between roughness metrics, they are fundamentally very similar; each presents a summary of local variability in elevations while filtering out larger wavelength topographic variability.", "For better comparison with the existing literature, herein we focus on σz as the main roughness metric used for grain size prediction; the other metrics are compared briefly for field patches in Section 3.7.", "Comparison of the relationships in Fig. 1A with that of Westoby et al. (2015), obtained over sediment of very different character, highlights that properties of sediment facies other than grain size influence surface roughness (see also the comparison in Fig. 15 of Vázquez-Tarrío et al., 2017).", "It is not surprising, therefore, that each study represented in Fig. 1 obtained a different empirical relationship.", "At a minimum, site-specific calibration of any σz-D50 relationship is required.", "However, a more generalizable approach would be to identify the individual factors that have caused these differences and present a suite of σz-D50 relationships, each one of which is appropriate for a different fluvial sediment facies.", "Such factors include:", "Survey error: low precision survey observations would increase σz and the sources of such error in SfM surveys (e.g., image network geometry, tie point quality, control measurement; see James et al., 2017) are very different from those in TLS surveys (e.g., registration errors, beam divergence and mixed pixels, range errors; see Smith, 2015);", "Particle irregularity: natural deviations of gravels from abstract shapes (e.g., spheres, prolates) would increase σz;", "Particle shape: sphere and prolate-shaped particles normally protrude above the bed more in comparison to oblates with the same a- and b- axes measurements;", "Grain packing structure: imbrication would affect grain orientation and protrusion (Buffington and Montgomery, 1999) and influence σz as particles do not lie flat, but are layered in the direction of the flow with one end elevated above the general surface level;", "Sorting: a well-sorted patch would exhibit a smaller σz compared to an otherwise identical poorly sorted patch as grains at the upper range of grain sizes would increase σz (Hodge et al., 2009a);", "Bedforms and form roughness: grain scale topographic variation may be superimposed upon longer scale topographic variability that is not filtered out by linear detrending (particularly at larger grid sizes) which would inflate σz for a given grain size.", "The aim of this study is to advance the use of using surface roughness as a proxy for grain size in high resolution topographic surveys by disentangling the confounding effects of survey error, particle shape, sorting, grain packing structure, and bedforms on empirical relationships.", "Through a mixture of > 300 laboratory and field SfM-MVS surveys, we: (i) isolate the effect of each influencing factor; (ii) identify limits of application for σz-D50 relationships; and (iii) produce a suite of empirical σz-D50 relationships for use with distinct fluvial sedimentary facies.", "A combination of laboratory and field-based SfM-MVS and grain size surveys were undertaken.", "Laboratory surveys were performed on artificial spheres and grains sampled from the field that had been prepared deliberately to isolate the effects listed above.", "Field surveys were then undertaken to investigate the influence of bedforms on the relationships observed.", "Field surveys were undertaken at two contrasting locations (Fig. 2).", "The River Cover is a moderate gradient river (slope = 0.025) that runs through the Coverdale catchment in the Yorkshire Dales.", "The River Cover is a tributary of the River Ure which eventually flows into the Yorkshire Ouse.", "The study reach was c. 900 m long and c. 1.5 km from the headwaters with a channel width of 5–10 m and an upstream catchment area of approximately 15 km2.", "The reach is characterised by a combination of gentle meanders with associated point bars and historically straightened sections with a gravel bed, where sediment sizes range from fine gravels (2 mm diameter) to boulders (> 256 mm diameter).", "The River Ure study area is 15 km downstream of the River Cover study site, and 2 km from the confluence of the Cover and Ure rivers.", "The River Ure study area has a larger upstream catchment area (~ 500 km2), wider channel (20–30 m) and lower channel gradient (0.003).", "The study area was focused on a large gravel bar, with an area of 5000 m2.", "The bar was characterised by a less diverse size range of sediments than the River Cover, with cobbles (< 256 mm) representing the largest sediments found.", "The sediments were well sorted towards the vegetated field, and less well sorted adjacent to the water edge.", "Field survey patches were established on exposed gravel bars throughout the study reaches.", "Thirty-one patches of 1 m2 were chosen.", "Immediately following SfM-MVS surveys, a manual areal sample of the a-, b- and c-axes of all pebbles represented on the surface of the patch was conducted.", "Grain b-axis sizes ranged from 9–300 mm (aside from a single grain at 5 mm) and all particles were rounded.", "To provide grains for laboratory surveys, a further 2000 rounded, fluvial particles were collected randomly from a 150 m2 area and each axis measured (with b-axis sizes from 9–111 mm).", "Wentworth's (1922) size classification based on particle b-axes was used to separate grains into size categories.", "All sediment was fluvial in origin.", "Pebbles were further divided into shape categories based on the classification of Sneed and Folk (1958) which uses three ratios of particle a- b- and c–axes to classify shape into three categories: spheres, oblates (disc-like) and prolates (rod-like).", "These ratios measure a particle's relative equancy (c/a), elongation (b/a) and ‘disc versus rod-like aspect’ (a − b/a − c).", "SfM-MVS surveys followed standard workflows, which are well-described in the geomorphological literature (e.g., Micheletti et al., 2015; Smith et al., 2015).", "The same survey methods were used in both field and laboratory settings.", "Each gravel patch was surrounded by a number of Ground Control Points (GCPs).", "These consisted of either yellow survey targets or crosshairs on bright red backgrounds.", "Each target was surveyed using a reflectorless total station (Leica TS15) in a local coordinate system.", "For the laboratory patches, all surveys were undertaken within an immobile array of GCPs that were surveyed prior to the photogrammetric surveys.", "The 3D co-ordinates of each GCP were recorded as an average over 10 distance measurements, with a 3D accuracy of 0.003 m.", "An average of 40 ground-based hand-held oblique photos were taken for laboratory patches and an average of 25 ground-based photos were taken for field patches.", "The survey range was typically ~ 2 m.", "The same camera was used as much as possible; however, changes in equipment were required due to camera failure.", "The majority of surveys were completed using a Canon PowerShot ISO200 (10 MP, 6 mm focal length).", "Several patches were surveyed using both FujiFilm FinePix J350 (16 MP, 5 mm focal length) and AV200 (14 MP, 5.7 mm focal length) models.", "Both cameras malfunctioned; while no direct comparisons were made between cameras on the same patch, previous studies suggest this would have minimal influence on the results (Thoeni et al., 2014; Eltner and Schneider, 2015; Micheletti et al., 2015; Prosdocimi et al., 2015; Mosbrucker et al., 2017).", "Photogrammetric surveys were processed using Agisoft PhotoScan Professional 1.2.6.", "Following bundle adjustment, three GCPs were located in the imagery for lab patches and five GCPs for field patches and the point cloud was scaled and oriented.", "Georeferencing errors were typically 0.79 mm for lab patches and 1.21 mm for field patches.", "MVS image matching algorithms were performed to produce final dense point clouds which were cropped to the measured gravels.", "For laboratory patches, this method resulted in edge effects as the sides of peripheral particles (i.e., particles located at the edge of the survey area) were visible in the point cloud.", "Thus, to better reproduce field conditions, further cropping was undertaken to remove peripheral pebbles.", "Using this method, sub-millimetre data resolution was obtained on all patches.", "All patches were linearly detrended prior to calculation of σz, yielding a single roughness value for each patch (as per Hodge et al. (2009b), Brasington et al. (2012), Westoby et al. (2015) and others).", "The median value was used to calculate rh as per Vázquez-Tarrío et al. (2017).", "The overall experimental design is outlined in Fig. 2.", "The patch surveys were undertaken on a range of grain sizes for different types of patch to isolate the six factors under investigation.", "Within the experimental design, each of the laboratory patches were surveyed three times to provide replication, and the average of these three values was used in linear regressions.", "For comparison with Eq. (1), 11 sets of polystyrene balls of diameters between 0.02 m and 0.12 m were painted to represent natural sediment colours (Fig. 2).", "Balls of the same diameter were arranged into a tightly packed patch in a levelled tray.", "Surveys were evaluated both before (‘Full hemispheres’) and after cropping the peripheral particles (as detailed in Section 2.2), as the non-cropped surveys provide a better comparison with theoretical calculations, while cropped surveys better simulate field conditions.", "To assess the effect of naturally occurring particle roughness, the collected particles classified as spherical in shape were used to manually create patches of different sizes.", "The σz-D50 relationship was then compared with the patches of smooth hemispheres.", "Patches based on each particle shape (sphere/oblate/prolate) were made for each particle size class for which a sufficient number of particles were available to create a patch.", "Relationships between σz and D50 for oblates and prolates were compared with the relationship for spheres.", "Ratios of c:b axes were in the range of 0.53–1.0 (spheres), 0.18–0.69 (oblates), and 0.30–1.0 (prolates).", "Imbrication is characterised by particles leaning on each other on a contact surface, with particles consistently dipping upstream, typically at an angle of approximately 36° (Laronne and Carson, 1976), although a range of dip angles has been observed in fluvial sediments.", "This analysis was undertaken on oblate- and prolate-shaped particles only as spherical particles, by definition, cannot be imbricated.", "Patches were arranged per size class, and pebbles were manipulated to re-create signs of imbrication on a row by row basis, with measured dip angles between 36° and 38°.", "Regression equations for imbricated oblates and prolates were compared with their non-imbricated counterparts.", "Sorting was highlighted by Brasington et al. (2012) as a key issue when using high resolution topographic data to derive grain size estimates.", "Many upland rivers are of glacial origin (Howard and Macklin, 2003), resulting in a large range of sizes, with the largest boulders being well above entrainment thresholds for their associated channels.", "Although laboratory analysis was undertaken on relatively small particles (with the largest particle having a b-axis measurement of 136 mm), the effect of sorting was evaluated.", "The sphere patches used in Factor (2) were well sorted due to their separation into size classes.", "A further 20 laboratory patches were created to evaluate the effect of sorting with particles of different sizes mixed throughout the patch (i.e., no clustering of small and large particles).", "To signify poor (-moderately poor) sorting, the inclusive graphic standard deviation, σI (Folk, 1968) of b-axes of each patch had to be > 0.8 (range 0.8–1.1).", "Field patches consisted on average of 53% oblate, 24% prolate and 23% sphere shaped particles.", "Particles measured ranged in b-axis size from 5–311 mm.", "The within patch range in b-axis size varied from a minimum of 74 mm to a maximum of 288 mm.", "Visual assessment of each patch revealed that patches were not imbricated, despite being of fluvial origin.", "Inclusive graphic standard deviation was again calculated per patch, ranging from 0.698–0.983.", "To test the effect of sorting on these patches, the data set was split in half, consisting of 16 moderately well-sorted patches (σI < 0.76), and 15 moderately sorted patches (σI > 0.76).", "Unlike the patches created manually in the laboratory, naturally occurring patches were assumed not to be on an underlying flat surface.", "To assess the effect of form roughness on σz, three methods of removing form roughness were compared to σz values calculated from a linearly detrended surface.", "CloudCompare (Girardeau-Montaut, 2016) was used to detrend the patch surface using:", "a 2.5D quadric curved surface fitted to the point cloud;", "a coarse DEM at 100 mm grid size; and", "a coarse DEM at 75 mm grid size.", "For (ii) and (iii), care was taken not to reduce the resolution so much that genuine grain-scale variability was removed during the process.", "In each case, the fitted surface was subtracted from the original point cloud and σz recalculated.", "Finally, alternative roughness metrics (2σloc and rh) were computed for field patches as described previously using a 0.2 m kernel/window size to reflect the maximum grain size.", "Linear regressions were performed in Stata to identify σz-D50 relationships and to provide comparison with the theoretical linear relationship of Eq. (1).", "Normality of residuals was evaluated using Shapiro-Wilk and skewness-kurtosis tests (D'Agostino et al., 1990); heteroscedasticity was evaluated using the tests of Breusch and Pagan (1979) and Cook and Weisberg (1983).", "The relationships for oblate and imbricated oblate particles showed non-normality of residuals; however, these were only minor and in the case of oblates arises from a single data point.", "For clear comparison with other relationships, linear regressions were retained for these patches.", "The relationship for the oblate patch was also recalculated without the outlier.", "A direct comparison of the σz-D50 relationship for ‘Full hemispheres’ with Eq. (1) showed a very close correspondence (Fig. 3).", "The gradient of the relationship was slightly lower, but overall differences are negligible with a significant relationship observed (R2 = 0.995; p < 0.001) (Table 2).", "However, when peripheral hemispheres were cropped (i.e., better reflecting a field situation as no particle sides are visible in the point cloud), systematically lower σz values were observed (Fig. 3), though there was a small increase in the R2 (0.997, p < 0.001).", "For comparison with field conditions, the data sets presented in subsequent sections have had the peripheral particles cropped.", "Gravels are more irregular in shape than the hemispheres used above (Fig. 2 and Fig. 3), resulting in a higher σz for a given D50.", "Nevertheless, a strong relationship is observed (R2 = 0.974; p < 0.001) with no intercept term and a gradient that is similar to Eq. (1).", "Patches of prolate-shaped (‘rod-like’) gravels behaved similar to spherical gravels with a slightly higher σz for a given D50 (Fig. 4).", "A similarly significant relationship was observed (R2 = 0.988; p < 0.001).", "However, oblate-shaped (‘disc-like’) gravels exhibited a much lower σz and considerable scatter in the σz-D50 relationship for larger grains (though with R2 = 0.776; p < 0.001).", "This result is a consequence of oblate-shaped gravels having minimal vertical expression.", "Removing the clear outlier for oblates in Fig. 4 improved the relationship (R2 = 0.936; p < 0.001) and lowered the gradient term (8.47).", "Imbricated prolate patches resulted in a higher σz than predicted theoretically for hemispheres (Eq. (1)) and for otherwise identical non-imbricated prolate patches (Fig. 5).", "However, overall, there was little difference observed between imbricated and non-imbricated prolates, and a strongly significant relationship was again observed (R2 = 0.949; p < 0.001).", "Imbrication exhibited a larger effect on oblates resulting in an inflation of the σz as the now inclined disc-shaped particles have greater expression of the b-axis in the vertical dimension (and indeed a greater vertical expression in general).", "Consequently, the model fit improved (R2 = 0.966; p < 0.001).", "When compared with well-sorted spherical gravels, poorly sorted laboratory patches exhibited a much larger σz for any given D50 (Fig. 6A) and no σz-D50 relationship was observed.", "This difference results from small particles being obscured by surrounding larger particles and are thus not represented in the point cloud such that roughness no longer reflects D50.", "The same situation is observed in field patches (Fig. 6B), though σz values are even higher than poorly sorted laboratory patches, reflecting the additional factor of form roughness (see Section 3.6 below).", "For moderately well-sorted field patches, a significant linear relationship was observed, though with a lower R2 than all well-sorted patches described above (R2 = 0.710; p < 0.001).", "Different grain size percentiles (D50 and D84) and particle axes (b- and c-axes), were examined for relationships among the patches exhibiting a range of grain sizes both in the laboratory and in the field.", "Table 3 identifies significant relationships between σz and the 84th percentile of c-axis measurements for all such patches (Fig. 7).", "This relationship is most notable for the poorly-sorted laboratory patches for which all other grain size metrics showed no significant relationship; however, two outliers account for the poor relationship with D84 in this case (when removed R2 = 0.63, p < 0.001).", "Fig. 6 shows σz and D50 values for field patches that had only been detrended linearly (i.e., within-patch form roughness had not been removed).", "There was no overlap in σz between the laboratory and field patches with field patches having much larger σz values.", "This difference suggests that larger scale undulations at the patch scale (1 m2) were inflating σz, as the patches otherwise were identical.", "When a 2.5D quadric curved surface was fitted, the relationship between D50 and σz did not improve (R2 = 0.701; p < 0.001) (Fig. 8A).", "However, minor improvements were noticed when a coarse DEM was subtracted from the point cloud (Fig. 8B).", "DEMs with a resolution of 100 mm and 75 mm resulted in an R2 of 0.742 and 0.736, respectively.", "The σz values were reduced to a level similar to (but still slightly higher than) the poorly sorted laboratory patches, suggesting that the higher σz values of field patches were associated with form roughness.", "For the poorly sorted field patches, no relationships between D50 and σz were observed for any type of detrending.", "While the quadric detrending further improved the relationship between σz and the 84th percentile of c-axis (R2 = 0.351; p < 0.020), no improvement was observed for the DEM-detrending methods.", "For comparison with previous studies relating roughness and grain size, Table 4 shows the performance of 2σloc and rh on the field patches (Fig. 9).", "Very few differences were observed and the three roughness metrics were highly correlated (Spearman's rs > 0.87 for each pair of metrics).", "Extracting grain size data from surface roughness metrics is increasingly popular in geomorphology as high resolution survey techniques have become more readily available.", "A number of empirical relationships have emerged in the literature, but there has been little synthesis of these findings and no attempt has been made to explain the very different relationships observed (Fig. 1; Table 1).", "This study demonstrated that while grain size information can be obtained accurately from SfM data (compare the ‘Full Hemispheres’ relationship with the theoretical line in Fig. 3), sediment facies type has a pronounced effect on the results obtained.", "This finding builds on previous work that indicated similar differences for a much smaller number of gravel patches (e.g., Heritage and Milan, 2009).", "The approach is most challenging with oblate-shaped gravels and on poorly sorted sediments, where c-axis percentiles are better represented by roughness (Fig. 7).", "For the first time, we have isolated several factors influencing σz-D50 relationships to provide plausible explanations for variability in the existing literature.", "Differences between theoretical and observed σz-D50 relationships are not simply a function of survey method (Fig. 3); when standardised hemispherical shapes were surveyed using SfM-MVS, only a very minor deviation from theoretical values was observed.", "Inherent SfM precision is not a limiting factor and any inaccuracies encountered would also occur when using TLS data.", "The differences between the observed relationships for different facies are much larger than the precision limitations of either method at the plot scale.", "This finding is encouraging and demonstrates that the use of surface roughness as a proxy for grain size is technically possible.", "However, the lower σz observed for the hemispheres with peripheral particles removed indicates that high resolution surveys do not fully capture the interstitial surfaces.", "This inherent underestimation of σz may have been obscured from previous studies, as Fig. 3 shows that it is counterbalanced by the inflation of σz caused by particle irregularities.", "Particle shape affected σz primarily for oblate-shaped gravels, which displayed lower σz for a given D50 compared to both the theoretical calculation and other shapes.", "Oblate-shaped particles are relatively flat, plate-like surfaces that exhibit a reduced vertical expression relative to spheres or prolate-shaped particles with the same b-axis length.", "This result adds support to previous suggestions that roughness is more closely related to the c-axis than the b-axis, as fluvially transported grains typically come to rest with their c-axis orientated vertically (Gomez, 1993; Heritage and Milan, 2009).", "However, despite this effect, particle shape is not the major influence on σz-D50 relationships, as Brasington et al. (2012) examined a range of shapes including platy fluvial gravels and rounded beach sediment.", "Imbrication caused an increase in σz for oblate-shaped gravels because the individual particles support each other and thus reach higher local elevations for a given grain size.", "Such an increase was not observed for prolates, as the increased elevation at the top was balanced by the obscuring of the lower end of the particle and thus reducing the topographic variability within the patch.", "None of the previous studies explicitly mentioned that their patches were imbricated.", "Although with all but Westoby et al. (2015) examining water-worked sediments, some degree of imbrication would have been likely.", "All σz values observed in the laboratory patches were smaller than the values obtained from a literature meta-analysis (Fig. 10).", "This higher σz indicates that other factors such as particle sorting and form roughness are inflating the σz of previously reported field studies.", "All the previous studies showed higher σz for a given D50 than the laboratory-based analysis and the theoretical calculation (Fig. 10).", "The data presented here identifies particle sorting as the major problem when using surface roughness to estimate grain size.", "Poor sorting of gravels caused a large increase in σz for a given D50 as a result of a much wider range of grain sizes and a subsequent breakdown in the relationship for poorly sorted gravels.", "In this case, smaller particles lower the D50 of the patch but are not proportionally represented in the point cloud.", "While no fines or sand were included in this study, the same issue of poor representation in point clouds is even more pronounced where they are present.", "The presence of large grains within a patch strongly affects σz and suggests a consistent methodological bias towards larger particles in poorly sorted facies.", "Larger particles take up a larger surface area within the patch and hence comprise a higher proportion of the points in the point cloud.", "While several studies report strong relationships between roughness and higher grain percentiles, specifically D84 (Aberle and Nikora, 2006; Baewert et al., 2014; Schneider et al., 2015; Woodget et al., 2016; Vázquez-Tarrío et al., 2017), in this study roughness metrics could not predict D84 in poorly sorted patches.", "However, more significant relationships were obtained when particle c-axis data (c-axis D84) were predicted in poorly sorted oblate field patches.", "Particle sorting also had similar and pronounced effects on the relationship between σz and D50 in the literature.", "Brasington et al. (2012) examined relatively well-sorted patches compared to the work of Westoby et al. (2015) on glacial moraine that was poorly sorted.", "Of interest is the significant σz-D50 relationship observed by Schneider et al. (2015) in glacial streams (R2 = 0.91; taking only directly measured values) which was surprising because glacial material is often very poorly sorted despite having some fluvial influence (Carrivick et al., 2013).", "The line-by-number grain size sampling method used by Schneider et al. (2015) may explain this difference, as this sampling method may lead to bias towards larger grain sizes (Bunte and Abt, 2001).", "In the one location where Schneider et al. (2015) used grid-by-number sediment sampling, they observed a lower D50.", "Likewise, Smart et al. (2004) observed very different relationships when different grain sampling methods were used (Table 1).", "We undertook areal sampling of all surface particles by hand which yields very different results from the Wolman method (Bunte and Abt, 2001) but does provide an accurate representation of grains present on the surface and was best-suited for comparison with laboratory patches.", "The closest comparisons are the work of Westoby et al. (2015) and Bertin and Friedrich (2016) which used photo-sieving methods to represent all grains in the patches.", "Certainly, grain size sampling technique requires some standardisation, as different methods yield different D50 values (see Graham et al., 2012).", "The presence of methodological problems in sediment reporting must be addressed before standardised methods can be adopted.", "For example, naturally occurring patches are not one particle deep; therefore the degree of burial would also have an effect on the σz of a patch and needs further consideration.", "Likewise, some particles may be buried in the presence of sand and fines.", "Analysis of surface roughness variability alongside hiding functions may yield further insight.", "Second, interactions between the different factors examined herein also require further analysis.", "Third, the effect of SfM survey range requires examination.", "Data from UAVs (e.g., Woodget et al., 2016; Vázquez-Tarrío et al., 2017) will exhibit lower precision and resolution (Smith and Vericat, 2015) than that presented here.", "Differences seen in Fig. 1 will arise from a combination of facies variability and survey factors (e.g., point precision and density, image blur, georeferencing errors).", "Precision limitations will set a minimum grain size for which these methods can be applied reliably; for ground-based surveys this should encompass the full size range of gravels.", "Fourth, where the b-axis is not orientated vertically, a roughness-only approach would be challenging.", "The most robust method of grain size estimation might be to combine a surface roughness approach with photosieving approaches that could be performed on orthophotographs produced using SfM.", "In combination, these methods might be able to represent all three dimensions of variability of gravel particles, thereby yielding a more robust grain size estimate (see also Steer et al. (2016) for an alternative approach involving 3D geometrical fitting algorithms to obtain grain size and shape).", "While three different roughness metrics have been used in the literature, all three are highly correlated and all represent a related metric of local elevation variability subject to different detrending methods.", "The findings here support those of Baewert et al. (2014), who observed a large change in roughness value for different detrending lengths.", "Removal of sub-grid topographic variability improved regressions slightly, but came at the expense of potentially filtering out large grains.", "As expected, the reduction in σz values achieved through these detrending methods brought field values close to the σz observed for poorly sorted lab patches (for a given D50).", "The various relationships identified in Table 2 indicate that the nature of gravel facies has some influence on measured σz for a given grain size.", "More accurate grain size estimates could therefore be obtained if a suite of empirical relationships were used to represent different gravel facies.", "However, use of surface roughness as a proxy for D50 is inappropriate in poorly sorted gravels; this is a substantial limitation of the approach presented here.", "That said, stronger relationships with σz were observed for higher grain size percentiles (D84) in poorly sorted gravels, both for the c-axis and b-axis (if two extreme values were removed) and thus subsequent SfM-derived grain size estimates may still be of value for use in flow resistance formulae, for example.", "Based on the empirical research in this study, Fig. 11 presents a new decision tree that can be utilised by practitioners to identify which empirical equation is most suited to characterising river bed D50 from SfM surveys along river reaches or for specific landforms.", "This process could potentially be automated, with gravel facies type (e.g., particle shape, degree of sorting) identified automatically.", "To achieve this level of automation, a much wider variety of roughness metrics would need to be applied to point clouds than is conventionally performed (Smith, 2014).", "For example, eigenvalue ratios identify clustering of normal vector orientations (Woodcock, 1977; McKean and Roering, 2004) and the inclination index of Qin et al. (2012) may potentially discriminate imbricated patches.", "Such automation is worthy of further investigation.", "Prior to such automation, further work is needed to validate empirical relationships in Table 2 and expand the suite of equations in Fig. 11 by incorporating several other factors such as particle angularity, degree of surface armouring, packing density and burial.", "Indeed, Aberle and Nikora (2006) found σz increased with successive armour development.", "The idealized end-member patches used herein are unlikely to be replicated in reality; Fig. 11 is intended as a demonstration of a potential approach.", "As with other such studies, it is unlikely that these relationships could be reliably extrapolated to consider rivers exhibiting much larger grain sizes.", "Most importantly, sorting effects need to be better understood as poorly sorted sediments are common in many environments.", "For such a method to be of use to practitioners, a set of sampling and reporting protocols would be needed to ensure consistent patch sizes and sedimentological reporting (e.g., degree of imbrication, sorting, angularity).", "Nevertheless, the gains of such a suite of equations could be considerable (Fig. 11).", "High resolution topographic surveys of gravel facies can be interrogated to extract grain size information.", "Previous studies have identified site-specific empirical relationships; however, there is a large variation in such relationships in the published literature.", "This research has investigated the reasons for differences between published datasets and evaluated the impacts of several factors on σz-D50 relationships.", "Through a series of laboratory and field SfM surveys, sources of variability in surface roughness have been identified for the first time.", "Results indicated that the survey technique itself is capable of providing accurate grain size estimates.", "A suite of empirical equations was developed, reflecting the importance of gravel facies-type in the expression of grain size in surface roughness.", "Further refinement will be needed to improve the method for application to poorly sorted gravel patches and to validate σz-D50 relationships on other facies types.", "The extraction of grain size information from SfM-MVS surveys would be of great benefit to river restoration practitioners.", "Estimating grain size using patch scale roughness could be used to aid ecological surveys where species have preferences towards certain grain sizes (e.g., Williams and Mundie, 1978; Kondolf and Wolman, 1993), or provide a detailed baseline survey of the sedimentological nature of an area prior to river restoration (Kondolf et al., 1996; Merz et al., 2006).", "Using SfM-MVS methods to measure patch scale roughness and consequently estimate grain size is a less expensive and comparatively simple method of obtaining results that are of the same quality as TLS outputs and have thus begun to increase in popularity (e.g., Westoby et al., 2015; Woodget et al., 2015).", "This can substantially decrease the time and financial cost of thorough post-restoration geomorphic monitoring (Marteau et al., 2016).", "A combination of surface roughness and photosieving methods, both of which can be extracted from SfM-MVS surveys, may ultimately provide the most reliable grain size estimates over the largest range of gravel facies." ]	[ "Factors influencing roughness-grain-size relationships isolated in 300 SfM patches", "SfM can reproduce theoretical roughness-grain-size relationship very closely.", "Different relationships observed for spheres, oblates and prolates", "Poorly sorted patches are the main limitation though c-axes can be estimated.", "Predictions can be improved by taking into account sediment facies." ]

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